Signaling Pathways

(1)

Project-team INRIA-UPMC-CNRS REO Laboratoire Jacques-Louis Lions, CNRS UMR 7598, Université Pierre et Marie Curie, Place Jussieu 4, Paris Cedex 05, France

Abstract

Multiple signaling processes are characterized by bursts of calcium ions that moves to specific locations for optimal activation of cell activity. Intracellular calcium regulates numerous protein functions in tiny cellular domains ( < 1 μm) and small time scales ( < 1 ms). For example,Ca_V1.2 channels form signaling clusters in plasmalemmal nanodomains. Fluorescence microscopy is aimed at imaging in real time communication within and between cells. Calcium fluxes through Ca ${}^{++}$ channels and gap junctions can be imaged once constitutive proteins (e.g., connexin) have been tagged with tetracysteines [1429].

“… , because of the interconnection of all things with one another.” (G. Leibniz, Philosophical Writings, 1670)

Multiple signaling processes are characterized by bursts of calcium ions that moves to specific locations for optimal activation of cell activity. Intracellular calcium regulates numerous protein functions in tiny cellular domains ( < 1 μm) and small time scales ( < 1 ms). For example,Ca_V1.2 channels form signaling clusters in plasmalemmal nanodomains. Fluorescence microscopy is aimed at imaging in real time communication within and between cells.¹ Calcium fluxes through Ca ${}^{++}$ channels and gap junctions can be imaged once constitutive proteins (e.g., connexin) have been tagged with tetracysteines [1429].

Cyclic nucleotide signaling is compartmentalized. The location of most signaling complexes triggered by cyclic adenosine monophosphate (cAMP) that contain protein kinase-A is determined by A-kinase (PKA)-anchoring proteins (Sect. 10.8.4), although others contain guanine nucleotide-exchange factor activated by cAMP (EPAC1 and -2 or RapGEF3 and -4; Sect. 9.4.1).

Between-protein interactions determine the location and composition of each node of signaling pathways. A prototypical signaling pathway is the cAMP–PKA axis based on the production of cAMP that activates cAMP-dependent protein kinase-A. This signaling cascade can be insulated or can interact with other pathways. Signaling precision, specificity, and coordination rely on the multiple modes used by signaling axes to insulate or collaborate as well as cross-regulate [1430].

Cyclic nucleotide-dependent kinases include cAMP-dependent protein kinase-A and cGMP-dependent protein kinase-G. Protein kinase-G-binding proteins and cognate phosphodiesterases govern the subcellular PKG distribution. Activated PKG phosphorylates (activates) PDE5 tethered to the IP₃R1–PKG complex (but not non-attached PDE5), thereby enabling the spatial and temporal regulation of cGMP signaling by hydrolyzing cGMP that inhibits IP₃R1-mediated calcium release in platelets [1431].

11.1 cAMP Signaling

Cyclic adenosine monophosphate is produced by both transmembrane and bicarbonate-sensitive, soluble adenylate cyclases. Plasma membrane-bound adenylate cyclases are regulated by both stimulatory agonists that act via the Gα_s subunit of the trimeric guanine nucleotide-binding (G) protein and inhibitory agonists that operate via either the Gα_i or Gβγ subunits.

On the other hand, cAMP signaling can terminate either via cAMP hydrolysis by phosphodiesterases or cAMP efflux from the cell through ATP-binding cassette ABCc4 transporter.

This second messenger controls numerous cellular functions (Table 11.1). The diversity of action of this diffusible messenger can be explained by its location and subsequent metabolism in subcellular compartments. The organization and function of the cAMP signaling, i.e., the spatiotemporal regulation of cAMP, is indeed associated with cortical nanodomains immediately beneath the plasma membrane. The compartmentation of second messenger cAMP allows the spatial segregation of cAMP signaling events.² The cAMP subcellular compartmentation has been particularly described in cardiomyocytes [1433]. In cardiomyocytes, the multiproteic complex formed by cAMP, PKAs, and PDEs attracts G proteins, adenylate cyclases, A-kinase anchoring proteins, and phosphoprotein phosphatases.

Table 11.1

Functions of cAMP signaling (Source: [10]; CREB: cAMP-responsive element-binding protein; MC₄: type-4 melanocortin receptor; PPP1R1b: protein phosphatase-1, regulatory (inhibitory) subunit-1B; SiM1: Single-minded-1 [hypothalamic transcription factor]).

Effect	Mechanism
Neurotransmission	Phosphorylation of PPP1R1b by PKA to coordinate
	the dopamine and glutamate signaling
Glycogenolysis	Phosphorylation of phosphorylase kinase by PKA in
	skeletal muscle induced by adrenaline
Glucagon synthesis	Activation of CREB
Heat	Production control by the noradrenaline–cAMP axis
Lipolysis	Stimulation of hormone-sensitive lipase
Diet	Reduction of food intake via the MC₄–SiM1 axis
Inflammation	Inhibition of macrophages and mastocytes
Oocyte maturation	Suppression of spontaneous Ca ${}^{++}$ oscillations

The cAMP signaling pathways require many components, as cAMP effectors cause divergent responses. Second messenger cAMP diffuses from its synthesis site at the plasma membrane to subcellular compartments. Targets of cAMP include: (1) cAMP-dependent protein kinase-A (PKA), which mediates most of the actions of cAMP; (2) cyclic nucleotide-gated ion channels (CNG); and (3) guanine nucleotide-exchange factors RapGEF3 and RapGEF4³ that regulate Rap1 activity.

The cAMP–PKA pathway can [10]: (1) stimulates gene transcription via phosphorylation of cAMP response element-binding protein (CREB), thereby supporting gluconeogenesis in hepatocytes; (2) activates ion channels, such as neuronal, ionotropic, AMPA-type glutamate receptors (AMPAR) and cystic fibrosis transmembrane conductance regulator (CFTR),⁴ as well as other types of carriers such as aquaporin-2 in collecting ducts of nephrons for water reabsorption; and (3) primes the activity of various enzymes that control the cell metabolism, such as fructose (2,6)-bisphosphate 2-phosphatase,⁵ hormone-sensitive lipase,⁶ and phosphorylase kinase.⁷ Protein kinase-A phosphorylates the regulatory (inhibitory) subunit-1A of protein phosphatase-1 (PPP1R1a),⁸ thereby fostering PP1 activity. In platelets, PKA phosphorylates vasodilator-stimulated phosphoprotein (VASP) associated with filamentous actin formation, thereby attenuating actin-dependent processes involved in clotting. In insulin-secreting β cells, PKA phosphorylates (inhibits) salt-inducible kinase-2 (SIK2), which phosphorylates CREB-regulated transcription coactivator, thereby preventing its nuclear import and limiting the activity of the transcriptional factor CREB.

Other effectors are components of other signaling axes, such as cGMP-dependent phosphodiesterase PDE1a,⁹ phospholamban,¹⁰ ryanodine receptors,¹¹ and Ca_V1.1 and Ca_V1.2 channels.¹²

In addition, PKA phosphorylates cytoskeletal proteins and contributes to the transfer of ion carriers as well as translocation and fusion of vesicles with the apical membrane during the onset of acid secretion by gastric oxyntic cells.¹³

The cAMP concentration is balanced by adenylate cyclases (ACase) and phosphodiesterases (PDE), particularly members of the PDE4 family. In bacteria and amoebae such as Dictyostelium discoideum, cAMP also targets members of a family of 4 cAMP receptors (CAR1–CAR4).¹⁴

The cAMP-dependent signaling cascades are activated by G-protein-coupled receptors that can lead to opposing effects. Moreover, the coupling between cAMP pathways and other signaling pathways participates in distinct effects of cAMP signaling.

The 3 β-adrenergic receptors act via distinct cAMP signaling pathways in neonatal cardiomyocytes of mice.¹⁵ β1- and β2-adrenergic agonists produce different effects on myocardium contractility [1434].¹⁶

Subcellular compartmentation of cAMP and colocalization of components of the cAMP cascade is implicated in β-adrenergic stimulation ofCa_V1.2a channel [1435]. A molecular complex composed of ryanodine RyR2 channel, FK506-binding proteins FKBP12.6, PKA, protein phosphatases PP1 and PP2, and A-kinase anchor protein AKAP6, controls the functioning of ryanodine channels of the sarcoplasmic reticulum. In failing human hearts, these calcium channels are phosphorylated by PKA and dissociates [1436].

11.1.1 Crosstalk between Calcium and cAMP Signalings

Signalings initiated by calcium ion and cyclic adenosine monophosphate interfere. Membrane-bound, Ca ${}^{++}$ -regulated adenylyl cyclases colocalize with store-operated Ca ${}^{++}$ channels (SOC) and are influenced by Ca ${}^{++}$ entry. Calcium-stimulated adenylyl cyclase-8 interacts with the pore component of SOC channels,Orai1 protein, to coordinate subcellular changes in both Ca ${}^{++}$ and cAMP messengers [1437].

11.1.2 Crosstalk between the ERK and cAMP Signaling Pathways

Crosstalk between the cAMP and ERK pathways occurs via Raf enzyme. Elevated cAMP level activates or inhibits ERK via bRaf or cRaf, respectively. These kinases are expressed in a cell type-specific manner. Inhibition of cRaf is achieved via phosphorylation by protein kinase-A. Conversely, augmented cAMP concentration activates bRaf and hence ERK kinase. In addition, protein Tyr phosphatases dephosphorylate (inactivate) ERK enzymes. Activity of these phosphatases requires a distinct docking site that contains a serine residue, which can be phosphorylated by PKA, thereby impeding ERK binding. Uncoupling PTP from ERK thus modulates the cAMP activity via Raf on ERK kinase.

Phosphodiesterases degrade cAMP messenger. The PDE4 family is aimed at interacting with ERK kinases. Activated ERK acts on PDE4s to regulate cAMP signaling either negatively or positively depending on the expression pattern and localization of long and short PDE4 isoforms [1438].¹⁷ Phosphorylation by ERK of PDE4 long isoform inhibits PDE4 and thus increases cAMP level. Furthermore, PDE4 long isoform is phosphorylated (inactivated) by PKA kianse. Consequently, PKA activation can relieve inhibition of phosphorylated ERK on PDE4 long isoforms. On the other hand, ERK phosphorylation of PDE4 short forms causes activation. Super-short isoform is only slightly responsive to ERK phosphorylation with a slight inhibition and without feedback from cAMP as it is not targeted by PKA enzyme.

However, in smooth muscle cells, growth factors activate ERK and subsequently PKA via an autocrine process, which then stimulate PDE4d long forms as activated ERK also activates phospholipase-A2. The latter generates prostaglandin-E2 via cyclooxygenase, which activates adenylate cyclase in an autocrine fashion, leading to cAMP generation and afterward PKA phosphorylation of PDE4d long isoforms.

β2-Adrenoceptor coupling to Gs activates adenylate cyclase, but also triggers its phosphorylation by G-protein receptor kinases and recruitment of cytosolic β-arrestin, which hinders the association of β2AR with Gs subunit. Moreover, activated PKA phosphorylates β2AR, thereby not only facilitating its uncoupling from Gs, but also priming its coupling to Gi that leads to ERK activation.

The PDE4 isoforms interact with β-arrestin. The PDE4–βArr complex is recruited to β2-adrenoceptor. This process delivers active PDE4 to the site of cAMP synthesis. The PDE4–βArr complex controls a pool of membrane-anchored PKAs that phosphorylate β2ARs by lowering local cAMP levels. The recruited PDE4 can thus serve to regulate the switching of coupling of β2AR from Gs to Gi, thereby activating ERK [1438].

11.1.3 cAMP Response Element-Binding Protein and Cofactors

The cAMP signaling pathway activates cAMP response element-binding proteins. Transcription factors of the CREB family (CREB1, CREB3, and CREB5; CREB2 is ATF4; in humans, other genes encode CREB-like proteins CREBL1, CREBL2, and CREB3L1 to CREB3L4) bind to cAMP response elements of DNA, then recruit coactivators and regulate the transcription of target genes. Protein kinase-A operates in protein synthesis via CREB factors. Factor CREB is related in structure and function to cAMP response element modulator (CREM) and activating transcription factor-1 (ATF1). In neurons, CREB factors can be involved in long-term memories and neuron survival.

Transcription factor CREB recruits 2 classes of coactivators: (1) transcriptional coactivators and histone acetyltransferases (as well as polyubiquitin ligase and adaptor) CREB-binding protein (CBP) and P300 and (2) CREB-regulated transcription coactivators (CRTC1–CRTC4).¹⁸ Under basal conditions, CRTC1 and CRTC2 are phosphorylated by SNF-related kinases (SNRK or SNFRK) and bind to 14-3-3 proteins that sequester CRTCs in the cytoplasm.

Augmented intracellular cAMP levels separate PKA catalytic subunits from PKA regulatory subunits. PKA catalytic subunits then phosphorylates SNF-related kinases, hence preventing CRTC phosphorylation. Dephosphorylated CRTC1 and CRTC2 are thus released from 14-3-3 proteins. Factors of the CRTC family move to the nucleus to bind to CREB and recruit CBP or P300 agents. Histone acetyltransferases CBP and P300 then interact with RNA polymerase-2 that is recruited by CRTC-interacting protein Nono,¹⁹ which is required for cAMP-dependent activation of CREB target genes [1439]. Partner Nono complexes with CRTC2 on cAMP-responsive promoters.

Cytoplasmic retention of CRTC by 14-3-3 proteins allows integration of converging signalings. In hepatocytes, glucose homeostasis is regulated by hormones such as insulin and glucagon, and within the cell by energy status. Glucagon enhances glucose delivery from the liver, as it stimulates the transcription of gluconeogenic genes via CREB factor. When cellular ATP level is low, energy-sensing AMPK inhibits hepatic gluconeogenesis. Antagonist hormonal and energy-sensing pathways converge on CRTC2 coactivator [1440]. Phosphorylated CRTC2 is targeted by competitive hormone and energy-sensing pathways to modulate glucose output via CREB-mediated hepatic gene expression. Sequestered in the cytoplasm under feeding conditions, CRTC2 is dephosphorylated and transported to the nucleus where it elicits CREB-dependent transcription. Conversely, activated AMPK represses the gluconeogenic program as it promotes CRTC2 phosphorylation. In pancreatic islet cells, phosphorylated CRTC serves as a convergent node for cAMP and calcium-signaling pathways to CREB-dependent transcription [1441]. Synergy of these pathways on cellular gene expression is mediated by a module made of calcium-regulated phosphatase PP3 and Ser/Thr kinase SIK2 that leads to CRTC2. Circulating glucose and gut hormones trigger calcium and cAMP pathways that disrupt CRTC2–14-3-3 complexes. Calcium influx increases calcineurin activity and cAMP inhibits SIK2 activity.

11.1.4 Adenylate Cyclases

Nine adenylate cyclases catalyze the synthesis of the second messenger cAMP (Table 11.2). These 9 differentially regulated, membrane-bound adenylate cyclases are isoforms of type-1, G-protein-responsive, transmembrane adenylate cyclases (tmAC or _mAC). Adenylate cyclase-10 and its isoforms that are encoded by the ADCY10 gene constitute a distinct type of adenylate cyclases.

Table 11.2

Adenylate cyclase distribution and regulators (Source: [889]; Cam: calmodulin; CamK: calmodulin-dependent kinase).

Type	Distribution	Activators	Inhibitors
AC1	Brain,	Gs,	Gi, Gβγ
	adrenal glands	Ca ${}^{++}$ –Cam
AC2	Brain	Gs, Gβγ, PKC	Gi
AC3	Brain,	Gs	Gi,
	olfactory epithelium	Ca ${}^{++}$ –Cam	CamK2
AC4	Ubiquitous	Gs, Gβγ, PKC
AC5	Brain, heart,	Gs	Gi, Ca ${}^{++}$
	adrenal glands
AC6	Brain, heart,	Gs	Gi, Ca ${}^{++}$ , PKA
	kidney, liver		PKA, PKC
AC7	Ubiquitous	Gs, Gβγ, PKC
AC8	Brain, lung,	Gs,	Gi, Gβγ
	pancreas	Ca ${}^{++}$ –Cam
AC9	Brain, heart, cochlea,	Gs	Ca ${}^{++}$ –PP3
	skeletal muscle

11.1.4.1 Soluble Adenylate Cyclase Isoforms

A distinct category (type-2) of adenylate cyclase is constituted by soluble enzymes (sAC). They depend on Mn ${}^{++}$ ion. They are modulated by Ca ${}^{++}$ and HCO₃ ⁻ ion in a pH-independent process. They are insensitive to heterotrimeric G protein and other regulators of membrane-bound enzymes.

Calcium and bicarbonate synergistically activate sAC [1442]. In fact, Mg ${}^{++}$ , Mn ${}^{++}$ , and Ca ${}^{++}$ increase adenylate cyclase activity in a dose-dependent manner [1443]. Whereas human orthologs of sAC responds minimally to HCO₃ ⁻ in the absence of divalent cations, HCO₃ ⁻ stimulates Mg ${}^{++}$ -bound sAC, but inhibits Mn ${}^{++}$ -bound sAC in a dose-dependent manner.

Soluble adenylate cyclase has approximately 10-fold lower affinity for its ATP^Mg substrate [1444]. It is a predominant source of cAMP in testis and sperm cells. Soluble adenylate cyclase is an evolutionary conserved bicarbonate sensor. In mammals, it is involved in bicarbonate-induced, cAMP-dependent processes in sperm that enable fertilization. This ubiquitous protein also participates in multiple other bicarbonate- and carbon dioxide-dependent mechanisms, e.g., diuresis, breathing, blood flow, and cerebrospinal fluid and aqueous humor formation.

Alternatively spliced isoforms include: (1) 187-kDa, full-length (sAC_FL) and (2) 53-kDa, truncated, highly active, of relatively low abundance (_tsAC) variants [1445].

Soluble adenylate cyclase is associated with various intracellular organelles (centrioles, nucleus, and mitochondria, as well as mitotic spindle and midbodies) [1442]. Cytoplasmic sAC colocalizes with microtubules, but not with microfilaments [1443]. Soluble adenylate cyclase resides throughout the nervous system (dorsal root ganglia, spinal cord, cerebellum, hypothalamus, and thalamus) [1445].²⁰

Soluble adenylate cyclase may act as a cellular sensor of pH in epididymis and kidney, a CO₂ and HCO₃ ⁻ sensor in airway cilia, a mediator of oxidative burst in response to tumor-necrosis factor in human neutrophils, and a modulator of the cystic fibrosis transmembrane conductance regulator (CFTR) in the corneal endothelium and human airway epithelium [1445].

11.1.4.2 Transmembrane Adenylate Cyclase Isoforms

Many cell types express more than one adenylate cyclase species. However, Ca ${}^{++}$ –calmodulin-stimulated adenylate cyclases AC1, are restricted to neurons and secretory cells.

Adenylate cyclases are activated by Gs proteins and inhibited by Gi proteins. Adenylate cyclases are regulated by different other signaling effectors, particularly Ca ${}^{++}$ ion. Like AC1, AC3 and AC8 are also activated by Ca ${}^{++}$ –calmodulin [1446]. The Ca ${}^{++}$ –PP3 complex inhibits AC9 enzyme. Calcium ion and protein kinase-C repress AC6 subtype. Calcium-inhibited AC5 mainly localizes to the striatum and heart. Isozymes AC2, AC4, and AC7 are insensitive to Ca ${}^{++}$ , but stimulated by protein kinase-C. Adenylate cyclases AC5 and AC6 are highly synthesized in the heart.

Like G-protein-coupled receptors and glycosyl-phosphatidylinositol-anchored proteins, adenylate cyclases, especially Ca ${}^{++}$ -regulated ones, can be found in membrane rafts and caveolae. Certain regulatory complexes are made of G-protein-coupled receptors, like β2-adrenergic receptors, with K_IR3 channels and adenylate cyclase [1447]. These complexes are not disturbed by receptor activation or functioning of Gα subunit. However, Gβγ interferes with the formation of the dopamine receptor–K_IR3 channel complex, but not with the maintenance of the complex.

Parathyroid hormone stimulates cAMP formation and sensitizes inositol trisphosphate receptors via cAMP second messenger. Adenylate cyclases are closely apposed to IP₃R receptors. Upon stimulation by cAMP following adenylate cyclase activation, IP₃R2 that directly links to adenylate cyclases AC6 is able to release huge amounts of calcium from intracellular stores [1448]. This massive calcium release functions as a switch (all-or-none process). A graded response results from the recruitment of activated complexes, but not modulation of a given complex. Two modes of cAMP signaling then exist: (1) binary mode that depends on local cAMP delivery, during which cAMP synthesized by AC6 activates IP₃R2; and (2) analog that is much more influenced by cAMP degradation, as local cAMP gradients target signaling effectors remote from adenylate cyclases. In addition, the IP₃R2–AC6 complex is inhibited by calcium. Therefore, local calcium concentration can fluctuate due to this negative feedback loop.

11.1.5 Phosphodiesterases

Cyclic nucleotide phosphodiesterases hydrolyze cAMP and cGMP cyclic nucleotides to AMP and GMP, respectively. They thus control the rate of degradation of cAMP and cGMP second messengers and terminate their signaling (Table 11.3). Phosphodiesterases bind metal ions ( Mg ${}^{++}$ , Mn ${}^{++}$ , and Zn ${}^{++}$ ) [1449]. Calmodulin is a Ca ${}^{++}$ -dependent regulator of some types of phosphodiesterases (Tables 11.4 to 11.7).

Table 11.3

Substrate specificity of cyclic nucleotide phosphodiesterases. Despite homology of their catalytic domains, slight structural differences determine the type of substrates of phosphodiesterases. Some phosphodiesterases are selective hydrolases, others have a dual substrate specificity.

Substrate(s)	PDE subtypes
cAMP	PDE4, PDE7–PDE8
cGMP	PDE5–PDE6, PDE9
cAMP, cGMP	PDE1–PDE3, PDE10–PDE11

Table 11.4

Phosphodiesterase distribution and substrates (Part 1; Sources: [889, 1450, 1451]; Cam: calmodulin; CamK: calmodulin-dependent kinase; SMC: smooth muscle cell; vSMC: vascular smooth muscle cell).

Type	Distribution	Substrates
PDE1a	Ubiquitous	cGMP > cAMP
	PDE1a1 in lung and heart; PDE1a2 in brain
PDE1b	Brain	cGMP > cAMP
	PDE1b1 in neurons, lymphocytes, SMCs
	PDE1b2 in macrophages and lymphocytes
PDE1c	Brain, blood vessels	cGMP = cAMP
PDE2a	Brain, heart,	cGMP = cAMP
	adrenal cortex, liver
	Platelets, macrophage subtypes, endothelial cell subsets; thymocytes
	PDE2a1 is cytosolic, PDE2a3 and PDE2a2 are membrane-bound
PDE3a	Heart, kidney,	cGMP = cAMP
	vSMCs, platelets
PDE3b	Brain, kidney,	cAMP
	vSMCs, adipocytes,
	T lymphocytes, macrophages, hepatocytes, β cells

Table 11.5

Phosphodiesterase distribution and substrates (Part 2; Sources: [889, 1450, 1451]; Cam: calmodulin; CamK: calmodulin-dependent kinase; SMC: smooth muscle cell; vSMC: vascular smooth muscle cell).

Type	Distribution	Substrates
PDE4a	Ubiquitous	cAMP
	PDE4a1 links to membrane; AKAP-bound PDE4a5 in membrane ruffles
PDE4b	Ubiquitous	cAMP
PDE4c	Lung, testis,	cAMP
	several cell lines mainly
	of neuronal origin
PDE4d	Ubiquitous	cAMP
PDE5a	SMC, platelets,	cGMP
	brain, lung, heart,
	kidney, skeletal muscle
	PDE5a1 and PDE5a2 are widespread, PDE5a3 is specific to vSMCs

Table 11.6

Phosphodiesterase distribution and substrates (Part 3; Sources: [889, 1450, 1451]; Cam: calmodulin; CamK: calmodulin-dependent kinase; SMC: smooth muscle cell; vSMC: vascular smooth muscle cell).

Type	Distribution	Substrates
PDE6a	Rods, pineal gland	cGMP
PDE6b	Rods, pineal gland	cGMP
PDE6c	Cones	cGMP
PDE6d	Rods	cGMP
PDE6g	Rods	cGMP
PDE6h	Cones	cGMP
PDE7a	Brain, heart, kidney,	cAMP
	skeletal muscle,
	endothelial cells, immunocytes
	PDE7a1 in immunocytes, PDE7a2 in heart
PDE7b	Brain, heart, liver,	cAMP
	pancreas, skeletal muscle
	PDE7b1 is widespread, PDE7b2 in testis, PDE7b3 in heart

Table 11.7

Phosphodiesterase distribution and substrates (Part 4; Sources: [889, 1450, 1451]; Cam: calmodulin; CamK: calmodulin-dependent kinase; SMC: smooth muscle cell; vSMC: vascular smooth muscle cell).

Type	Distribution	Substrates
PDE8a	Widespread	cAMP
PDE8b	Brain, thyroid	cAMP
	PDE8b1 in thyroid, PDE8b3 in brain and thyroid
PDE9a	Brain, kidney, spleen,	cGMP
	digestive tract, prostate
	PDE9a1 is nuclear, PDE9a5 cytosolic
PDE10a	Brain, heart, thyroid,	cAMP < cGMP
	pituitary, testis
	(cytosolic PDE1Oa1–PDE10a3 variants)
PDE11a	Pituitary, adrenal glands,	cAMP = cGMP
	thyroid, salivary glands, liver, skeletal muscle, prostate, testis
	PDE11a3 is specific to testis; PDE11a4 level is the highest in prostate

Cyclic nucleotide phosphodiesterases differ according to their three-dimensional structure, kinetic properties, synthesis cell types, subcellular localization, modes of regulation, and inhibitor sensitivities. Initially, the multiple forms of PDE were classified into 3 categories: cAMP-targeting PDEs, cGMP-targeting PDEs, and PDEs, according to their affinities for cyclic nucleotides. They were afterward categorized into major categories, such as Ca ${}^{++}$ –calmodulin-stimulated PDE, cGMP-stimulated PDE, cGMP-inhibited PDE, and cAMP-stimulated PDE, according to their regulatory and kinetic properties. Because the list of new PDE isozymes expands, a new nomenclature has been proposed based on the primary structure. In fact, cyclic nucleotide phosphodiesterases constitute 11 families, each family containing several isoforms and splice variants. In fact, 21 different transcripts generate via alternative transcriptional start sites and alternative splicing more than 100 functional PDE enzymes.

Certain members of the PDE family induce vasodilation either by acting directly in vascular smooth muscle cells such as those of pulmonary arteries [1452]²¹ or via endothelial cells such as those of the aorta [1453]²² (Tables 11.8 and 11.9).

Table 11.8

Function of PDEs (Part 1; Source: [1450]: ANP: atrial natriuretic peptide).

Type	Effect
Calcium- and calmodulin-activated PDE1 phosphodiesterases
Phosphorylation by PKA (PDE1a and PDE1c)
Phosphorylation by CamK2 (PDE1b)
PDE1a	Vasomotor tone regulation
PDE1b	Dopamine signaling, immunocyte activation and survival
PDE1c	Vascular smooth muscle cell proliferation
cGMP-activated PDE2 phosphodiesterases
PDE2	Crosstalk between cGMP and cAMP pathways (inhibition),
	inhibition by ANP of aldosterone secretion,
	phosphorylation by PKA of cardiac Ca_V1.2a channels,
	long-term memory,
	vascular permeability in inflammation
cGMP-inhibited PDE3 phosphodiesterases
Phosphorylation by PKA
PDE3a	Regulation of cardiac contractility, platelet aggregation,
	vascular smooth muscle contraction, oocyte maturation,
	renin release
PDE3b	Cell proliferation, inhibitory effects of leptin,
	signaling by insulin and renin
Phosphorylation by PKA and ERK
PDE4	Cerebral function, monocyte and macrophage activation,
	neutrophil infiltration, vascular smooth muscle proliferation,
	vasodilation, cardiac contractility, fertility

Table 11.9

Function of PDEs (Part 2; Source: [1450]).

Type	Effect
Phosphorylation by PKA and PKG
PDE5	Regulation of vascular smooth muscle contraction,
	NO–cGMP signaling in platelets (aggregation)
PDE6	Ocular phototransduction
PDE7	Activation of T lymphocytes and inflammatory cells
PDE8	T-lymphocyte activation
PDE9	Cerebral NO–cGMP signaling
PDE10	Cerebral functions
PDE11	Sperm development and function

Protein PDE2 is stimulated by cGMP. Subtypes PDE4, PDE7, and PDE8 are specific for cAMP [1454, 1455]. Phosphodiesterases limit the diffusion of second messengers, subsequently influencing the activity of cyclic nucleotide-gated ion channels, RapGEF3 and RapGEF4 regulators (Sect. 9.4.1.6), and PKA and PKG kinases.

11.1.5.1 Phosphodiesterase-1 Family

Three genes (Pde1A–Pde1C) encode Ca ${}^{++}$ –calmodulin-dependent members of the PDE1 family. The 3 resulting gene products (PDE1a–PDE1c) differ in their substrate affinities, specific activities, activation constants for calmodulin, tissue distribution, molecular weights, and regulation by Ca ${}^{++}$ ion and phosphorylation. Complementary DNA segments encode cardiac PDE1 isozyme PDE1a1, cerebral 60-kDa PDE1a2 and 63-kDa PDE1b1, and 70-kDa PDE1c [1456]. In addition, alternative splicing creates diverse N- and C-termini. In humans, several splice variants of PDE1s have been identified with different Michaelis constants (K _M) for a given substrate and Ca ${}^{++}$ sensitivity (PDE1a3 and PDE1c1–PDE1c5) [1457] (Table 11.10). Nonetheless, the bulk structure of PDE1 isoforms is conserved with 4 domains: 2 calmodulin-binding domains, an inhibitory motif, and a PDE1 catalytic sequence.

Table 11.10

Kinetic properties of PDE1 isoforms (Source: [1457]; K _Cam: association constant for calmodulin). The activation of PDE1a by Ca ${}^{++}$ –calmodulin relieves auto-inhibition.

Subtype	Species	Molecular	${\mathtt{K}}_{\mathrm{{M}_{cAMP}}}$	${\mathtt{K}}_{\mathrm{{M}_{cGMP}}}$	K _Cam
		weight (kDa)	(μmol)	(μmol)	(nmol)
PDE1a1	Bovine (lung)	58	42	2.75
	Bovine (heart)	59	40	3.2	0.1
	Dog (heart)	68	2.8	2.1
PDE1a2	Bovine (brain)	60–61	32	2.7	1
PDE1a3	Human	61	51	3.5
PDE1b1	Bovine (brain)	63	12	1.2	1
PDE1c1	Mouse	72	3.5	2.2
PDE1c2	Rat		1.2	1.1
PDE1c3	Human	72	0.57	0.33
PDE1c4/5	Mouse	74	1.1	1.0

The calmodulin-stimulated phosphodiesterases-1 are ubiquitous. In particular, the 3 PDE1 isoforms are expressed in the central nervous system and many peripheral neurons, but to different degrees according to the region. Different PDE1 isoforms differentially reside in the heart and blood vessels as well as macrophages and T lymphocytes [1450]. Most PDE1 isoforms are cytosolic.

Stimulation of PDE1 enzymatic activity requires a physiological concentration of Ca ${}^{++}$ and calmodulin, once both molecules have complexed. Inactive calmodulin and PDE1 exist separately at low Ca ${}^{++}$ concentration. When Ca ${}^{++}$ concentration rises, calmodulin binds to Ca ${}^{++}$ and becomes active. Active calmodulin then associates with PDE1 (Ca ${}^{++}$ –Cam–PDE1 complex).

The inhibitory domain of PDE1, which is conserved among all PDE1 isoforms, maintains the enzyme in a low activity state in the absence of Ca ${}^{++}$ ions; full activation is restored by Ca ${}^{++}$ –calmodulin [1457]. Like Ca ${}^{++}$ -sensitive adenylate cyclases, phosphodiesterase-1 is activated by Ca ${}^{++}$ influx.²³ Unlike Ca ${}^{++}$ -sensitive adenylate cyclases that are exclusively regulated by the capacitative Ca ${}^{++}$ entry through transient receptor potential channels in non-excitable cells,²⁴ PDE1 is activated almost exclusively by Ca ${}^{++}$ influx from the extracellular space [1457].²⁵

Phosphorylation of PDE1a and PDE1c by cAMP-dependent protein kinase PKA and of PDE1b by calmodulin-dependent kinase CamK2 reduces the affinity of PDE1 isoforms for calmodulin and, thus, their sensitivity to Ca ${}^{++}$ ions [1457]. On the other hand, PDE1 isoforms are dephosphorylated (re-activated) by Ca ${}^{++}$ –calmodulin-dependent protein phosphatase PP3 (or calcineurin).

PDE1a

Cardiac PDE1a1 isoform, cerebral PDE1a2, and pulmonary and ocular PDE1a isozymes share almost identical kinetic and immunological properties, but are differentially activated by calmodulin [1456]. Calmodulin concentration in mammalian brain is approximately 10 times higher than that in mammalian heart. Consequently, cardiac PDE1a1 and ocular PDE1a isozymes have a higher affinity for calmodulin than cerebral PDE1a2 subtype. Pulmonary PDE1a isoform has the highest apparent affinity for calmodulin, because it contains calmodulin as a subunit. In addition, when calmodulin concentration increases, Ca ${}^{++}$ concentration required for half-maximal activation decreases. Differential affinity for Ca ${}^{++}$ and calmodulin of tissue-specific isozymes may result from adaptive regulation in respective tissues as well as fine-tuned control. Moreover, several acidic phospholipids, such as lysophospholipids phosphatidylinositol, and phosphotidylserine, as well as unsaturated fatty acids and gangliosides, can activate PDE1a in a Ca ${}^{++}$ -independent manner.

Isozymes PDE1a1 and PDE1a2 have a higher affinity for cGMP than cAMP (Table 11.11) [1456].

Table 11.11

Affinity of PDE1a1 and PDE1a2 for cGMP and cAMP (Source: [1456]).

Type	${\mathtt{K}}_{\mathrm{{M}_{cAMP}}}$	${\mathtt{K}}_{\mathrm{{M}_{cGMP}}}$
	(μmol)	(μmol)
PDE1a1	40	3.2
PDE1a2	35	2.7

Enzyme PDE1a interacts with apolipoprotein apoA1, neurocalcin-δ,²⁶ neuronal calcium sensor NCS1,²⁷ and hippocalcin.²⁸ Cardiac PDE1a1 and cerebral PDE1a2 are phosphorylated by cAMP-dependent protein kinase-A (Ser120) that attenuates its affinity for calmodulin [1456]. Phosphorylation is inhibited by Ca ${}^{++}$ and calmodulin. Isozyme PDE1a2 can be dephosphorylated by the calmodulin-dependent PP3 phosphatase.

The central nervous system contains the highest PDE1a activity. Yet, it is also detected in human lymphocytes and monocytes that circulate in blood. The PDE1A gene gives rise to many alternatively spliced transcripts (PDE1a1–PDE1a6 and PDE1aL42). 5′-Splice variants of the PDE1A gene generate 59-kDa PDE1a1 and 61-kDa PDE1a2 isozymes [1456].

PDE1b

Isoform PDE1b1 is detected in the central nervous system, lung, heart, smooth and skeletal muscle, and olfactory epithelium. In addition to neurons and smooth muscle cells, among other cell types, cytosolic PDE1b1 localizes to lymphocytes, monocytes, macrophages, and spermatids [1456]; cytosolic PDE1b2 in macrophages and lymphocytes [1450].

PDE1b1

Brain PDE1b1 isoform is phosphorylated by calmodulin-dependent protein kinase CamK2 that decreases PDE1b1 affinity for calmodulin, hence an increase in Ca ${}^{++}$ concentration is required for enzyme activation by calmodulin [1456]. Conversely, PDE1b1 can be dephosphorylated by calmodulin-dependent protein phosphatase PP3 that increases PDE1b1 affinity for calmodulin, thereby reducing Ca ${}^{++}$ concentration for its calmodulin-mediated activation.²⁹ When cAMP concentration augments, cAMP-dependent protein kinase PKA phosphorylates (activates) protein phosphatase inhibitor PPI1. Phosphorylated PPI1 can inhibit protein phosphatase PP1 that reverses effect of CamK2 autophosphorylation. On the other hand, PPI1 is dephosphorylated (inactivated) by PP3 phosphatase. Consequently, PP1 is reactivated and cAMP can then exert its inhibition on PDE1b1 activity.

Isoform PDE1b1 has a basal affinity for cAMP and cGMP. In the presence of Ca ${}^{++}$ –calmodulin, PDE1b1 affinity rises 2- to 3-fold for both cAMP (K _M 12 μmol) and cGMP (K _M 1.2 μmol) [1456].

PDE1b2

Granulocyte–macrophage colony-stimulating factor (CSF2) that is involved in the differentiation of the myeloid lineage upregulates PDE1b2 isoform during monocyte-to-macrophage differentiation. On the other hand, upregulation of PDE1b2 is suppressed during the differentiation of macrophages to a dendritic cell phenotype. T lymphocytes can also produce PDE1b2 [1456]. In addition, PDE1b2 is expressed strongly in the spinal cord and slighly in the putamen and caudate nucleus, thyroid, thymus, small intestine, and uterus.

PDE1c

Isoform PDE1c is expressed in the cilia of olfactory sensory neurons, where olfactory signal transduction takes place [1456]. Yet, PDE1c is elevated in pulmonary artery smooth muscle cells in both idiopathic and secondary pulmonary arterial hypertension. It also resides in cytosol of human cardiomyocytes as well as cells of the central nervous system, pancreas, bone, and testis. Several splice variants exist [1456]. Splice variants of PDE1c have different affinities for cAMP and cGMP (human native PDE1c1 ${\mathtt{K}}_{\mathrm{{M}_{cAMP} }}$ 0.9 ± 0.2μmol; ${\mathtt{K}}_{\mathrm{{M}_{cGMP} }}$ 1.2 ± 0.2μmol).

Activity of PDE1c is inhibited by protein kinase-A. Subtype PDE1c interacts with various types of adenylate cyclases (AC1, AC3, and AC5–AC9) and calmodulin-like protein-3 and atrial natriuretic peptide receptor-A precursor [1456].

11.1.5.2 Phosphodiesterase-2 Family

Phosphodiesterases-2 are encoded by a single gene (Pde2A). Three splice variants have been observed (PDE2a1–PDE2a3). Soluble PDE2a1 is cytosolic, whereas PDE2a1 and PDE2a3 bind to the plasma membrane [1450]..

Members of the PDE2 family target both cAMP and cGMP messengers. They are produced in various cell types of the brain and heart, as well as in platelets, endothelial cells, adrenal glomerulosa cells, and macrophages [1450]. Expression of PDE2 is upregulated during the differentiation of monocyte to macrophage.

In endothelial cells, PDE2a is synthesized under basal conditions only in small vessels and capillaries, but not in large vessels. In the brain, PDE2a abounds in specific regions and cell types.

Members of the PDE2 family are responsible for mutual inhibition between the cGMP and cAMP pathways. In particular, in adrenal glomerulosa cells, PDE2 mediates inhibition exerted by atrial natriuretic peptide on aldosterone secretion.³⁰ In these cells, elevation of cGMP mediated by ANP activates PDE2 that, in turn, lowers cAMP stimulated by adrenocorticotropin. In platelets, NO causes cGMP accumulation that activates PDE2 and reduces cAMP agent.³¹

In human cardiomyocytes, PDE2 regulates Ca_V1.2a channel. In addition, PDE2 intervenes in the cAMP response to catecholamine stimulation. It also mediate the inhibition of NO on cAMP agent. Like in platelets, the activation of PDE2 by cGMP is antagonized by the inhibition of PDE3 enzyme.

11.1.5.3 Phosphodiesterase-3 Family

Members of the PDE3 family hydrolyze both cAMP and cGMP messengers. They are encoded by 2 genes (Pde3A and Pde3B). Variants have been identified only for the PDE3a isoform (PDE3a1–PDE3a3) [1450].

Both PDE3a and PDE3b are regulated by phosphorylation in response to hormonal stimulation in several cell types. In platelets, adrenaline and prostaglandins exert a feedback via PKA to activate PDE3a isoform.

Insulin, insulin-like growth factor IGF1, and leptin use the PI3K pathway to phosphorylate PDE3b by activated PKB kinase [1451] (Table 11.12).³²

Table 11.12

Pancreatic β-cell phosphodiesterase (Source: [1451]). Membrane-bound PDE3b regulates the cAMP pool that modulates insulin secretion. Isoform PDE3b opposes insulin release. Activity of PDE3b in β cells may be regulated by insulin, insulin-like growth factor-1 (IGF1), leptin, and glucose. In adipocytes, insulin activates PDE3b to support its antilipolytic effect. Autocrine regulator insulin binds to its receptor on secreting β cell to inhibit its own release (negative feedback). Factor IGF1 potently activates PDE activity using the PI3K–PKB axis. Leptin may prevent augmentation by glucagon-like peptide GLP1 of glucose-induced insulin release due to reduced cAMP concentration.

Regulator	Target	Product	Catabolism
		Metabolism	Effect
Ca ${}^{++}$	PDE1
	ACase	cAMP	AMP (PDE1)
			PKA → PDE3b
	Exocytosis		Insulin release
Glucose		ATP	K_ATP,
			membrane depolarization,
			Ca ${}^{++}$ influx
			Activation of PDE1/3b
Insulin, IGF1,	PDE3b
leptin
NO	GCase	cGMP	Inhibition of PDE3b
			GMP (PDE5)

Subtype PDE3a is relatively highly expressed in platelets, vascular smooth muscle cells, cardiomyocytes, and oocytes. In platelets, PDE3a contributes to the regulation of aggregation. On the other hand, PDE3b is a major PDE in adipose tissue, liver, and pancreas, in addition to the cardiovascular apparatus [1450]. Isoform PDE3b is also synthesized in T lymphocytes and macrophages, in addition to pancreatic β cells and adipocytes.

Members of the PDE3 family are also involved in the regulation of cardiac contractility and vasomotor tone. Enzyme PI3Kγ can associate with PDE3b to control cardiac contractility. Both PDE3a and PDE3b are synthesized in vascular smooth muscle cells to modulate the vasomotor tone.

11.1.5.4 Phosphodiesterase-4 Family (PDE4)

Four cAMP-specific PDE4 subtypes (PDE4a–PDE4d), encoded by 4 genes (Pde4A-Pde4D), hydrolyze cAMP to AMP agent. More than 20 different PDE4 isoforms have been detected; they are created via alternative start sites and alternative splicing. The PDE4 isozymes have multiple promoters and transcription factors [1459].³³ Many of the PDE4 variants have a cell type-specific expression. In addition, several variants have distinct subcellular localizations [1450]. Therefore, PDE4 isoforms control spatially distinct pools of cAMP messenger.

Phosphodiesterases-4 are phosphorylated (activated) by PKA kinase. In most cells, in particular immunocytes that possess PDE4 subtypes, PDE4b and PDE4d predominate. Enzymes of the PDE4 family can homodimerize, but not heterodimerize.

Upstream conserved regions (UCR1–UCR2) of PDE4 enzymes influence outcome of phosphorylation of the catalytic unit. They regulate the catalytic activity and can interact with each other.³⁴ More precisely, ERK kinases phosphorylate enzymes of the PDE4B, PDE4C, and PDE4D subfamilies. Consequently, ERK kinases: (1) prevent the activity of long alternatively spliced variants that have both UCR1 and UCR2 motifs; (2) activate short variants that possess only UCR2; and (3) do not influence the action of supershort variants that have a truncated UCR2, thereby being catalytically inactive [1460]. Catalytically inactive PDE4 dead-short isoforms lack both UCR1 and UCR2 and have truncated N- and C-termini of their catalytic units.

Phosphatidic acid binds to long PDE4 isoforms (PDE4_L) and primes their activation [1460]. Reactive oxygen species can trigger the phosphorylation of PDE4_L isoforms at 2 sites: (1) phosphorylation by ERK at the C-terminus of the catalytic unit and (2) that at the N-terminus of the catalytic unit, which does not modify the PDE4 activity, but switch the inhibitory phosphorylation by ERK to activation [1460].

Multiple other interactors include β-arrestin-1 and -2, PDE4d-interacting protein (or myomegalin), receptor for activated C-kinase RACK1,³⁵ several A-kinase anchoring proteins, and protein Tyr kinases such as members of the SRC family [1450].

PDE4a

The isoform-specific N-terminus controls the subcellular localization of phosphodiesterases. In the absence of its N-terminus, membrane-associated PDE4a1 isoform lodges in the cytosol.

PDE4d

The PDE4D gene encodes 11 splice variants (PDE4d1–PDE4d11) due to alternative splicing. These isoforms can be classified into long (PDE4d_L, i.e., PDE4d3 to PDE4d5, PDE4d7 to PDE4d9, and PDE4d11), short (PDE4d_S, i.e., PDE4d1), and supershort (PDE4d_SS, i.e., PDE4d2, PDE4d6, and PDE4d10) categories [1460]. Each isoform is characterized by an isoform-specific N-terminal region. The catalytic unit comprises 3 subdomains that are coordinated by 2 metal ions ( Mg ${}^{++}$ and Zn ${}^{++}$ ). Widespread PDE4d isoforms can reside in the cytosol and associate with membranes.

Phosphorylation of PDE4d_L by ERK1 and ERK2 on catalytic unit (inhibitory) antagonizes that of PKA on UCR1 domain (stimulatory), and conversely the action of PKA neutralizes the effect of ERK enzymes [1460]. These kinases can thus organize feedback loops.

The N-terminal region enables the recruitment of PDE4d isoforms by scaffold proteins, such as β-arrestin, AKAPs, disrupted in schizophrenia DISc1,³⁶ receptor of activated protein kinase-C RACK1, and nuclear distribution gene-E homolog (NuDe)-like protein NDeL1,³⁷ thereby determining sites of distinct PDE4 isoforms (local cAMP sinks) to compartmentalize cAMP signaling [1460].

Myomegalin, or phosphodiesterase-4D-interacting protein (PDE4dIP),³⁸ of the Golgi body and centrosome interacts directly with PDE4d enzymes.

Isoform PDE4d5 that is detected in cardiomyocytes and vascular smooth muscle cells has an additional binding site for β-arrestin in its N-terminus. The βArr–PDE4d5 complex anchors Ub ligase DM2 [1460]. The subsequent transient ubiquitination of PDE4d5 further enhances the βArr–PDE4d5 interaction (autoamplification). This transient ubiquitination can result from the activation of receptors, such as β2-adrenoceptor and vasopressin V₂ receptor, that recruit the βArr–PDE4d5–DM2 complex, thereby delivering a cAMP-degrading complex to the plasma membrane, where is synthesized cAMP messenger.

Unlike β2-adrenoceptor that attracts the βArr–DM2–PDE4d5 complex, β1-adrenoceptor directly interacts with PDE4d8 [1460].

In vascular smooth muscle cells, activation of PDE4d increases its affinity for Mg ${}^{++}$ and decreases cAMP concentration. Isoform PDE4d also controls cAMP level in respiratory conduits. Parasympathetic control of the smooth muscle tone of airways involves: (1) muscarinic M₁ and M₃ receptors coupled to phospholipase-C and Ca ${}^{++}$ as well as (2) M₂ receptors inhibitory for adenylate cyclase. Released acetylcholine from parasympathetic nerves induces smooth muscle contraction, with a control by M₂ muscarinic receptors to limit the bronchoconstriction magnitude.

Multiprotein signaling complexes focalize the enzyme activity that transduce actions of many signaling agents. Isoform PDE4d and PKA form a complex coordinated by A-kinase anchoring proteins. In cardiomyocytes, AKAP6 assembles a self-regulatory cAMP signaling module with PKA and PDE4d3 [1461]. Enzyme PDE4 is recruited by β-arrestin close to G-protein-coupled receptors [1462].³⁹

In cardiomyocytes, AKAP9 and PDE4d complex withK_V7.1 channel to control the basal and β-adrenoceptor-activated channel activity (i _Ks current) [1460].

Protein AKAP7 that anchors PKA to the basolateral plasma membrane of epithelial cells and interacts withCa_V1.2a channels in skeletal muscle cells and cardiomyocytes, thereby facilitating Ca_V1.2a phosphorylation by protein kinase-A, interacts directly with PDE4d3 enzyme. The AKAP7–PDE4d complex may regulate the activity of AKAP-tethered PKA on aquaporin-2 + vesicles, thereby regulating aquaporin-2 transfer and water permeability [1460].

Cardiomyocytes and arterial smooth muscle cells use distinct phosphodiesterase PDE4d splice variants to regulate PKA activity [1463]. In rat cardiomyocytes (hypertrophied or not), PDE4d3 participates in the PKA–AKAP complex, whereas in rat aortic smooth muscle cells, PDE4d3 is not detected in PKA–AKAP complex, but PDE4d8 associates with PKA–AKAP complexes. Both PDE4d8 and PKA are recruited to the leading edge of migrating vascular smooth muscle cells.

In cardiomyocytes, PDE4d3 interacts with the ryanodine RyR2 receptor [1460]. This interaction may yield a negative feedback to limit β-adrenoceptor-primed PKA phosphorylation of RyR2 receptor.

Prostaglandin-E2 is a major pro-inflammatory agent in the cardiovascular apparatus and a potent inducer of cAMP production (Gs–ACase–cAMP–PKA axis), which remains confined along the plasma membrane, via stimulatory G protein coupled to type-E prostaglandin EP4 receptor, the most abundant EP subtype in the myocardium. In addition, PGE2 attenuates the cardiac contractility (negative inotropic effect) caused by adrenergic stimuli [1464]. Prostaglandin-E2 activates PKA that phosphorylates (activates) PDE4d isozyme that degrades cAMP, thereby impeding the transfer of cAMP from the plasma membrane to the sarcoplasmic reticulum. On the one hand, catecholamines stimulate Gs-coupled β-adrenergic receptors, hence cAMP signaling that initiates phosphorylation of PKA substrates on the plasma membrane, sarcoplasmic reticulum, and myofibrils, to support cardiomyocyte contractility. β-Adrenoceptor associates with different PDE4d subtypes, but, once it is liganded, PDE4d isoforms dissociate from activated β-adrenoceptors to allow cAMP diffusion from the plasma membrane to intracellular organelles [1464]. On the other, cAMP produced in response of PGE2 cannot access the sarcoplasmic reticulum to foster calcium signaling and cardiomyocyte contraction. In particular, PGE2 precludes phosphorylation by PKA of phospholamban primed by β-adrenoceptors. Therefore, a Gs-coupled prostaglandin receptor is able to prevent the intracellular signaling initiated by a Gs-coupled catecholamine receptor via the control of cAMP transport that results from intracellular cAMP degradation. Moreover, PGE2 prevents PDE4 dissociation from β-adrenoceptors [1464]. These 2 types of Gs-coupled receptors — EPs and βARs — colocalize in many cell types, such as neurons and astrocytes, in addition to cardiomyocytes [1464]. In microglial cells, EP promotes and βAR represses inflammation.

Phosphodiesterase-4D complexes with SH3 and multiple ankyrin repeat domain-containing protein SHAnk2 and cystic fibrosis transmembrane conductance regulator (CFTR) that controls phosphorylation by PKA of CFTR [1460].

Subtype PDE4d3 can connect to RapGEF3 and -4, which activate Rap1 and Rap2 GTPases [1460]. Scaffold AKAP6 can tether to PDE4d3, PKA, ERK5, and RapGEF3 and -4 agents. Isozyme PDE4d undergoes inhibitory and stimulatory phosphorylations by ERK and PKA, respectively.

Isoform PDE4d4 interacts directly with certain SH3 domain-containing proteins, such as SRC family kinases (Abl, Fyn, Lyn, and Src) as well as PI3K and fodrin [1460]. It also links to spectrin and, then, may control the phosphorylation by PKA of the microtubule-stabilizing Tau protein.

Inhibitors that are PDE4-selective and inhibit all PDE4 isoforms are used to treat chronic obstructive pulmonary disease and fibrosis, among other diseases [1460].

11.1.5.5 Phosphodiesterase-5 Family (PDE5)

Phosphodiesterase-5 that possesses high affinity-binding sites for cGMP is characterized by a relative specificity for cGMP messenger at low levels. A single Pde5 gene generates 3 variants (PDE5a1-PDE5a3) due to differentially regulated promoters.

Phosphodiesterase-5 lodges in the brain, heart, lung, kidney, liver, skeletal muscle, pancreas, gastrointestinal tract, and placenta [1450].⁴⁰

In the cardiovasular system, PDE5a is synthesized in vascular endothelial and smooth muscle cells, cardiomyocytes, as well as platelets.

Phosphodiesterase-5A participates in the regulation of vascular tone. Endothelial PDE5a resides in or near caveolin-rich membrane rafts. The vasodilatory NO–sGC–cGMP–PKG pathway is associated with caveolae. Agents NOS, sGC, PKG1, and PKA, actually localize to or near caveolae. Following cGMP catabolism by PDE5a, PKG1 activity decays, hence NOS3 phosphorylation (activation) by PKG1 (Ser1177) [1465]. In addition, NOS3 can be strongly phosphorylated by PKG2 kinase.

Phosphodiesterase-5 is phosphorylated not only by PKG, but also by PKA. The latter stabilizes its catalytic activity by enhancing the affinity of cGMP binding [1450]. This modification prolongs PDE5 activation ( feedback loop initiated by cGMP synthesis via guanylate cyclases). Messenger cGMP operates as a PDE5 feedforward activator.

11.1.5.6 Phosphodiesterase-6 Family (PDE6)

Members of the PDE6 family are photoreceptor phosphodiesterases, as they contribute to the conversion of a light signal into a photoresponse. They are encoded by 3 genes (Pde6A–Pde6C). In addition, PDE6γ and PDE6δsubunits modulate PDE6 activity and localization.

11.1.5.7 Phosphodiesterase-7 Family (PDE7)

Members of the PDE7 family, like PDE4 and PDE8, is highly selective for cAMP, especially at low cAMP concentrations. In humans, 3 splice variants exist (PDE7a1–PDE7a3).

They are synthesized in the brain, heart, lung, kidney, muscle, spleen, and thymus, as well as various types of immunocytes [1450]. However, PDE7b2 and PDE7b3 are restricted to testis and heart, respectively.

11.1.5.8 Phosphodiesterase-8 Family (PDE8)

Members of the PDE8 family are encoded by 2 genes (Pde8A–Pde8B). Both isoforms (PDE8a–PDE8b) have a very high affinity for cAMP (the highest cAMP affinity among PDEs; K _M 40–150 nmol for cAMP, K _M > 100 μmol for cGMP [1466]) that confers a specificity for this messenger.

Both PDE8a and PDE8b share C-terminal catalytic domain common to all PDEs, but they differ from other PDEs by their N-terminus. The N-terminus contains a receiver domain (Rec) and a close Period, ARNT, and Sim motif (PAS), a regulatory sequence of several regulators of circadian rhythm.

PDE8a

Subtype PDE8a possesses several variants produced by alternative splicing and alternative start sites [1450]. Isoform PDE8a2 is a spliced variant of the most abundant variant PDE8a1 without the PAS domain; PDE8a3 is a truncated protein without the PAS and REC sequences; PDE8a4 and PDE8a5 are identical truncated proteins that are longer than PDE8a3 and also lacks the PAS and REC regions. Various PDE8b variants also arise from alternative splicing.

Expression of Pde8A mRNA is widespread with its highest levels in the heart, kidney, small intestine, colon, spleen, ovary, and testis [1450]. Protein PDE8a1 has been detected in T lymphocytes. Production of PDE8b is more restricted, mainly in the brain and thyroid. However, alternatively spliced isoform PDE8b1 is detected only in the brain, although PDE8b3 is produced in the brain and thyroid.

Activity of PDE8a depends on divalent cations, either Mg ${}^{++}$ or Mn ${}^{++}$ ion. It can be phosphorylated by protein kinase-A and -G. Enzyme PDE8a modulates excitation–contraction coupling in ventriculomyocytes, as its decreases calcium transients and calcium spark frequency [1466].

PDE8b

In humans, full length PDE8b (PDE8b1) contains 885 amino acids. It has a high affinity for cAMP (K _M 101 nmol) [1467]. Several splice variants of PDE8b exist in humans, with specific tissue expression. In particular, variants lacking the PAS domain have been observed.

11.1.5.9 Phosphodiesterase-9 Family (PDE9)

Ubiquitous members of the PDE9 family have the highest affinity for cGMP messenger ( ${\mathtt{K}}_{\mathrm{{M}_{cGMP}}}$ 70–170 nmol). The single family member PDE9a coexist with numerous variants after mRNA processing, but only nuclear PDE9a1 and cytosolic PDE9a5 have been characterized [1450].

11.1.5.10 Phosphodiesterase-10 Family (PDE10)

Phosphodiesterase-10 is encoded by a single PDE10A gene. Four variants PDE10a1–PDE10a4) have been identified. Phosphodiesterase-10 hydrolyzes both cAMP and cGMP in vitro, with a higher affinity for cAMP than for cGMP substrate [1450]. In vivo, PDE10 may function as a cAMP-inhibited cGMP phosphodiesterase.

Phosphodiesterase-10 is mainly synthesized in the central nervous system. It also produced in the thyroid and pituitary gland as well as skeletal and cardiac muscles [1450].

Variant PDE10a2 can be phosphorylated by cAMP-dependent protein kinase-A, thereby moving from the Golgi body to the cytosol [1450].

11.1.5.11 Phosphodiesterase-11 Family (PDE11)

A single gene encodes members of the PDE11A family. Four variants (PDE11a1–PDE11a4) have been detected, among which 3 isoforms are truncations of varying lengths of the longest PDE11a4 variant. They hydrolyze both cAMP and cGMP nucleotides ( ${\mathtt{K}}_{\mathrm{{M}_{cAMP}}}$ 1 –6 μmol; ${\mathtt{K}}_{\mathrm{{M}_{cGMP}}}$ 0.5–4 μmol) [1450].

In humans, Pde11A1 mRNA is prominent in skeletal muscle and prostate; Pde11A3 mRNA is found specifically in testis; and Pde11A4 mRNA is highly expressed in prostate, but also in the hypophysis, heart, and liver [1450]. In fact, PDE11a variants localize to epithelial, endothelial, and smooth muscle cells, but at the highest levels in the kidney, adrenal gland, colon, skin, and prostate.

11.2 cGMP Signaling

Cyclic guanosine monophosphate (cGMP) is a second messenger implicated in cell growth, smooth muscle cell relaxation, homeostasis, and inflammation, among other tasks [1468].

Two well-known activators of cGMP signaling exist (Fig. 11.1): atrial natriuretic peptide⁴¹ and nitric oxide. Natriuretic peptides bind to a transmembrane receptor, the particulate guanylate cyclase (pGC; Table 11.13).⁴² Nitric oxide acts via soluble guanylate cyclase (sGC), especially in smooth muscle cells.⁴³

Fig. 11.1

cGMP signaling pathways are involved in cellular functions (vasodilation, clotting, angiogenesis, etc.). Signaling is induced by natriuretic peptides and nitric oxide, via particulate guanylate cyclase (pGC) and soluble guanylate cyclase (sGC). Effectors are cGMP-dependent protein kinases-1 (PKG1) and phosphodiesterases (PDE; adapted from [1468]).

Table 11.13

Types of guanylate cyclases (GC or GuCy; ANPR: atrial natriuretic peptide receptor; GCA: guanylate cyclase activator; GCAP: guanylate cyclase-activating protein; HStaR: heat stable enterotoxin receptor; NPR: natriuretic peptide receptor; RetGC: retinal guanylate cyclase; RoSGC: rod outer segment membrane guanylate cyclase).

Type	Gene	Other aliases and name
Particulate guanylate cyclases
GC2a	GUCY2A	GuCy2a, GCa
		NPR1, NPRa, ANPRa, ANP_A
GC2b	GUCY2B	GuCy2b, GCb
		NPR2, NPRb, ANPRb, ANP_B
GC2c	GUCY2C	GuCy2c, GCc, HStaR
GC2d	GUCY2D	GuCy2d, GCd, RetGC1, RoSGC1
GC2e	GUCY2E	GuCy2e, GCe
GC2f	GUCY2F	GuCy2f, GCf, RetGC2, RoSGC2
GC2g	GUCY2G	GuCy2g, GCg
Soluble guanylate cyclase subunits
GC1α1	GUCY1A3	GC^Sα1 (α3)
GC1β1	GUCY1B3	GC^Sβ1 (β3)
GC1α2	GUCY1A2	GC^Sα2
GC1β2	GUCY1B2	GC^Sβ2
Activators
GCA1a	GUCA1A	GCAP1
GCA1b	GUCA1B	GCAP2
GCA1c	GUCA1C	GCAP3
GCA2a	GUCA2A	GCAP-I, guanylin
GCA2b	GUCA2B	GCAP-II, uroguanylin

At least 3 types of cGMP-binding effectors transduce cGMP signals: (1) cGMP-modulated cation channels; (2) cGMP-dependent protein kinases (PKG or cGK);⁴⁴ and (3) cGMP-regulated phosphodiesterases (PDE).⁴⁵ Protein kinase-G1 stimulates myosin phosphatase. It can also interact with inositol (1,4,5)-trisphosphate receptor-associated PKG1β (IRAG), inhibiting intracellular Ca ${}^{++}$ release. Agent IRAG may be involved in the anti-platelet effects of exogene nitric oxide. Vasodilator-stimulated phosphoprotein (VASP) activated by PKG1 inhibits platelet adhesion to endothelial cells.

In vascular smooth and cardiac striated myocytes, cGMP level is determined by activity of 3 types of guanylate cyclases: (1) receptor for nitric oxide sGC; (2) receptor for A- and B-type natriuretic peptides GCa; and (3) receptor for C-type natriuretic peptide GCb. In vascular cells, cGMP is hydrolyzed by phosphodiesterases, mainly PDE5, as well as dual-substrate cGMP–cAMP-hydrolyzing phosphodiesterases PDE1 and PDE2.

In vascular smooth muscle cells, cGMP and its effector PKG1 diminish contraction state, as PKG1 phosphorylates regulators of calcium handling and cytoskeleton. Moreover, PKG1 stimulates growth of vascular smooth muscle cells, as it stimulates sumoylation of the ELk1 transcription factor.

In cardiomyocytes and cardiac fibroblasts, cGMP–PKG1 signaling can counteract trophic action of hormones and growth factors and impedes remodeling in response to pressure overload by repressing some cascades involved in maladaptive cardiac hypertrophy, such as Gq-coupled receptors via PKG1-mediated activation of RGS2 regulator of G-protein signaling. Pulmonary arterial hypertension is in fact associated with an increased expression of PDE5 phosphodiesterase.

Nitric oxide is an intercellular signaling molecule in most cell types, having diverse activities (blood flow regulation, neurotransmission, immune response; Sect. 11.10). Guanylate cyclase-coupled receptor activation by NO binding reversibly triggers a conformational change that transduces the signal with a ligand-concentration dependent intensity. Downstream effectors include kinases, phosphodiesterases, and ion channels. Receptor deactivation follows NO unbinding.

Cyclic guanosine monophosphate is also linked to Rac GTPase for the regulation of actin cytoskeleton during cell migration. Constitutively active Rac increases the activity of transmembrane guanylate cyclases in a cell subjected to a chemotactic signal [1469]. Consequently, concentration in second messenger cGMP rises up to tenfold. Rac effector Ser/Thr P21-activated kinases (PAK1 and PAK2) bind and stimulate the activity of guanylate cyclases. The Rac–PAK–GC–cGMP pathway is involved in fibroblast migration induced by platelet-derived growth factor and lamellipodium formation.

11.3 Signaling via Cell Junctions

11.3.1 Elastin–Laminin Receptor

Certain transmembrane receptors couple the cells to the extracellular matrix and can transduce applied loadings. Elastin strongly increases calcium influx and inhibits calcium efflux in aortic smooth muscle cells [1470]. It also increases sodium influx in monocytes. Elastin-κ induces a vasorelaxation mediated by the elastin–laminin receptor (ELR) and endothelial NO production [1471].

The elastin–laminin receptor is located on both endothelial and smooth muscle cells. Both laminin and elastin bind to cells via the elastin–laminin receptor. The elastin–laminin receptor is a heterotrimer that recognizes several hydrophobic domains on collagen-4, elastin, and laminin. It forms a transmembrane complex with other proteins.

Cyclic stretch of the matrix of cell culture (30-mn duration), which mimics pressure pulse effects on the vessel wall, inhibits the expression of Fos transcription factor, as well as the proliferation of coronary vascular smooth muscle cells grown on elastin matrices [1472]. These effects depend on ELR signaling. On the other hand, cells do not exhibit changes in gene expression or proliferation when the matrix is simply stretched.

11.3.1.1 Collagen Signaling

Heterodimeric integrins operate as receptors for cell adhesion to various constituents of the extracellular matrix ( collagens, fibronectin, fibrinogen, laminins, osteopontin, tenascins, thrombospondins, and vitronectin) or cellular receptors such as cell adhesion molecules (e.g., VCAM1 and ICAM). Among integrins, β₁-integrins coupled with α₁, α₂, α₁0, and α₁1 subunits that link to collagens via their I domain owing to divalent metal Mg ${}^{++}$ cation are the so-called collagen receptors. In addition, α_Xβ₂-integrin may also act as a collagen receptor [1473]. Collagen binding is not necessarily specific, as α₁ and α₂ subunits tether laminins, but with a lower affinity.

Integrin-α₁ and -α₂β₁ are ubiquitous, in particular on the surface of smooth muscle and endothelial cells, whereas α₁₀– and α₁₁β₁-integrins have a more restricted expression [1473]. Integrin-α₂β₁ is the single collagen-binding integrin in platelets (GP1a–GP2a). Integrin-α₁₀β₁ is located with collagen-2 on cartilage cells as well as striated myocytes (heart and skeletal muscle). Integrin-α₁₁β₁ is detected on mesenchymal cells during embryogenesis and in muscles in adults.

Integrin-α₁β₁ preferentially attaches to collagen-4 of basement membrane and collagen-13, α₂β₁– and α₁₁β₁-integrin to fibrillar collagens, and α₁₀β₁-integrin to collagen-2 [1473].

After activation by collagen of integrins, integrins recruit cytoplasmic Tyr kinases, in particular focal adhesion kinase. Cytoplasmic tail of β₁-integrin interacts with paxillin and talin that complex with focal adhesion kinase. Autophosphorylation of FAK enables it to phosphorylate its effectors that are mainly cytoskeletal proteins, such as paxillin, talin, α-actinin, tensin, zyxin, VASP, and vinculin. These mediators then recruit adaptors (GRB2, SHC1, and BCAR1 [CAS]), PI3K, and cytosolic Tyr kinases like Src enzyme.

Signaling by FAK also activates small GTPases. Activated Ras then initiates a cascade of Ser/Thr kinases, such as ERK and JNK, to remodel the cytoskeleton. Vinculin and paxillin are scaffold proteins that yield connections to microtubules.

Depending on cell type, different pathways are activated by collagen-binding integrins. Vascular smooth muscle cells do not proliferate in response to collagen. Only collagenase-degraded collagen simulates FAK activation and causes cleavage of paxillin and talin, as well as FAK by calpain.

In platelets, collagen receptor glycoprotein-6, a major thrombocyte aggregation agent, mediates FAK phosphorylation by protein kinase-C [1473].⁴⁶ In many cell types, α₁β₁-integrin stimulation provokes collagen-1 synthesis.

Another type of collagen receptors, glycoprotein-6 (GP6), is a member of the immunoglobulin superfamily that is constitutively associated with Fcγ. It is synthesized only in platelets and their precursors.

11.3.1.2 Collagens, Adhesion Molecules, and Matrix Metallopeptidases

The synthesis and activation of the 26 known matrix metallopeptidases are tightly controlled. Matrix metallopeptidases are released by cells as proenzymes that are afterward activated in the extracellular space mainly by serine peptidases. Yet, MMPs are often sequestered in a deactivating complex with tissue inhibitors of metallopeptidases.

In several cell lines, MMP expression is primed by collagen. Production of MMP1, MMP13, and mt1MMP as well as activation of proMMP2 is triggered by α₂β₁-integrins [1473].

In human fibroblasts, MMP1 expression depends on PKCζ and P38MAPK kinases. In keratinocytes, proMMP1 complexes with α₂β₁-integrin, hence competing with collagen. In vascular smooth muscle cells, newly formed adhesive contact between collagen and integrins induces production of proMMP1 that is then secreted and binds to integrin to become active. Activated MMP1 then cleaves collagen-1, thereby lowering its integrin-binding affinity. Focal adhesion complex then disassembles and the cell moves and forms novel cell–matrix adhesions. However, MMP can connect to plasmalemmal receptors that do not bind collagen.

Certain MMPs that are located on plasmalemmal receptors do not bind collagens such as MMP2 that binds to α_Vβ₃-integrin, MMP9 to CD44,⁴⁷ and MMP7 to heparan sulfate proteoglycans.

Membrane-associated metallopeptidases cause ectodomain shedding of various transmembrane proteins, such as cytokines, growth factors, receptors, and adhesion molecules. They then contribute to the regulation of cell proliferation, migration, and, hence, inflammation and cancer progression.

Structurally related, membrane-associated adamlysins also provoke ectodomain cleavage of various substrates. In particuler, ADAM17 causes shedding of transforming growth factor-α, a mitogen that interacts with the epidermal growth factor receptor, as well as other HER family agonists (members of the EGF superfamily), such as amphiregulin and heparin-binding EGF-like growth factor (HB-EGF), thereby controlling the access of generated soluble ligands to receptors of the HER family. Adamlysin ADAM17 also cleaves cytokines and cytokine receptors. Therefore, ectodomain shedding by ADAM17 participates in the regulation of cell proliferation, inflammation, and cancer progression. Ectodomain cleavage is primed once ADAM17 is phosphorylated (activated) by P38MAPK and ERK kinases.

Tissue inhibitors of metallopeptidases not only target matrix metallopeptidases, but also inhibit adamlysins. In basal conditions, ADAM17 resides in the plasma membrane as dimers associated with tissue inhibitor of metallopeptidase TIMP3, which inhibits ADAM17 enzyme. Upon activation of the ERK or P38MAPK pathway, the density of ADAM17 monomers at the cell surface rises and its association with TIMP3 lowers [1475].

11.3.1.3 Collagen Receptors in Wound Healing

In cells at the wound margin, collagen tethers to α₂β₁-integrin and activates focal adhesion kinase and elicits laminin deposition that triggers FAK signaling via α₃β₁– and α₆β₄-integrin in cells more distant from the wound edge [1473].

External regulators, particularly transforming growth factor-β, also modulate keratinocyte migration by promoting collagen and integrin synthesis. Following wound closure, proliferation of dermal fibroblasts terminates with apoptosis that depends on collagen-1 as well as α₁– and α₂β₁-integrin.

11.3.2 Adhesion Molecules

Integrins, cadherins, and other adhesion molecules interact with growth factor receptors. Cell adhesion is necessary for activation of growth factor receptors, and growth factors are required to stimulate cell adhesion or motility. However, adhesion molecules can trigger ligand-independent activation of growth factor Tyr kinase receptors, translating environmental cues into intracellular signals [1476]. Conversely, growth factors can act on adhesion molecules for adhesion-independent signaling. The receptors for PDGF and VEGF, among others, are activated by integrins [1477, 1478]. The EGF receptor associated with E-cadherin can be tyrosine phosphorylated and then activates MAPK and Rac without EGF stimulation [1479].

11.3.2.1 Integrin Receptors

Integrins mediate cell adhesion to the extracellular matrix and transmit signals that stimulate cell spreading, retraction, migration, and proliferation, etc., i.e., processes that range from cell survival to death on the cell level (Vols. 1 – Chap. 7. Plasma Membrane and 2 – Chap. 6. Cell Motility). On the tissue scale, integrins contribute to tissue development, immunity, wound healing, hemostasis and thrombosis, as well as carcinogenesis.

Integrins are heterodimeric, adhesion, and bidirectional signaling receptors that sense chemical and mechanical agents from inside and outside the cell, form integrin clusters, and transmit cues from both side of the plasma membrane, as they connect components of intra- and extracellular medium (Vol. 1 – Chaps. 7. Plasma Membrane and 8. Cell Environment).

On the one hand, activation of bidirectional signaling integrins is regulated by binding of cytoskeletal protein talin and focal adhesion protein kindlin-2 to distinct sites of cytoplasmic tails of β subunit (in–out signaling) [1480, 1481]. Kindlin-2 acts synergistically with talin to activate α_2Bβ₃-integrins. Moreover, kindlin-2 interacts with integrin-linked kinase complex and mediates its recruitment to focal adhesions to regulate cell spreading and actin cytoskeleton organization.

On the other hand, upon ligand binding, integrins transduce signals into cells by recruiting proteins to their cytoplasmic tails (out–in signaling). Integrin out–in signaling relies on Gα₁₃ subunit of heterotrimeric guanine nucleotide-binding protein that directly binds to β₃-integrin cytoplasmic domain. Interaction between G13 and α_2Bβ₃-integrin upon ligand binding to integrin as well as GTP loading on G13 activate Src kinase and RhoA GTPase [1482]. This signaling cascade causes cell spreading and prevents cell retraction.

Integrin adhesome integrates the entire set of interactions involved in integrin-mediated adhesion and signaling, especially responses to mechanical stresses. Integrin adhesome encompasses 6 main types of constituents: (1) actin–integrin set with actin regulators, adaptors, and adhesion molecule-associated proteins; (2) protein Ser/Thr kinases and phosphatases; (3) protein Tyr kinases and phosphatases; (4) monomeric GTPases of the RHO superfamily; (5) phosphoinositides regulated by phosphoinositide kinases and phosphatases; and (6) peptidase calpain and ubiquitin ligase CBL that degrade integrin adhesome proteins.

Integrin adhesome can be considered as a highly dynamic, robust signaling component, as its response depends on stimulus type and it retains its function after experiencing internal and external perturbations.

Small Rap1 GTPase induces the formation of an integrin-activation complex that binds to integrins. It can then mediate protein kinase-C activity associated with the integrin activation, using adaptor talin, a PKC substrate. Small Rap1 GTPase provokes the migration of talin toward the plasma membrane. GTPase Rap1 and protein kinase-Cα then induce, via the Rap1 effector Rap1-interacting adaptor molecule (RIAM) and with recruited talin, the formation of an integrin-activation complex that binds to and activates integrins [1483].

Integrin-mediated cell adhesion regulates numerous cellular responses. Integrins mediate either activation or inhibition of anchorage-dependent receptors. Integrins contribute to signaling from receptor Tyr kinases. On the other hand, integrins can also hinder receptor Tyr kinases [1484].

Signaling cooperation exists between integrins and growth factor receptors. In particular, α_Vβ₃-integrin binds to the platelet-derived growth factor receptor and vascular endothelial growth factor receptor-2. The collagen-activated α₁β₁-integrin attenuates epidermal growth factor receptor signaling via the activation of PTPn2 phosphatase. Phosphatase PTPn2 regulates cell proliferation. Cell adhesion to collagen induces PTPn2 translocation to the cell cortex, where PTPn2 colocalizes with and is activated by α₁-integrin.

Intracellular protein Tyr kinase SYK, a signaling effector of immune receptors, is also involved in integrin-mediated responses. Crosstalk between integrins and immune receptor signaling enables cooperation from the early stage. Both immune pathways and integrin-mediated signaling also work with the same effectors, phospholipase PLCγ2, and lymphocyte cytosolic protein LCP2 adaptor. Integrin-triggered signaling during interactions between lymphocytes and antigen-presenting cells implicates not only kinase SYK, but also transmembrane adaptors such as TYRO protein Tyr kinase-binding protein (TYROBP)⁴⁸ Integrin-β₃ clusters bound to fibrinogens on platelet plasma membrane (whereas β₁-integrins link to collagen) phosphorylate (activate) SYK kinase.

11.3.2.2 Catenins

Overexpressed catenin-δ1 disrupts stress fibers and focal adhesions , decreases RhoA functioning, and increases the activity of CDC42 and Rac1, thereby promoting cell migration. Catenin-δ1 binds Vav2 agent.

Catenin-δ1 interacts with transcriptional factor Kaiso [1485].⁴⁹ Both catenin-δ1 and Kaiso are increased at the wound border with respect to endothelial cells away from the wound border [1486]. C-terminal Src kinase (CSK) is involved in VE-cadherin signaling [1487]. The association of VE-cadherin and CSK in endothelial cells is increased with elevated cell density, inhibiting cell growth.

11.3.3 Discoidin Domain Receptors

Discoidin domain receptors (DDR) are receptor Tyr kinases that possess a single transmembrane region and a discoidin motif in their extracellular domain. They form homodimers upon ligand engagement.

Activation of DDR1 or DDR2 by collagen primes a sustained phosphorylation that allows binding of adaptors. Paralog DDR1 autophosphorylates once it is bound to various types of collagens, i.e., collagen-1 to -6 and -8, whereas DDR2 is only activated by fibrillar collagens [1473]. In addition, DDRs have other ligands that can activate these receptors together with or without collagen.

Isoform DDR1 undergoes proteolysis into a membrane-anchored β subunit and a soluble, extracellular α subunit. Five isoforms of DDR1 arise from alternative splicing. Long DDR1c isoform corresponds to the full-length protein, whereas DDR1a and DDR1b isoforms lack 37 or 6 amino acids in the kinase domain, respectively. Both DDR1d and DDR1e are truncated variants that lack the entire kinase region or part of it and the ATP-binding site [1473]. Splice variant DDR1b is the predominant isoform during embryogenesis.

11.3.4 Signaling via Focal Adhesions

Focal adhesions are also important sites of signal transduction. Their components propagate signals that arise from activated integrins following their association with matrix proteins, such as fibronectin, collagen, and laminin. The interaction of integrins with matrix ligands can either generate or modulate signals for motility, cell division, differentiation, and apoptosis [1488].

Integrins and paxillin are implicated in signal transduction. Paxillin binds to β-integrin cytoplasmic tail, vinculin, or other cytoskeletal and signaling proteins [1489]. Paxillin recognizes integrin sequences distinct from α-actinin-binding sites. Paxillin binding is independent of its association with focal adhesion kinase, although both bind to the same region of β₁-integrin. Paxillin yields multiple docking sites for activated FAK and Src kinases [1490]. Various regulatory proteins, such as calpain-2, protein kinase-C, FAK, and Src, control the assembly of focal adhesion [1491]. Focal adhesion disassembly involves microtubules and dynamin that interact with focal adhesion kinase [1492].

11.3.5 Signaling via Gap Junctions

Gap junctions allow communication between adjoining cells. Messenger ATP is released by multiple types of stimuli (mechanical stress, osmotic pressure changes, rise in intracellular concentration of inositol trisphosphate, decay in extracellular calcium ion level, etc.).

Manifold mechanisms of ATP release include vesicular exocytosis, active transport via ABC transporters, diffusion via stretch-activated channels, voltage-dependent anion channels, pores opened by P2X₇ receptors, and connexin hemichannels.

Connexin hemichannels, normally closed, are paths for ATP, NAD⁺, glutamate, prostaglandins, etc. Hemichannels open following membrane depolarization and mechanical stimulation. Inositol trisphosphate activates hemichannel composed of connexin 43. Decreases in extracellular Ca ${}^{++}$ and Mg ${}^{++}$ levels potentiate or trigger the opening of hemichannels, releasing particularly ATP and glutamate.

Connexin-32 and -43 have 2 and 1 calmodulin interaction sites, respectively. A Ca ${}^{++}$ -binding site exists for hemichannels made of connexin-32. Connexin hemichannels open when the cytosolic calcium concentration rises [1493]. However, [Ca ${}^{++}$ ]_ielevation following release from cell storage compartments can close gap junctions.

11.4 Interactions between Ion Channels and Small GTPases

Heterotrimeric GTPases that are activated by heptahelical plasmalemmal receptors (GPCRs or 7TMRs) can regulate ion channels. Small (monomeric) GTPases (Sect. 9.3) that are stimulated by cytoplasmic guanine nucleotide-exchange factors can interact directly with ion channels to regulate their activity (Tables 11.14 to 11.16).

Protein RhoA associates withK_V1.2 channel to suppress its activity [1494].

Protein Rab11a binds to epithelial, Ca ${}^{++}$ -selective members of the transient receptor potential superfamily of cation channels TRPV5 and TRPV6 [1495].

Small Rem GTPase complexes with auxiliary β subunit ofvoltage-gated Ca_V channel (Sect. 9.3.25), thereby inhibiting Ca_V channels. Small Rho GTPase also regulates Ca_V1.2a channels in ventriculomyocytes [370]. Specific GDP-dissociation inhibitor RhoGDIα decreases basal activity of Ca_V1.2a channels, but not expression level, without influencing inward rectifier and transient outward K⁺ channels (K_V4). Inhibition of RhoA, but not Rac1 or CDC42, impedes Ca_V activity, i.e., RhoA^GDP, but not RhoA^GTP, precludes Ca ${}^{++}$ import via Ca_V channels.

Table 11.14

Regulation of ion channels by small guanosine triphosphatases of the RAB superfamily (Source: [925]; CFTR: cystic fibrosis transmembrane conductance regulator; ENaC: epithelial Na⁺ channel; SLP: synaptotagmin-like protein; TRPV: transient receptor potential vanilloid channel). Ubiquitous Rab proteins constitute the largest branch of the small GTPase class. With monomeric GTPases of the ARF superfamily, they contribute to intracellular vesicular transport.

Small GTPase	Channel	Effect
Rab3	ENaC	Inhibition via effectors
Rab4	CFTR	Inhibition (basal and cAMP-stimulated activity)
	ENaC	Inhibition via effectors
Rab5	CFTR	Enhanced insertion
Rab7	CFTR	Lysosomal degradation
Rab9	CFTR	Exocytosis
Rab11	CFTR	Endosomal recycling
Rab11a	TRPV5/6	Enhanced translocation
Rab27a	CFTR	Inhibition via effectors
	ENaC	Munc13-4 and SLP5

Table 11.15

Regulation of ion channels by small guanosine triphosphatases of the RAS hyperfamily (Source: [925]; hRas: Harvey rat sarcoma viral oncogene homolog; kRas: Kirsten rat sarcoma viral oncogene homolog). Four major Ras isoforms constitute the P21RAS subfamily (hRas, kRasA, kRasB, and nRas). Effectors of Ras GTPases include Raf kinase (via adaptor GRB2 and RasGEF SOS recruited by RTKs, Src kinases attracted by other receptor types, or transactivation), RalGDS, RIN1, and phosphatidylinositol 3-kinase (PI3K). Members of the RAS hyperfamily can have isoform-specific effects on ion channel activity as well as different effects on distinct channels. Monomeric GTPases Rap1a and Ras have opposite effects on atrial K⁺ channels coupled to muscarinic M₂ receptors and voltage-gated Na⁺ channels (as well as ^Nmethyl ^Daspartic acid [NMDA] and α-amino 3-hydroxy 5-methylisoxazole 4-propionic acid [AMPA]-type glutamate receptors). Subtype hRas inhibits inward rectifier K_IR1 channel via mitogen-activated protein kinase (MAPK) signaling by supporting removal of channel from the plasma membrane, but excites Ca_V3 channel. Aldosterone increases density and activity of kRasA, an activator of epithelial Na⁺ channel (ENaC) via PI3K, as its product PI(3,4,5)P₃ interacts with ENaC channel. Ras-related GTPases of the RGK (Rad–GEM/KIR) family bind to β subunit of Ca_V1 channels, thereby inhibiting these channels, with or without attenuation of channel density.

Small GTPase	Channel	Effect
Ras	Atrial K_ACh	Inhibition
	Na_V	Activation
hRas	Ca_V3	Stimulation
	K_IR2	Reduced plasmalemmal insertion
	Na_V	Stimulation
kRas	ENaC	Activation via PI3K
Rap1	Na_V	Inhibition
	Atrial K_ACh	Activation
RGK	Ca_V1	Inhibition
Rem2	Ca_V2.2	Inhibition

Table 11.16

Regulation of ion channels by small guanosine triphosphatases of the RHO superfamily (Source: [925]; Ca_V: voltage-gated Ca ${}^{++}$ channel; ENaC: epithelial Na⁺ channel; NSC: non-selective cation channel; PI(4)P: phosphatidylinositol 4-phosphate; PP1: phosphoprotein phosphatase-1; RoCK: Rho-associated, coiled-coil-containing protein kinase; TRPC: transient receptor potential canonical channel; VRAC: volume-regulated anion channel). The superfamily of Rho GTPases includes Rho, Rac, and CDC42 that are involved in ^Factin polymerization, thus channel translocation. They can act via phospholipid messengers and adaptors such as phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂) that promotes channel insertion into the plasma membrane. Different members of the RHO superfamily, such as Rac1 and RhoA, have opposite effects on K_V11.1 channels. Inhibitory Rho and activatory Rac operate via phosphorylation and dephosphorylation, respectively.

Small GTPase	Channel	Effect
CDC42	Ca_V	Inhibition
Rac1	ENaC	Enhanced activity via RoCK and PI(4)P5K
	K_V11.1	Activation via PP1 and/or PP5
	Ca_V	Inhibition
Rho	VRAC	Sensitization
	NSC	Inhibition
RhoA	ENaC	Enhanced activity via RoCK and PI(4)P5K
	K_V11.1	Inhibition via kinases
	K_V1.2	Inhibition
	Ca_V1.x	Stimulation
	TRPC5	Increased density via PI(4)P5-kinase
	K_IR2.1–K_IR2.3	Inhibition
RhoQ	CFTR	Augmentation of density

Others small GTPases interact indirectly with ion channels. Small GTPases RhoA and kRas heighten activity of epithelial Na⁺ channel (ENaC) via PI(4)P5K and PI3K kinases, respectively [1497].

Protein Rac1 mediates rapid vesicular insertion from vesicles held in reserve just under the plasma membrane of transient receptor potential channel TRPC5 into the plasma membrane via PI3K and PI(4)P5-kinase-α following epidermal growth factor stimulation for Ca ${}^{++}$ influx [1498].

Small Rab27a GTPase regulates ENaC activity, using its effectors synaptotagmin-like protein SLP5 and transport cofactor of the MUNC family of syntaxin-binding protein Munc13-4 [1499].

Different types of small GTPases can have opposite effects. The hypothalamic neuropeptide thyrotropin-releasing hormone (TRH) inhibits the activity of K_V11.1 channels in the pituitary gland (hypophysis), whereas thyroid hormone triiodothyronine (T₃) antagonizes TRH action, hence stimulating K_V11.1 channel activity. Small RhoA GTPase stimulated by G13-protein-coupled receptors upon TRH binding is able to rapidly inhibit K_V11.1 channels. On the other hand, Rac activated by RacGEF stimulated by PI3K in T₃-primed nuclear hormone receptor signaling stimulates K_V11.1 channel [1500].

Neurotransmitters target voltage-gated calcium channels that trigger Ca ${}^{++}$ influx in neurons. Upon bradykinin excitation, heterotrimeric G-protein subunit G13 inhibits voltage-gated calcium channels. Protein G13 can couple to monomeric RhoA, Rac1, and CDC42 GTPases. Small Rac1 GTPase and/or CDC42 mediate inhibition of Ca_V by neurotransmitters [1501]. Lysophosphatidylcholine (3–50 μmol) generates a non-selective cation current (I_NSC) in a dose-dependent manner with a lag in guinea pig ventricular myocytes via Gi/o-protein-coupled receptor and small GTPase Rho [1502].

Small GTPases can operate by activating action or permissive effect. Hypo-osmotic stimulation of cells rapidly activates compensatory Cl⁻ and K⁺ fluxes to limit excessive cell swelling and, ultimately, restore original cell volume. Osmotic stress-induced cell swelling is associated with a rapid, transient remodeling of the actin cytoskeleton. In calf pulmonary artery endothelial as well as human intestinal cells, the Rho–RoCK–myosin light chain phosphorylation axis favors the swelling-triggered outwardly rectifying anion current through volume-regulated anion channels [372, 1504]. However, the Rho pathway sensitizes VRACs to cell swelling via actin filaments. It indeed exerts a permissive effect on Cl⁻ channel VRAC, i.e., swelling-induced VRAC opening requires functional, but not directly interacting Rho pathway [1506]. The activity of endothelial swelling-activated Cl⁻ channels also depends on tyrosine phosphorylation, as protein Tyr kinases promote Cl⁻ flux, whereas protein Tyr phosphatases preclude it [1507].

Small GTPases can enhance activity of plasmalemmal ion channel by heightening their density, as they are involved in cellular exocytosis and membrane insertion of ion channels. On the other hand, small GTPase RhoQ, but not CDC42 or RhoA, interacts with cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand CAL⁵⁰ that reduces plasmalemmal density in CFTRs by targeting CFTR for degradation in lysosome [966]. Active form of RhoJ (RhoJ^GTP) protects CFTR against CAL-mediated lysosomal degradation.

11.5 Calcium Signaling

Among the second messengers, calcium regulates cellular functions in all cell compartments on time scales ranging from milliseconds to days [1508]. The cell response to stimulation of certain receptor Tyr kinases and G-protein-coupled receptors depends on the amplitude and duration of calcium influx. Calcium flux into the cell can be modulated to ensure the suitable response.

A small calcium proportion binds to effectors, such as annexins, calmodulin, membrane-trafficking synaptotagmin, S100 proteins, and troponin-C (Vol. 5 – Chap. 5. Cardiomyocytes). The calcium-signaling effectors are involved in cellular transport (operating in time of the order of $\mathcal{O}$ [10 μs]), metabolism (operating in time of the order of $\mathcal{O}$ [s]), gene transcription (operating in time of the order of $\mathcal{O}$ [mn]), cell fate and motility (operating in time of the order of $\mathcal{O}$ [h]), and myocyte contraction (operating in time of the order of $\mathcal{O}$ [10 ms]), according to the location, timing, and calcium-bound molecules (Table 11.17). Calcium ions are mostly linked to buffers. Calcium buffers affect both the amplitude and the recovery time of Ca ${}^{++}$ fluxes. Calcium buffers have different expression patterns, motility, and binding kinetics. Calcium ions mediate or stimulate multiple cellular processes, such as the myocyte contraction.

Table 11.17

Calcium signaling (Source: [10]). The Ca ${}^{++}$ concentration in a cell at rest equals approximately 100 nmol It augments to at least 500 nmol in a stimulated cell. When the stimulus is removed, the Ca ${}^{++}$ concentration return to its resting level. Calcium ion is a universal second messenger that triggers many cellular processes over various time scales.

Target process	Time scale
Exocytosis	$\mathcal{O}$ [1 μs]
Cell contraction	$\mathcal{O}$ [1 ms]
Cell metabolism	$\mathcal{O}$ [1 s]
Gene transcription	$\mathcal{O}$ [1 mn]
Cell proliferation	$\mathcal{O}$ [1 h]

11.5.1 Calcium-Induced Calcium Release

Calcium-induced Ca ${}^{++}$ release (CICR) enables the mobilization of Ca ${}^{++}$ ions from its intracellular stores, primarily the endoplasmic reticulum. Calcium channels in the membrane include ryanodine and IP₃ receptors.

The CICR process transmits information from the plasma membrane to the endoplasmic reticulum via Ca ${}^{++}$ -releasing RyR and IP₃R receptors. The first mechanism relies on voltage-gated channels that open in response to membrane depolarization to carry a small amount of Ca ${}^{++}$ ions toward the cytosol (Table 11.18). Calcium ions then diffuses into the cytosol to reach the endoplasmic reticulum and activate RyR and/or IP₃R channels. The second mechanism is based on the IP₃ axis, after synthesis of IP₃ initiated by activated plasmalemmal receptors.

Table 11.18

Calcium-induced Ca ${}^{++}$ release (CICR) and Ca ${}^{++}$ signal types [10]). Elementary Ca ${}^{++}$ events, such as Ca ${}^{++}$ sparks and puffs are produced by ryanodine (RyR) and inositol (1,4,5)-trisphosphate (IP₃R) receptors, respectively. Fall in Ca ${}^{++}$ in the endoplasmic reticulum opens plasmalemmal, store-operated CRAC channels. Resulting Ca ${}^{++}$ entry through CRAC channels refills intracellular stores and, thus, enables prolonged IP₃-triggered Ca ${}^{++}$ oscillations. The additional role of CICR is to set up cytoplasmic Ca ${}^{++}$ waves from initiation sites that propagate through the cytosol to form the global Ca ${}^{++}$ signal.

Trigger	Target	Effect
Ca ${}^{++}$	RyR, IP₃R	Ca ${}^{++}$ sparks
		Intracellular Ca ${}^{++}$ wave
IP₃	IP₃R	Ca ${}^{++}$ puffs
		Intracellular Ca ${}^{++}$ wave

In addition, Ca ${}^{++}$ released from endoplasmic reticulum RyR and IP₃R channels diffuses along the membrane of the endoplasmic reticulum and can then stimulates neighboring channels to release further Ca ${}^{++}$ ions, thereby creating regenerative waves [10]. Oscillatory Ca ${}^{++}$ signals with given oscillatory amplitude and frequency for discriminating signaling allow to raise cytoplasmic Ca ${}^{++}$ transiently, thereby avoiding cell damage caused by sustained elevated Ca ${}^{++}$ elevation.

Repetitive cytoplasmic Ca ${}^{++}$ oscillations triggered by IP₃-mediated Ca ${}^{++}$ release from intracellular stores arise from activated plasmalemmal receptors coupled to the phospholipase-C axis that enables regenerative Ca ${}^{++}$ release and are supported by store-operated Ca ${}^{++}$ entry through Ca ${}^{++}$ release-activated Ca ${}^{++}$ channels. Upon stimulation of type-1 cysteinyl leukotriene receptors (CysLT1) by leukotriene-C4, large-amplitude Ca ${}^{++}$ oscillations (rapid, large, all-or-nothing baseline Ca ${}^{++}$ spikes) and CRAC channel activity result from action of transmembrane endoplasmic reticulum stromal interaction moleculeStIM1 sensor and CRAC channel activator [1509]. On the other hand, stimulated FCεR1 receptor by antigens or immunoglobulin-E causes activation by protein Tyr kinase of phospholipase-Cγ and slow Ca ${}^{++}$ oscillations developed after a longer delay on an elevated background Ca ${}^{++}$ rise using both StIM2 and StIM1 agents [1509]. Therefore, different stimuli can recruit and activate different combinations of StIM proteins to sustain cytoplasmic Ca ${}^{++}$ signals.

The kinetics of IP₃ production (fast, transient elevation vs. slow, prolonged rise in IP₃ concentration) and its baseline concentration differ according to the stimulus type. Sensor StIM2 is less efficient, for a given CRAC channel-formingOrai1 density, in Ca ${}^{++}$ entry. Protein StIM2 has an approximately 2-fold lower affinity for Ca ${}^{++}$ than StIM1 [1509]. Molecule StIM2 requires a smaller Ca ${}^{++}$ drop in the endoplasmic reticulum lumen for its activation. Whereas StIM1 activates CRAC channels during strong stimulation, StIM2 is involved in moderate store depletion. Large and quick Ca ${}^{++}$ release may potentiate calmodulin activity that prevents StIM2 from activating Orai1 channel.

11.5.2 Calcium Signaling and Myocyte Contraction

Myocyte contraction illustrates the role of calcium ions in cell functioning. Several protein sets are involved in such a process: (1) plasmalemmal calcium channnels for influx of calcium ions from and efflux to the extracellular space; (2) calcium channnels in the membrane of intracellular calcium stores, mainly the sarcoplasmic reticulum, to ensure a sufficient amount of calcium into the cytosol for suitable activity; (3) second messenger to release calcium from its intracellular stores; (4) the mitochondrial machinery to synthesize the energy source ATP; (5) buffers; (6) sarcomere effectors; and (7) regulators such as kinases and phosphatases (Fig. 11.2). Other molecules participate in myocyte contraction, especially to coordinate the deformation of the cell compartments. Dystrophin associates with cytoplasmic syntrophin and forms the dystrophin–glycoprotein complex (DGC), which links plasmalemmal β-dystroglycan. The latter binds α2-laminin–merosin of the basal lamina. Dystrophin thus stabilizes the sarcolemma, especially during contraction.

Fig. 11.2

Calcium messengers for myocyte contraction (Source: [1510]). Calcium influxes are due to Ca ${}^{++}$ channel (Ca Ch), indirectly by nicotinic acetylcholine receptor (NAChR), which drives Na⁺ influx, thereby stimulating Ca ${}^{++}$ influx by the sodium–calcium exchanger (NCX), to dihydropyridine receptor (DHPR) of the plasmalemma (PL) and ryanodine receptor of the sarcoplasmic reticulum (SR). A fraction of imported calcium enters in mitochondria (mitoc) by Ca ${}^{++}$ uniporter (Ca uni). It stimulates nitric oxide synthase (NOS) and mitochondrial creatine kinase (miCK), generating NO and ATP. ATP is indirectly exported from phosphocreatine via cytosolic creatine kinase (cyCK) for contraction and ion ATPase activity. Mitochondrial Ca ${}^{++}$ is exported by NCX and mitochondrial permeability transition pore (miPTP). Cytosolic Ca ${}^{++}$ efflux is done by Ca ${}^{++}$ ATPases of the plasma membrane (PMCA) and the sarcoplasmic reticulum (SERCA), and by NCX. Na⁺ is exported by sodium–potassium ATPase. Nitric oxide can form with superoxide (O2 − ), produced by the respiratory chain, peroxynitrite (ONOO − ).

11.5.3 Instantaneous and Long-Lasting Responses to Calcium

Calcium signaling is characterized by instantaneous and long-lasting responses. Intracellular calcium controls various events according to its spatial and temporal distributions and magnitude. Cytosolic and plasmalemmal calcium sensors, with high affinity for Ca ${}^{++}$ (binding Ca ${}^{++}$ even at small increments in concentration above resting levels) and distinct targets, transduce calcium signals into functional changes. Ubiquitous calmodulin (Cam) is a specific cytosolic Ca ${}^{++}$ sensor. The calcium–calmodulin complex activates calmodulin-dependent protein kinases. Calmodulin regulates other targets, such as PP3 phosphatase and phosphodiesterases.

Once released in the cytosol, Ca ${}^{++}$ binds to calmodulin. The Ca ${}^{++}$ –Cam complex then interacts with other proteins for instantaneous response. Instantaneous reaction begins once the cytosolic concentration [Ca ${}^{++}$ ]_i rises

([Ca ${}^{++}$ ]_i ∼ 10^{− 4} [Ca ${}^{++}$ ]_e),

and ends as soon as [Ca ${}^{++}$ ]_i returns to its basic level. The release of cAMP is initiated by ligand-loaded G-protein-coupled receptors. Messenger cAMP is produced from adenosine triphosphate after the activation of adenylate cyclase by the receptor-activated Gs protein [1511] (Fig. 11.3).⁵¹

Messenger cAMP travels in the cytosol and accumulates at specific sites [1513]. The AKAP proteins then recruit PKA to locations of cAMP production, where phosphorylation is confined to a subset of potential substrates. Agent cAMP controls the Ca ${}^{++}$ influx. In turn, Ca ${}^{++}$ activates cAMP synthesis.

Sustained contractions of smooth muscle cells triggered by Ca ${}^{++}$ influx during the instantaneous response result from a long-lasting response. When [Ca ${}^{++}$ ]_i increases, the Ca ${}^{++}$ –Cam complex responsible for the transient reaction interacts with the membrane Ca ${}^{++}$ pump to augment its functioning. Moreover, Ca ${}^{++}$ -activated protein kinase-C (PKC) enhances the Ca ${}^{++}$ pump efficiency. The Ca ${}^{++}$ efflux thus compensates Ca ${}^{++}$ influx [1514]. Calcium recycling is improved with an increased submembrane Ca ${}^{++}$ concentration, which activates membrane-bound PKC (Fig. 11.3). Furthermore, activation of phospholipase-C (PLC) generates inositol trisphosphate (IP₃) and diacylglycerol (DAG) from phosphatidylinositol diphosphate (PIP₂). Messenger IP₃ induces Ca ${}^{++}$ release from the endoplasmic reticulum. Diacylglycerol remains in the membrane and, as long as its membrane concentration is sufficient,⁵² PKC is fixed by DAG to the membrane and is activated. Protein kinase-C thus acts as a transducer during the long-lasting reaction.

Fig. 11.3

Second messengers (Source: [1514]). The activated receptor activates phospholipase-C (PLC), which cleaves membrane-bound phosphatidylinositol bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG). Messenger IP₃ travels to its receptor, a calcium (Ca) channel, on the surface of the endoplasmic reticulum (ER). Calcium is thereby released from its ER store. Cytosolic calcium is then available for binding to calmodulin (Cam) or various cytoskeletal proteins. Protein phosphorylations are catalyzed by Ca–Cam-dependent protein kinases. Mediator ATP is converted into cyclic adenosine monophosphate (cAMP) by adenylate cyclase (AC), stimulated by stimulatory G protein (Gs) and inhibited by inhibitory G protein (Gi). Messenger cAMP activates cAMP-dependent protein kinase-A (PKA). Effectors DAG and Ca ${}^{++}$ activate protein kinase-C (PKC) at the plasma membrane. Protein phosphorylation by PKC produces cell responses.

11.5.4 Calcium Signaling Components

Several Ca ${}^{++}$ -regulated proteins maintain low intracellular concentration of Ca ${}^{++}$ ( ${[\mathrm{{Ca}}^{++}]}_{\mathrm{i}}$ ) and couple changes in ${[\mathrm{{Ca}}^{++}]}_{\mathrm{i}}$ to physiological responses. They include Ca ${}^{++}$ membrane carriers and Ca ${}^{++}$ effectors. According to the Ca ${}^{++}$ -binding site, Ca ${}^{++}$ effectors are grouped into different families.

Effectors characterized by an EF-hand motif ⁵³ can be categorized into 2 primary subclasses of proteins: (1) subclass of EF-hand Ca ${}^{++}$ sensors (e.g., calmodulin) that transduce Ca ${}^{++}$ signals and (2) subclass of Ca ${}^{++}$ signal modulators (e.g., calbindin, a 29-kDa calretinin) that modulate the shape and/or duration of Ca ${}^{++}$ signals.

The superclass of EF-hand proteins that include more than 60 families can be subdivided into 2 classes: (1) class of canonical EF-hand proteins (12-residue canonical EF-hand loop), such as calmodulin, calpain, PP3, recoverin, spectrin, troponin-C, etc.; and (2) class of pseudo-EF-hand proteins, such as S100 and S100-like proteins. Once bound to Ca ${}^{++}$ , EF-hand proteins regulate their cellular targets.

11.5.4.1 Channels, Receptors, and Effectors of Calcium Flux

Calcium effects and fluxes result from the combined action of buffers, ion channels, pumps, and exchangers, signaling effectors, and transcription factors (Tables 11.19 to 11.25). Activated receptors can prime entry into the cell of Ca ${}^{++}$ ions from the extracellular space and formation of second messengers that release Ca ${}^{++}$ ions from its intracellular stores. Different cues activate various types of Ca ${}^{++}$ channels, such as receptor-operated channels, second messenger-operated channels, store-operated channels, thermosensors, and stretch-activated channels. Several types of Ca ${}^{++}$ channels belong to the superfamily of transient receptor potential protein. They regulates slow cellular processes (smooth muscle cell contraction and cell proliferation).

Table 11.19

Calcium signaling components. (Part 1) Calcium channels (Source: [1508]). Calcium channels are associated with channel regulators, such as triadin, junctin, sorcin, FKBP12, phospholamban, IP₃R-associated PKG substrate IRAG, and adenosylhomocysteinase-like-1. In particular, the ryanodine receptor-2 complex includes stabilizers, such as FKBP12.6, calmodulin, and PP1 and PP2 phosphatases via scaffold proteins spinophilin (PP1_r9b) and PP2_r3a, respectively, kinases attached by A-kinase anchoring proteins, and calsequestrin anchored by membrane-bound junctin and triadin. Calcium pumps are characterized by their affinities (functioning thresholds), transport rates, and opening duration. Secretory pathway Ca ${}^{++}$ ATPases could be responsible for Ca ${}^{++}$ sequestration into Golgi compartments.

Voltage-gated channels	Ca_V1.1–Ca_V1.4
	Ca_V2.1–Ca_V2.3
	Ca_V3.1–Ca_V3.3
Receptor-operated channels	NMDA-type Glu receptors
Ligand-gated ion channels	(GluNR1, GluNR2A–GluNR2D)
	AMPA-type Glu receptors
	ATP receptor (P2X₇)
	nACh receptor
	5HT₃
Second messenger-operated channels	Cyclic nucleotide–gated channels
	(CNGA1-CNGA4, CNGB 1, CNGB 3)
	Arachidonate-regulated Ca ${}^{++}$ channel
	(i _ARC)
Transient receptor potential ion channels	TRPC1–TRPC7
	TRPV1–TRPV16
	TRPM1–TRPM8
	TRPML, TRPNI

Table 11.20

Calcium signaling components. (Part 2) Calcium channels (Cont.; Source: [1508]).

Inositol trisphophate receptors	IP₃R1–IP₃R3
Ryanodine receptors	RyR1-RyR3
Polycystins	PC1–PC2
Calcium pumps	Plasma membrane Ca ${}^{++}$ ATPases
	(PMCA1–PMCA4)
	Sarco(endo)plasmic reticulum
	Ca ${}^{++}$ ATPases
	(SERCA1–SERCA3)
	Secretory-pathway Ca ${}^{++}$ ATPases,
	or Golgi pumps (SPCA1–SPCA2)
Plasmalemmal Na⁺–Ca ${}^{++}$ exchangers	NCX1–NCX3
	NCKX1–NCKX4
Mitochondrial Ca ${}^{++}$ channels	Na⁺–Ca ${}^{++}$ exchangers
	Ca ${}^{++}$ uniporter
	H⁺–Ca ${}^{++}$ exchanger
	Permeability transition pore

Table 11.21

Calcium signaling components. (Part 3) Receptors (Source: [1508]). G-Protein-coupled receptors are associated with G-protein component Gα subtype Gq, G11, G14, and G16, as well as Gβγ dimer. They are controlled by regulators of G-protein signaling RGS1, RGS2, RGS4, and RGS16. They stimulate phospholipase-Cβ. Receptor Tyr kinases can activate phospholipase-Cγ.

Components	Types
G-protein-coupled receptors	Muscarinic receptors (M₁–M₃)
	α1-Adrenoceptors (A–C)
	Endothelin receptors (ET_A–ET_B)
	Angiotensin receptor (AT₁)
	Bradykinin receptors (B₁–B₂)
	Histamine receptor (H₁)
	Serotonin receptors (5HT_2A–5HT_2C)
	Leukotrine receptors (BLT, CysLT₁–CysLT₂)
	Ca ${}^{++}$ -sensing receptor (CaR)
	Prostanoid receptor (PGF2α)
	Thrombin receptor (PAR₁)
	Bombesin receptors (BRS₁–BRS₂)
	Cholecystokinin receptors (CCK₁–CCK₂)
	Metabotropic glutamate receptors (mGlu1, mGlu5)
	Luteinizing receptor (LSH)
	Neurotensin receptor (Nts₁)
	Oxytocin receptor (OT)
	Substance-P receptor (NK₁)
	Substance-K receptor (NK₂)
	Substance-B receptor (NK₃)
	Thyrotropin-releasing hormone receptor (TRHR)
	Vasopressin receptors (V_1A–V_1B)
Receptor Tyr kinases	Epidermal growth factor receptors
	(HER1–HER4)
	Platelet-derived growth factor receptors
	(PDGFRα–PDGFRβ)
	Vascular endothelial growth factor receptors
	(VEGFR1–VEGFR3

Table 11.22

Calcium signaling components. (Part 4) Calcium effectors (Source: [1508]).

Phospholipase-C	PLCβ(1–4), PLCγ(1–2), PLCδ(1–4),
	PLCε, PLCζ
and inositol trisphosphate
Ca ${}^{++}$ -binding proteins	Calmodulin
	Troponin-C
	S100A1–S100A14, S100B, S100C, S100P
	Annexin-1–annexin-10
	Neuronal Ca ${}^{++}$ sensor
	Visinin-like proteins (ViLiP1–ViLiP3)
	Hippocalcin, recoverin
	K_V channel–interacting proteins (KChIP1–KChIP4)
	Guanylate-cyclase-activating proteins
	(GCAP1–GCAP3 or GCA1a–GCA1c;
	phototransduction)
	Calcium-binding proteins
	(caldendrins CaBP1_L and CaBP1_S
	and CaBP2–CaBP5)
Ca ${}^{++}$ -sensitive ion channels	Ca ${}^{++}$ -activated K⁺ channels
	(small SK, intermediate IK, and
	large-conductance BK channels)
	Cl⁺ channel (HClCA1)

Table 11.23

Calcium signaling components. (Part 5) Calcium-regulated enzymes (Source: [1508]; Cam: calmodulin, a portmanteau word for calcium-modulated protein). Proline-rich Tyr kinase PYK2 is a Ca ${}^{++}$ -sensitive protein that acts in osteoclast podosomes.

Ca ${}^{++}$ -regulated enzymes	Ca ${}^{++}$ –Cam-dependent protein kinases
	(CamK1–CamK4)
	Myosin light-chain kinase (MLCK)
	Protein kinase-C
	(PKCα, PKCβ1, PKCβ2, PKCγ)
	PYK2
	Phosphorylase kinase (PhK)
	Diacylglycerol kinase (DAGK)
	PIK3C3C (VPS34)
	IP₃ 3-kinase
	Protein phosphatase-3 (calcineurin)
	cAMP phosphodiesterase (PDE1a–PDE1c)
	Adenylate cyclases
	(AC1, AC3, AC5, AC6, AC8)
	Nitric oxide synthase (NOS1, NOS3)
	Miro (mitochondrial motility)
	Dual oxidases (DuOx1–DuOx2)
	Ca ${}^{++}$ -activated peptidases
	(calpain-1, calpain-2)

Table 11.24

Calcium signaling components. (Part 6) Calcium catalytic effectors and ion channel regulators (Source: [1508]; ^ADPribose: adenosine diphosphate–ribose; cADPR: cyclic adenosine diphosphate–ribose; FKBP12 and FKBP12.6: 12- and 12.6-kDa immunosuppressant FK506-binding protein; Miro: mitochondrial Rho GTPase). The cADPR hydrolase, or ^ADPribosyl cyclase-1 (also CD38), is a glycoprotein on the surface of many leukocyte types and at internal sites that functions in cell adhesion and calcium signaling. Aspartyl–asparaginyl β-hydroxylase (AspH), or junctin, is involved in calcium storage in and release from the endoplasmic reticulum (ER) as well as hydroxylation of aspartic acid and asparagine in epidermal growth factor-like domains of proteins. Peptidyl-prolyl cis-trans isomerase FKBP1a (FKBP12) and FKBP1b (FKBP12.6) are members of the immunophilin family that interact with multiple intracellular calcium release channels such as ryanodine receptors. Annexins are Ca ${}^{++}$ -dependent phospholipid-binding proteins involved in material exo- and endocytosis and organization of vesicles, as well as formation of Ca ${}^{++}$ channels.

Molecules	Effect
Enzymes
^ADPribosyl cyclase	Synthesis of Ca ${}^{++}$ -mobilizing
	messengers cADPR and NAADP
cADPR hydrolase	Cell adhesion and Ca ${}^{++}$ signaling
	Generation of NAADP
Regulators of monomeric and heterotrimeric GTPases
Guanine nucleotide-exchange factors	RAS signaling in the brain
RasGRF1	(learning and memory)
Regulators of G protein signaling	Signal termination
(GTPase-accelerating proteins)
(RGS1, RGS2, RGS4, RGS16)
Channel regulators
Triadin	Calcium-induced calcium release
Junctin	Calcium storage in and release from ER
Sorcin	Binding to annexin-7
FKBP12, FKBP12.6	Interactors of Ca ${}^{++}$ release channels
Phospholamban	Inhibition of SERCA pump
Molecular tranfer regulators
Synaptotagmin-1–synaptotagmin-3	Membrane trafficking,
Synaptotagmin-5–synaptotagmin-7	calcium sensors and regulators of
Synaptotagmin-9, synaptotagmin-10	neurotransmitter release and
	hormone secretion
Annexins	Intracellular transport
(annexin-A1–annexin-A13)	Formation of Ca ${}^{++}$ channels

Table 11.25

Calcium signaling components. (Part 7) Transcription factors and buffers (Source: [1508]; GRP78 and GRP94: 78- and 94-kDa glucose-regulatory protein [GRP78 is a.k.a. endoplasmic reticulum luminal Ca ${}^{++}$ -binding protein (BiP) and glucose-regulated 70-kDa heat shock protein HSPa5; GRP94 as 90-kDa heat shock protein-β1 (HSP90β1) and endoplasmin]; StIM: stromal interaction molecule).

Transcription factors	Nuclear factor of activated T-cells
	(NFAT1–NFAT4)
	cAMP response element-binding protein
	(CREB)
	Downstream regulatory element modulator
	(DREAM)
	CREB-binding protein (CBP)
Cytosolic buffers	Calbindin, calretinin, parvalbumin
Endoplasmic reticulum buffers	Calnexin, calreticulin, calsequestrin,
	GRP78/94
Endoplasmic reticulum sensor	StIM

Signal transmission can tolerate large variations in the expression level of signaling components that are observed in different cell types. Cells can either determine the correct density of signaling components via adequate synthesis and stabilization of produced mediators, or monitor the output of a pathway and use a feedback to adapt concentrations of signaling mediators. Cells monitor Ca ${}^{++}$ concentrations in the cytosol and endoplasmic reticulum and adjust concentrations of stromal interaction molecule (StIM) as well as of plasma membrane (PMCA) and endoplasmic reticulum (SERCA) Ca ${}^{++}$ ATPases, among other signaling components [1515]. Cells can sense the state of signaling pathways and use multiple parallel adaptive feedbacks.

11.5.4.2 Cell-Specific Calcium Signalosomes

Calcium signalosomes characterized by their spatiotemporal regulation are adapted to the main cell function (Table 11.26). A Ca ${}^{++}$ signalosome is composed of various types of effectors that can constitute Ca ${}^{++}$ signaling modules.⁵⁴ Inside the cell, Ca ${}^{++}$ ions can be stored in the endoplasmic reticulum (primarily) and mitochondria. During Ca ${}^{++}$ influx (in the cytoplasm) through voltage-gated Ca ${}^{++}$ channel (VGCC or Ca_V) as well as receptor (ROC), store (SOC), and second messenger (SMOC)-operated channels, Ca ${}^{++}$ ions interacts with buffers. Calcium sensors and transducers launch Ca ${}^{++}$ signaling using a set of effectors. The diversity of a given component heightens the number of signaling pathways.

Table 11.26

Cell-specific Ca ${}^{++}$ signalosomes (Source: [10]). Examples of cell-specific Ca ${}^{++}$ signalosomes with their own spatial and temporal regulation (IP₃R: inositol trisphosphate receptor; NCX: Na⁺–Ca ${}^{++}$ exchanger; ^NMDAGlu: NMDA-type glutamate receptor; PLC: phospholipase-C; PMCA: plasma membrane Ca ${}^{++}$ ATPase; RYR: ryanodine receptor; SERCA: sarco(endo)plasmic reticulum Ca ${}^{++}$ ATPase). Conventionally, whether the Ca ${}^{++}$ source is the extracellular space or intracellular stores, Ca ${}^{++}$ influx is referred to as Ca ${}^{++}$ entry and Ca ${}^{++}$ release, respectively.

Mediator	Atriomyocyte	Neuron	T lymphocyte
Receptors	ET₁, α1AR,	mGluR1, M₁	TCR
	ATR
PLC	PLCβ	PLCβ	PLCγ1
Entry	Ca_V1.2	Ca_V1.2/2.1/2.2	Orai1
channels		^NMDAGlu
Release	RyR2, IP₃R2	RyR2, IP₃R2	IP₃R1
channels
PMCA	PMCA1c/1d/2a	PMCA1a/2a/3a	PMCA4b
SERCA	SERCA2a	SERCA2b/3	SERCA2b/3
NCX	NCX1	NCX1/3
Buffers		Parvalbumin,
		calbindin-1
Sensors	Troponin-C,	Calmodulin	Calmodulin
	calmodulin

In the striated myocyte, the Ca ${}^{++}$ signalosome deliver rapid Ca ${}^{++}$ pulses for contraction. In T-lymphocytes, Ca ${}^{++}$ signalosome contains different components to generate much slower repetitive Ca ${}^{++}$ pulses for cell proliferation required to efficiently struggle against invading pathogens.

Muscle contraction is triggered by a global elevation in cytosolic Ca ${}^{++}$ concentration. On the other hand, the release of neurotransmitters is launched by a tiny, localized Ca ${}^{++}$ pulse delivered directly to storage vesicle by a Ca ${}^{++}$ sensor linked to exocytotic machinery. Between these 2 extreme cases, many spatiotemporal modalities of Ca ${}^{++}$ signaling exist.

The major cytosolic buffers in cells are parvalbumin (PAlb) and calbindin-1 (CalB1)⁵⁵ Parvalbumin localizes to fast-contracting muscles (at its highest levels) and in the brain (gabaergic interneurons, Purkinje cells, among others) and some endocrine glands. Parvalbumin is a slow-onset buffer [10]. It has relatively low activation and inactivation rates, thereby being unable to respond to a rapid influx of Ca ${}^{++}$ ions. However, it can buffer Ca ${}^{++}$ ions after an initial wave (delayed buffer).

Calbindins⁵⁶ are classified in subfamilies according to the number of Ca ${}^{++}$ -binding EF-hand sites. Calbindin-1, encoded by the vitamin-D-responsive CALB1 gene, resides in the kidney as well as neuroendocrine cells. It restricts the magnitude of the elementary Ca ${}^{++}$ cues generated near Ca ${}^{++}$ channels. Vitamin-D-dependent calbindin-D9k, or S100 calcium-binding protein-G (S100G), acts in enterocytes. In the kidney and intestine, calbindin facilitates Ca ${}^{++}$ reabsorption [10].

The major buffers in the lumen of the endoplasmic reticulum are calsequestrin (Csq) in the sarcoplasmic reticulum of myocytes and calreticulin (Crt), or calbindin-2 (CalB2) in the endoplasmic reticulum of non-myocytes. However, calreticulin operates as both a cytosolic and ER luminal buffer. It can indeed be detected in the nucleus and cytoplasm. Calreticulin is a low-affinity Ca ${}^{++}$ -binding protein. In addition to its buffer role, it acts as a chaperone in conjunction with the chaperone calnexin to ensure a correct folding and subunit assembly of glycoproteins, hence proper protein transfer and secretion [10]. When the Ca ${}^{++}$ concentration is too high, calreticulin and calnexin inhibit the sarco(endo)plasmic reticulum Ca ${}^{++}$ ATPase (SERCA pump); when the luminal Ca ${}^{++}$ concentration diminishes, this inhibition is relieved.

Calsequestrin allows the sarcoplasmic reticulum to store a hugh amount of calcium ions (each molecule of calsequestrin can bind 18 to 50 Ca ${}^{++}$ ions). It maintains an elevated Ca ${}^{++}$ concentration in the sarcoplasmic reticulum, which is much higher than in the cytosol. Calsequestrin possesses 2 isoforms: calsequestrin-2 is present in the myocardium and slow skeletal muscle and calsequestrin-1 in the fast skeletal muscle.

Major sensors comprise troponin-C (TnC), calmodulin (Cam), neuronal Ca ${}^{++}$ sensors (NCS), and S100 proteins. These sensors relay information to numerous effectors, such as Ca ${}^{++}$ -sensitiveK_Ca channels, Ca ${}^{++}$ -sensitive Cl⁻ channels, Ca ${}^{++}$ –calmodulin-dependent protein kinases, protein phosphatase-3 (or calcineurin), phosphorylase kinase (PhK),⁵⁷ myosin light-chain kinase (MLCK), and RasA4 activator.⁵⁸

11.5.4.3 Calcium Flux from and to Cell Organelles

A set of substances (inositol trisphosphate, cyclic ^ADPribose, nicotinic acid adenine dinucleotide phosphate, and sphingosine 1-phosphate) modulate calcium release from the endoplasmic reticulum and other organelles (Fig. 11.4). Inositol (1,4,5)-trisphosphate is produced from phosphatidylinositol (4,5)-bisphosphate (PIP₂) by different phospholipase-C isoforms. Isozyme PLCβ is activated by G-protein-coupled receptors, PLCγ by receptor Tyr kinases, PLCδ by calcium influx, and PLCε by Ras GTPase. Messenger IP₃ stimulates its receptor IP₃R at the membrane of the endoplasmic reticulum. Receptor IP₃R is modulated by phosphorylation by calcium–calmodulin-dependent kinase-2 and protein kinases PKA, PKC, and PKG, after possible recruitment of corresponding scaffold proteins.

Fig. 11.4

Calcium activities in cells. Ligand (L)-bound receptor (R) causes the entry of calcium from the extracellular space and the formation of second messengers, such as inositol trisphosphate (IP₃), cyclic ADP ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and sphingosine-1-phosphate (S1P). The second messenger releases calcium from its intracellular stores, the endoplasmic reticulum (ER; sarcoplasmic reticulum in the myocyte). Agent IP₃ is formed from phosphatidylinositol bisphosphate (PIP₂) by different isoforms of phospholipase C. It targets its receptor IP₃R. Metabolism-linked messengers cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), generated by ADP ribosyl cyclase from NAD and NADP, respectively, favors Ca ${}^{++}$ release from its stores. Ca ${}^{++}$ can also be transported into the cytosol via either ryanodine receptor (RR) associated with plasmalemmal voltage-dependent calcium channels, or by plasmalemmal, non-excitable store-operated channels (SOC; Orai1 channel) coupled to Ca ${}^{++}$ sensor stromal interaction molecule-1 (StIM1) in regions of the endoplasmic reticulum close to the plasma membrane. Most of the intracellular calcium binds to buffers; the remaining part targets effectors. Afterward, calcium ion leaves its effectors and buffers and is removed from the cell by various exchangers and pumps. Both Na⁺–Ca ${}^{++}$ exchanger (NCX) and plasma-membrane Ca ${}^{++}$ -ATPase (PMCA) expell Ca ${}^{++}$ in the extracellular medium, and sarco(endo)plasmic reticulum Ca ${}^{++}$ -ATPase (SERCA) pumps fill the stores. Mitochondria participate in the recovery. They quickly sequester Ca ${}^{++}$ via uniporters; Ca ${}^{++}$ is then slowly released into the cytosol to be extruded by SERCAs and PMCAs. (Source: [1508]).

Low concentration in inositol (1,4,5)-trisphosphate causes a minor Ca ${}^{++}$ flux from its store through a single IP₃R. This Ca ${}^{++}$ release then stimulates additional IP₃Rs that generate a Ca ${}^{++}$ burst. This short, fast flux provokes subsequent Ca ${}^{++}$ waves, as cytosolic Ca ${}^{++}$ concentration rises. Recruitment of Ca ${}^{++}$ egress can result from IP₃ concentration elevation. Local interaction between IP₃Rs then causes a rapid stimulation and slow inhibition by cytosolic Ca ${}^{++}$ . The IP₃R channels are initially randomly distributed (between-channel distance ∼ 1 μm). Rapid, reversible channel oligomerization triggered by low IP₃ concentration allows cooperativity [1516]. Oligomeric IP₃ receptors (generally tetramers) release Ca ${}^{++}$ from the endoplasmic reticulum in response to IP₃ and Ca ${}^{++}$ agents. Sensitivity of IP₃Rs is modulated by IP₃-mediated receptor clustering. At resting cytosolic Ca ${}^{++}$ concentration, clustered IP₃Rs can open independently with lower open probability, shorter open time, and less IP₃ sensitivity than isolated IP₃Rs. Increasing cytosolic Ca ${}^{++}$ concentration reverses the functional inhibition induced by channel clustering. Channel clustering reduces the distance between IP₃Rs to about 20 nm, so that clustered IP₃Rs are immediately exposed to Ca ${}^{++}$ released by their neighbors. Moreover, in a channel cluster, Ca ${}^{++}$ can counterbalance attenuated IP₃ sensitivity. At rest, IP₃ initiates IP₃R clustering. At the beginning, Ca ${}^{++}$ release remains restricted, but IP₃Rs are ready for Ca ${}^{++}$ excitation from adjacent channels for signal amplification. Furthermore, coupled gating enables simultaneous, prolonged open channel states.

Several mechanisms activate plasmalemmal Ca ${}^{++}$ channels, especially depleted endoplasmic reticulum (capacitative or store-operated Ca ${}^{++}$ entry. Signaling from the endoplasmic reticulum to the plasma membrane is initiated by membrane Ca ${}^{++}$ sensors, the stromal interaction moleculesStIM1 and StIM2, with Ca ${}^{++}$ -binding EF-hand motifs directed to endoplasmic reticulum lumen. Dissociation of Ca ${}^{++}$ from StIMs causes StIM aggregation and accumulation beneath the plasma membrane, where they interact with store-operated plasmalemmal Ca ${}^{++}$ channels of theOrai family (Orai1–Orai3). Transmembrane proteins StIM1 and StIM2 can strongly, rapidly, and directly interact with Orai1 that has an intrinsic ability to cluster [1517]. Whereas high, fast activation of Orai1 requires StIM1 or StIM2, Orai3 can be activated independently of both StIM1 and StIM2 proteins.

Calcium release-activated Ca ${}^{++}$ channels generate sustained Ca ${}^{++}$ signals as: (1) Ca ${}^{++}$ depletion from the endoplasmic reticulum triggers oligomerization of stromal interaction molecule-1 and its redistribution to junctions between endoplasmic reticulum and plasma membrane (ERPMJ; gap of 10–25 nm) and (2) CRAC channel subunit Orai1 accumulates in the plasma membrane. Non-stimulated Ca ${}^{++}$ -bound StIM1 moves freely throughout the endoplasmic reticulum membrane. Because StIM1 binds only a single calcium ion, its oligomerization is required for cooperative, efficient activity after Ca ${}^{++}$ store depletion. Furthermore, its oligomerization provokes StIM1 accumulation at ERPMJ [1518]. Oligomerization of StIM1 then drives store-operated Ca ${}^{++}$ influx after having triggered organization and activation of StIM1–Orai1 clusters at ERPMJ junctions.

Proteins Orai1 and StIM1 that regulate store-operated Ca ${}^{++}$ entry facilitate cell migration and tumor metastasis (Vol. 2 – Chap. 6. Cell Motility) by regulating focal adhesion turnover that involves protein phosphorylation and proteolysis [1519]. Elevated intracellular Ca ${}^{++}$ concentration can increase the activity of focal adhesion kinase and calcium-dependent peptidase calpain in focal adhesions. The former activates small Rac GTPase and the latter cleaves talin at adhesion sites. In addition, Ca ${}^{++}$ -sensitive myosin light-chain kinase and PP3 phosphatase intervene in focal adhesion turnover.

Sensor StIM1 has many binding partners. In fact, it regulate other types of store-operated channels such as members of the TRPC family. Proteins TRPC and Orai can form Ca ${}^{++}$ -selective, store- (SOC) and receptor-operated (ROC) calcium channels [1520]. Diacylglycerol-responsive channel TRPC can actually be activated by the Gq– and Gi–PLCβ axes. The TRPC–Orai complex participates in Ca ${}^{++}$ influx with or without activation of store depletion.

Phospholipase-Cγ has dual roles in regulating cellular calcium concentrations. It generates inositol trisphosphate, which releases calcium from intracellular stores. It binds to the transient receptor potential channel TRPC3 and promotes its insertion into the plasma membrane for calcium influx. The general transcription factor GTF2i outside the nucleus⁵⁹ inhibits calcium entry into cells by binding phospholipase-Cγ, antagonizing interaction of phospholipase-Cγ with the calcium channel TRPC3. This competition in favor of GTF2i for binding to PLCγ thereby suppresses surface accumulation of TRPC3 channels and hinders calcium influx across the plasma membrane [1521]. Dephosphorylated GTF2i could free PLγ and elevate the density of plasmalemmal TRPC3 receptor.

Ubiquitous Extended synaptotagmin-like proteins (ESyt) bind to Ca ${}^{++}$ in a phospholipid complex of intracellular membrane (ESyt1) and plasmalemmal (ESyt2 and ESyt3) components [1522]. They then serve as calcium sensors with multiple C2 domains. The C2 domain is a protein module used as calcium- and phospholipid-binding sites and/or as between-protein interaction domains. ⁶⁰

Calcium depletion from intracellular stores not only activates plasmalemmal store-operated channels, but also regulates formation of cAMP by adenylate cyclase [1523]. Ligand binding to receptors that are coupled to Gq or Gs subunits primes the PLC–IP₃ axis and Ca ${}^{++}$ release from endoplasmic reticulum store through IP₃R or excites ACase to trigger the cAMP–PKA pathway, respectively. Lowering concentration of free Ca ${}^{++}$ in the endoplasmic reticulum, whatever the cytosolic Ca ${}^{++}$ concentration, leads to recruitment of adenylate cyclases.Translocated Ca ${}^{++}$ sensor StIM1 that aggregates in a region near the plasma membrane can indeed activates ACase, either alone or in synergy with other ACase activators. Among 9 transmembrane ACases, AC1 and AC8 are major Ca ${}^{++}$ -activated isoforms, whereas AC5 and AC6 are inhibited by augmented cytosolic Ca ${}^{++}$ level, particularly after store-operated Ca ${}^{++}$ entry. Sensor StIM1 may then act as an attractor that facilitates recruitment and activation of ACases that can dimerize (e.g., AC2–AC5 heterodimers), without necessarily binding them. In addition, Ca ${}^{++}$ -dependent regulation of phosphodiesterases influences cAMP level.

11.5.5 Types of Calcium Signalings

Signaling pathways are characterized by their spatial distribution (cellular compartmentation), associated with involved molecular complexes, and temporal dynamics to keep the specificity of calcium signaling. Quick, brief, localized calcium transients are associated with fast responses; transient, repetitive, distributed calcium oscillations, which can generate calcium waves, trigger slow responses. Calcium flux oscillations of given amplitude and frequency are determined by the stimulus intensity. Furthermore, calcium is able to regulate its own signaling pathways, as it influences the functioning of Ca ${}^{++}$ channels.

Ca ${}^{++}$ influx for long-term effect can be triggered by inositol trisphosphate on plasmalemmal IP₃Rs and Orai1 channels (store-operated Ca ${}^{++}$ release–activated Ca ${}^{++}$ influx, (Vol. 3 – Chap. 3. Main Classes of Ion Channels and Pumps).

Some activated G-protein-coupled receptors trigger calcium influx (Table 11.20). In the nervous system, both metabotropic glutamate receptor mGluR1 coupled to Gα_q and mGluR5 coupled to Gα₁₁ activate PLCβ, but trigger different types of calcium signaling via IP₃R, generating a single Ca ${}^{++}$ transient and an oscillatory pattern, respectively [1508]. In pancreatic acini, muscarinic receptors, which are more sensitive to the inhibition of RGS, provoke small, localized Ca ${}^{++}$ transients, and cholecystokinin receptors cause large, distributed Ca ${}^{++}$ transients. Among other GPCRs, bradykinin and neurokinin-A receptors give a large, rapid calcium influx. Lysophosphatidic acid, thrombin, and histamine receptors trigger small, slow, persistent calcium fluxes.

The inhibitory subunit of protein phosphatase-1 catalytic subunit PP1_r9b ⁶¹ binds to actin, regulators of G-protein signaling, and G-protein-coupled receptors. Once bound to GPCRs, it reduces the intensity of calcium signaling by GPCRs, such as α1b-adrenergic receptors [1524]. On the other hand, the inhibitory subunit of protein phosphatase-1 PP1_r9a ⁶² that does not bind to α1b-adrenoceptors, increases the intensity of calcium signaling by α1b-adrenoceptors. Subunit PP1_r9b prevents binding of RGS2 to cytosolic PP1_r9a subunit. The latter binds to RGS2 that is thus removed from G-protein-coupled receptors. Agent RGS2 inhibits calcium signaling by GPCRs, especially α1b-adrenergic receptors. Conversely, PP1_r9a hinders binding of RGS2 to PP1_r9b associated with G-protein-coupled receptors. Therefore, PP1_r9a and PP1_r9b form a pair of antagonist regulators that tune the intensity of calcium influx by GPCRs (Fig. 11.5).

Fig. 11.5

A functional pair of antagonist regulators of GPCR-triggered calcium influx. Both spinophilin (i.e., inhibitory subunit PP1_r9b of protein phosphatase-1) and neurabin (PP1_r9a inhibitor) bind regulator of G-protein signaling RGS2, which hampers calcium influx by G-protein-coupled receptors. Subunit PP1_r9b bound to a GPCR prevents RGS2 binding to cytosolic PP1_r9a, thus precluding calcium influx. Conversely, PP1_r9a hinders RGS2 binding to PP1_r9b, removing RGS2 away from GPCR, and thereby favoring GPCR-mediated calcium influx.

11.5.6 Calcium-Mobilizing Mediators cADPR and NAADP

Calcium-mobilizing second messengers include cyclic ^ADPribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). The former is one of the Ca ${}^{++}$ signaling messengers associated with the NAD⁺ pathway. Its production is coupled to the cell metabolism, hence available chemical energy. The latter is one of the most potent Ca ${}^{++}$ signaling messengers that cause Ca ${}^{++}$ release from intracellular stores.

Two paralogous catalytic transmembrane proteins,^ADPribosyl cyclase-1 and -2, or cADPR hydrolase-1 and -2,⁶³ produce NAADP and cADPR, using their ectodomains. This type of enzyme uses NAD⁺ to manufacture cADPR and NADP to synthesize NAADP agent. Moreover, synthesis and degradation of cADPR is carried out by the same enzyme that is both a synthase and hydrolase. Hydrolysis of cADPR is inhibited by ATP or NADH [10]. Unlike cADPR, NAADP is degraded to NAAD by phosphatases such as alkaline phosphatase.

11.5.6.1 Cyclic ^ADPRibose Signaling

The cADPR-mediated control of Ca ${}^{++}$ release may occur indirectly via the activation of the sarco(endo)plasmic reticulum Ca ${}^{++}$ ATPase. Increased storage may sensitize release channels such as ryanodine-sensitive channels. Therefore, cADPR messenger operates as a modulator rather than a mediator of Ca ${}^{++}$ signaling [10].

Modulator cADPR may also enhance the sensitivity of ryanodine receptors. Catabolism of cADPR produces ADPR, a regulator of plasmalemmal melastatin-related TRPM2 transient receptor potential channel.

11.5.6.2 Nicotinic Acid–Adenine Dinucleotide Phosphate Signaling

Nicotinic acid–adenine dinucleotide phosphate (NAADP) uses a NAADP-sensitive store distinct from the endoplasmic reticulum, which may be a lysosome-related organelle. It does not interact with inositol trisphosphate and ryanodine receptors, but with a family of NAADP-regulated Ca ${}^{++}$ cation-selective 2-pore channels (TCP) encoded by the 2 TPCN genes (TCPN1–TCPN2 at least in mice).⁶⁴ Subtype TCP1 localizes to the endosomal membrane; TCP2 to the lysosomal membrane [10].

11.5.7 Calcium and Nervous Control of Blood Circulation

11.5.7.1 Neuronal Calcium Sensors

Members of the neuronal calcium sensor (NCS) family are involved in manifold neuronal signaling pathways. The mammalian set of neuronal calcium sensors contains NCS1,⁶⁵ 3 visinin-like proteins (ViLiP or VsnL1),⁶⁶ neurocalcin, hippocalcin (HpCa),⁶⁷ recoverin,⁶⁸ 3 guanylate cyclase-activating proteins (GCAP),⁶⁹ and 4 voltage-gated potassium channel-interacting proteins (KChIP1–KchIP4), with several spliced KChIP isoforms.⁷⁰

Table 11.27

Time scales of calcium-regulated processes in neurons (Source: [1526]).

Neurotransmission	< 1 ms
Channel activity	> 1 ms
Short-term plasticity	> 100 ms
Long-term potentiation	< 10 s
Long-term depression	> 10 s
Gene expression	> 10 s

The activity of the cardiovascular system is controlled by the nervous system. Neuron activities depend on the temporal feature of the calcium signal (Table 11.27). Local increases in intracellular calcium trigger neurotransmitter release less than 100 μs after Ca ${}^{++}$ influx. Ca ${}^{++}$ -binding synaptotagmin acts as a Ca ${}^{++}$ sensor for fast neurotransmission [1526].

11.5.7.2 Calcium Signaling in the Cardiovascular Apparatus

Calcium Signaling in Cardiomyocytes and the Nervous Control

Cardiomyocytes permanently bear calcium influxes and effluxes for contraction and relaxation. Adaptative responses associated with modifications in gene transcription then require changes in Ca ${}^{++}$ signaling. Calcium ions act indirectly on transcription factors. Phosphatase PP3 dephosphorylates the transcription factor nuclear factor of activated T cells NFAT3, which can enter the nucleus to induce gene transcription, in opposition to the PI3K pathway that inhibits glycogen synthase kinase-3, thus inactivating NFAT3 (Vol. 5 – Chap. 6. Cardiomyocytes).

Elevation of cytosolic calcium levels can be caused by angiotensin-2 and endothelin-1 via GPCR (Gq), and phospholipase-Cβ that generates inositol trisphosphate. Diacylglycerol can target via protein kinase-D histone deacetylases and may recruit the mitogen-activated protein kinase module to activate cAMP response element-binding protein.

The adrenergic pathway enhances calcium signaling via adenylate cyclases that produces cAMP messenger. The latter leads to phosphorylation by PKA of Ca_V1.2 channels and ryanodine receptors RyR2, as well as phospholamban that inhibits SERCA pumps. Calcium ions are involved in cardiac hypertrophy and congestive heart failure. Decay in SERCA activity, at least partially due to enhanced inhibition by phospholamban, is associated with a decline in β-adrenergic signaling.

Calcium Signaling in Vascular Smooth Muscle Cells

Resistance arteries regulate locally blood flow by constricting or relaxing in response to changes in hemodynamic stresses. In particular, increased intraluminal pressure causes gradual depolarization of arterial smooth muscle cells, in which the Ca ${}^{++}$ concentration subsequently rises and activates myosin light-chain kinase that causes contraction of stress fibers to maintain constant the flow rate ( autoregulation).

In arterial smooth muscle cells, opening of single or clusteredCa_V1.2b produces local elevations in intracellular Ca ${}^{++}$ concentration, the so-called Ca ${}^{++}$ sparklets at hyperpolarized membrane potentials, for which the open probability of Ca_V1.2b channels is very low.

Another Ca_V type — Ca_V1.3 —, with a voltage dependence of activation more negative than that of Ca_V1.2b channels, can also cause Ca ${}^{++}$ sparklets [1527]. Like Ca_V1.1 and Ca_V1.2 channels, Ca_V1.3 may operate with 2 gating modes: short ( ∼ 1.6 ms) and long ( ∼ 9.5 ms) open times. On the other hand, Ca_V1.4 channels may not function with a bimodal gating.

Randomly activating solitary Ca_V1.2b channels acting in a low-activity mode open rarely and create limited Ca ${}^{++}$ entry (low-activity Ca ${}^{++}$ sparklets) [1527]. On the other hand, single or clusters of Ca_V1.2b channels operating in a high-activity mode generate persistent Ca ${}^{++}$ sparklets that depend on protein kinase-C (high-activity or persistent Ca ${}^{++}$ sparklets). Protein kinase-Cα coerces discrete clusters of Ca_V1.2b channels to work in a high open probability mode, thereby engendering subcellular domains of nearly continuous Ca ${}^{++}$ entry. Calcium sparklets also depends on activities of protein phosphatases PP2 and PP3.

Like Ca_V1.2 channels, Ca_V1.3 can produce persistent Ca ${}^{++}$ sparklets. However, Ca_V1.2 channels, not Ca_V1.3 channels, give rise to Ca ${}^{++}$ sparklets in mouse arterial smooth muscle cells [1527]. The voltage dependences of activation and inactivation of Ca ${}^{++}$ flux in arterial myocytes ressemble to those of Ca_V1.2, but not Ca_V1.3 currents. Moreover, transcripts for Ca_V1.2, but not Ca_V1.3 protein, are observed in human arterial smooth muscle cells.

Membrane depolarization increases Ca ${}^{++}$ entry via low- and high-activity Ca ${}^{++}$ sparklets. Low- and high-activity Ca ${}^{++}$ sparklets modulate local and global Ca ${}^{++}$ concentration and effect in arterial smooth muscle cells [1528].

Signaling responsible for endothelium-dependent regulation of vascular smooth muscle tone relies on calcium sparklets in the vascular endothelium of resistance arteries generated by TRPV4 cation channels [1529]. Gating of a single TRPV4 channels within a 4-channel cluster causes vasodilation due to amplification resulting from cooperation of adjoining TRPV4 channels as well as via activation of endothelialintermediate (IK) andsmall (SK) conductance, Ca ${}^{++}$ -sensitive K⁺ channels. Intermediate conductance (IK) channels colocalize with TRPV4 close to myoendothelial gap junctions. The ionic flux and associated current through activated IK and SK channels can then spread to surrounding smooth muscle cells using myoendothelial gap junctions. Hyperpolarization of smooth muscle cell membrane subsequently induces a vasodilation.

11.5.8 Calcium Signaling and Immunity

Resting lymphocytes have a low concentration of intracellular calcium ions. Upon commitment of antigen receptors, calcium enters into lymphocytes from the extracellular space mainly via store-operated calcium channels. The latter comprises 2 major components: pore-forming calcium release-activated calcium modulator CRACM1 and endoplasmic reticulum-resident sensor of stored calcium, the stromal interaction StIM1 molecule. Upon antigen recognition by lymphocytes, phosphorylated (activated) phospholipase-C generates diacylglycerol and inositol trisphosphate from phosphatidylinositol (4,5)-bisphosphate. Inositol trisphosphate binds to its receptor on the surface of the endoplasmic reticulum to release Ca ${}^{++}$ from its stores. Calcium influx with protein kinase-C activated by diacylglycerol initiates quick remodeling of actin cytoskeleton to promote T-cell motility, adhesion, and formation of the immunological synapse. Store depletion stimulates SOC channels, such as low-conductance, Ca ${}^{++}$ -selective calcium release-activated calcium channels for a sustained response that is necessary for the maintenance of immunological synapses [1530].

Other channels can be involved such as canonical transient receptor potential channels. Channel TRPC1 is able to form diverse channels via homo- or heteromeric interactions with TRPC3, TRPC4, and TRPC7 that are relatively selective or non-selective to Ca ${}^{++}$ [1530]. Some TRPC channels are activated by diacylglycerol.

Diacylglycerol also excites the Ras–MAPK cascade that activates transcription AP1 factor. The latter cooperates with transcription factors NFAT and NFκB to control gene expression. Calcium entry by CRAC channels can activate the Ras–MAPK pathway to enhance AP1 activation [1530]. In activated lymphocytes, Ca ${}^{++}$ signaling also targets JNK and CamK kinases. A prolonged increase in cytosolic Ca ${}^{++}$ concentration via CRAC channels is required for activation of phosphatase PP3 that dephosphorylates NFAT factor. Dephosphorylated NFAT enters the nucleus. A persistent elevation in Ca ${}^{++}$ concentration is needed to prevent PP3 ejection from the nucleus [1531]. Nuclear import of NFAT is impeded by GSK3 kinase. Therefore, NFAT transcriptional activity depends on receptor stimulation and NFAT intranuclear concentration.

Several other cytosolic and nuclear molecules modulate intracellular NFAT location, such as PP3 inhibitors (AKAP, calcineurin (PP3)-binding protein [CaBin], calcineurin homologous protein [CHP], or calcium-binding protein P22, Down syndrome critical region gene product DSCR1, cytoplasmic scaffold proteins Homer-2 and Homer-3, and NFAT kinases GSK3, CK1, and DYRK) [1531, 1532].

Calcium signaling is necessary for T-cell proliferation and cytokine secretion. Composition of CRAC channels in T lymphocyte can vary with its differentiation status. Homologs CRACM1, CRACM2, and CRACM3 are involved at various degrees (CRACM2 to a lesser extent than other types),⁷¹ as well as StIM1, in Ca ${}^{++}$ influx in developing, mature, and activated T lymphocytes [1533]. T lymphocytes also express Ca_V channels that can participate in response to stimulated T-cell receptors. Phosphatase PP3 that is activated by Ca ${}^{++}$ –calmodulin complex intervenes in the development of CD4 + , CD8 + , double-positive thymocytes into CD4 + , CD8 − and CD4 − , CD8 + , single-positive thymocytes. Non-store-operated cation channel TRPM7 for Ca ${}^{++}$ and Mg ${}^{++}$ ions with an intrinsic kinase activity operates in thymocyte maturation.

Calcium signaling is important in mastocytes that work at the interface of innate and adaptive immune responses. Crosslinking of immunoglobulin-E receptor FcεR1 that is the main activation mechanism of mastocytes initiates intervention of SRC family kinases Lyn and Fyn that trigger the Lyn–SYK–LAT–PLC and Fyn–GAB2–PI3K–PKC cascades to prime Ca ${}^{++}$ flux and control degranulation. Agents StIM1 and CRACM1 are involved in mastocyte degranulation. Melastatin-related transient receptor potential channel TRPM4, a Ca ${}^{++}$ -activated non-selective cation channel, diminishes the driving force of Ca ${}^{++}$ influx through CRAC channels by modulating the membrane potential [1534]. Both StIM1 and CRACM1 are also needed for leukotriene and cytokine secretion as CRAC channels and increase in cytosolic Ca ${}^{++}$ concentration are involved in the secretion of pro-inflammatory lipid mediators and activation of NFAT and NFκB that regulate the synthesis of cytokines [1535].

11.5.9 Calcium Signaling and Intracellular Transport

Exocytosis comprises multiple Ca ${}^{++}$ -dependent steps that also involve Ca ${}^{++}$ -dependent nanomotors, cytoskeleton, and SNARE proteins down to the formation of a fusion pore, an aqueous channel that connects the vesicle lumen to the extracellular space. Their controlled opening and closure regulate secretion of the vesicle content. Calcium transients are major players of this process; they are associated with fixed and mobile Ca ${}^{++}$ buffers, in addition to various types of carriers (channels, pumps, and exchangers).

11.5.9.1 Fast Secretors

In fast secretors such as neurons, pore formation and transmitter release follow (delay $\mathcal{O}$ [1 μs]– $\mathcal{O}$ [1 ms]) elevation of cytoplasmic Ca ${}^{++}$ concentration. Calcium transients originate from Ca ${}^{++}$ influx through voltage-gated Ca ${}^{++}$ channels. Plasmalemmal voltage-gated Ca ${}^{++}$ channels close to the site of fusion are activated during the prefusion stage in neurons and neuroendocrine cells. Transient calcium signals then propagate throughout the cell, particularly around subplasmalemmal vesicles (speed ∼ 10 μm/s) [1536].

11.5.9.2 Slow and Non-Excitable Secretors

Slow secretors such as alveolar type-2 pneumocytes that slowly release pulmonary surfactant (choline-based phospholipids and surfactant proteins) stored in secretory vesicles, the so-called ellipsoidal lamellar granules or lamellar bodies (length 300–400 nm; width 100–150 nm, ≤ 10/cell), which have a scattered intracellular distribution rather than organized in cortical clusters, using Ca ${}^{++}$ and the actin cytoskeleton.

Lamellar granules fuse with the plasma membrane in a sequential process (duration ∼ 20 mn). Each fusion steps is followed by a transient rise of localized cytoplasmic Ca ${}^{++}$ concentration (decay half-life 3.2 s) that originate at the site of lamellar body fusion [1536]. The major calcium source is the extracellular space, hence the name fusion-activated Ca ${}^{++}$ entry, although Ca ${}^{++}$ ions are stored in lamellar granules. Besides, type-2 pneumocytes lack voltage-gated Ca ${}^{++}$ channels.

In isolated rat alveolar type-2 cells, fusion-activated Ca ${}^{++}$ entry follows initial fusion pore opening with a delay of 200 to 500 ms [1537].

Calcium-induced Ca ${}^{++}$ release may amplify Ca ${}^{++}$ influx. A moderate overall increase in cytoplasmic Ca ${}^{++}$ concentration (slightly above resting values; ∼ 320 nmol/l) is required to induce fusion of lamellar granules with the plasma membrane.

Calcium influx may results from currents through channels of the plasma membrane or channels of the lamellar granule membrane. Whatever its location, channel activation may results from membrane mechanical stress or action of membrane chemical merger. In fact, fusion-activated Ca ${}^{++}$ entry results from activity of vesicle-associated Ca ${}^{++}$ channels [1537]. Ionotropic P2X₄ receptors indeed reside on lamellar granules.

An actin coat forms around a lamellar granule after fusion; its contraction enables full extraction of the vesicular content. In fact, after fusion of a lamellar granule with the plasma membrane, surfactant, a water-insoluble complex of lipids and proteins remains within the fused vesicle.

During the postfusion phase, when the lumen of the lamellar granule becomes a part of the extracellular space, an elevated cytoplasmic Ca ${}^{++}$ concentration leads to fusion pore dilation. A fusion-activated Ca ${}^{++}$ entry supports the postfusion phase of surfactant secretion [1537].

11.5.10 Calcium Signaling and Cell Fate

Promyelocytic leukemia protein (PML),⁷² controls calcium signaling at the endoplasmic reticulum, near mitochondria, i.e., at signaling regions involved in endoplasmic reticulum-to-mitochondrion Ca ${}^{++}$ transport and in induction of apoptosis [1538].⁷³

Multiple isoforms of human PML transcripts arise from alternative splicing. Post-translational modifications add further diversity in PML structure and function. These isoforms have both cytoplasmic and nuclear locations. Promyelocytic leukemia protein localizes to nuclear bodies (a.k.a. nuclear dots, PML bodies, and Kremer bodies; number 10–30 per nucleus; size 0.2–1 μm). It resides also in the nucleoplasm as well as in the nucleolus during cell senescence and stress. It also lodges at sites of contact between the endoplasmic reticulum and mitochondria, where it connects to the IP₃R calcium channel, protein kinase-B, and protein phosphatase-2 [1538].

Protein PML can then modulate phosphorylation of IP₃R and hence calcium release from the endoplasmic reticulum to regulate calcium mobilization into the mitochondrion. It can then trigger early, transcription-independent apoptosis program.

11.6 Oxygen Delivery and Hypoxia Transduction

The cardiovascular and ventilatory apparatus cooperate under the control of the nervous system to ensure the delivery of oxygen and nutrients needed throughout the organism. Oxygen supply is indeed necessary for efficient energy production. Cells use the tricarboxylic acid cycle and oxidative phosphorylation in the mitochondria as well as glycolysis for energy production under aerobic conditions (Vol. 1 – Chap. 4. Cell Structure and Function). Cell survival depends on O₂ delivery.

Oxygen is an electron acceptor during mitochondrial respiration, but the respiratory chain functions within a narrow range of O₂ concentrations. Oxygen is required to generate energy, but energy generation produces potentially toxic oxidants such as reactive oxygen species (Sect. 10.6.1).⁷⁴

Acute hypoxia and hyperoxia disturb cell life. However, the organism adapts to conditions of low or high oxygen by triggering a cascade of events to reprogram cellular oxygen requirements. In particular, multiple mechanisms allow the cell to respond to decreased oxygen levels. The hypoxia response provokes activation of multiple signaling pathways involved in regulation of cell respiration, metabolism, and survival. Under hypoxia, cells shift from an aerobic mode of energy production to an anaerobic mode. They raise glucose uptake and inhibit enzymes leading to the tricarboxylic acid cycle, hence shifting ATP generation from mitochondrial respiration that consumes oxygen to anaerobic glycolysis outside mitochondria (Pasteur effect). They also decrease the metabolic rate.

Hypoxia at high altitudes induces 3 main responses: (1) neurotransmitter release by the carotid body to increase breathing; (2) pulmonary vascular constriction in poorest oxygenated regions of the lung; and (3) erythropoietin production (up to several 100-fold increase) by renal interstitial fibroblasts to augment erythropoiesis and hemoglobin concentrations in blood. The hypoxia response comprises 2 axes: HIF-dependent and -independent pathways.

11.6.1 Hypoxia-Inducible Factor Axis

The main transcription factor that regulates transcriptional responses to hypoxia is hypoxia-inducible factor (Sect. 10.9.2) that was first found to interact with erythropoietin gene. Hypoxia-inducible factor is a DNA-binding heterodimer.⁷⁵

The HIF1α–HIF1β dimer, i.e., HIF1 transcription factor, binds to hypoxia response elements in the regulatory regions of target genes, thereby controlling the expression of a huge number of genes. These target genes vary according to the cell type (canonical hypoxic signaling). Both HIF2α and HIF3α are also produced in response to hypoxia. Oxygen-regulated HIF2α also dimerizes with HIF1β. HIF2α is produced not only in endothelial cells, but also in parenchymal and interstitial cells of many organs, such as cardiomyocytes, renal interstitial cells, hepatocytes, duodenal epithelial cells, and astrocytes.⁷⁶ Subunit HIF3α inhibits HIF1αcomponent. Under O₂-independent conditions, HIF1α production can be enhanced by the PI3K–PKB–TOR pathway.

Hypoxia-inducible factor regulates the expression of several genes involved in iron homeostasis, such as transferrin, thereby coordinating erthyropoiesis with iron availability. It also favors cell survival [1544] (Table 11.28). It indeed adapts the cellular metabolism to hypoxia by priming glycolysis to compensate energy loss due to reduced oxidative phosphorylation and enhances glucose uptake (Fig. 11.6).⁷⁷ HIF also intervenes in mitochondrial respiration,⁷⁸ and regulates intracellular pH.⁷⁹

Table 11.28

Hypoxia-inducible factor intervenes in cell survival via a set of pathways. HIF activation favors cell protection from oxygen deprivation. Glucose transporters and glycolytic enzymes favor anaerobic ATP production. Under hypoxia, HIF1 upregulates the gene expression for [1545]: (1) glucose transporters GluT1 and GluT3, which increase intracellular glucose uptake; (2) glycolytic enzymes that convert glucose into pyruvate; (3) lactate dehydrogenase-A that transforms pyruvate into lactate; (4) pyruvate dehydrogenase kinase-1 that phosphorylates (inactivates) pyruvate dehydrogenase (that transforms pyruvate into acetylCoA, which enters into the mitochondrial tricarboxylic acid cycle); and (5) cytochrome-C oxidase subunit CyOx4-2 that replaces CyOx4-1 and then increases the efficiency of mitochondrial respiration.

↓ cell apoptosis
modulation of activity of hypoxia-regulated microRNAs
↓ ROS production
↓ inflammation
↑ antioxidant enzymes (heme oxygenase-1)
↑ inducible nitric oxide synthase
↑ cyclooxygnease-2
↑ glucose uptake (GluT)
↑ glycolysis
↓ ATP depletion
↑ oxygen delivery
↑ erythropoiesis
↑ angiogenesis
↑ extracellular matrix regulation

Fig. 11.6

Hypoxia tolerance by reprogramming basal metabolism from oxidative to anaerobic ATP production via inhibition of oxygen-sensitive prolyl hydroxylase domain-containing protein PHD1 that lowers oxygen consumption (Source: [1546]). Subunit HIF2α augments the expression of pyruvate dehydrogenase kinases (PDK) that inhibit the pyruvate dehydrogenase complex (PDC), which controls entry of glucose-derived pyruvate into the mitochondrion. Inside the mitochondrion, pyruvate enters the tricarboxylic acid cycle (TCA) that together with fatty acid oxidation indirectly generates ATP using enzymes of the electron transport chain (ETC) in the presence of oxygen, as well as reactive oxygen species (ROS) produced by the electron transport chain. (When myocytes continue to consume oxygen despite the limited oxygen supply, they generate excessive ROS amounts.) Subunit HIF1α upregulates pyruvate dehydrogenase kinase PDK1 isoform. Subunit HIF2α increases the expression of the transcription factor peroxisome proliferator-activated receptor PPARα, which elevates PDK4 to reduce mitochondrial respiration in striated myocyte.

Subunit HIF1α improves oxygen delivery not only by incresing erythropoiesis, but also stimulating synthesis of various compounds, such as vasoactive molecules (nitric oxide synthase), growth factor (VEGF), and extracellular matrix regulators (urokinase-type plasminogen activator receptor, collagen prolyl 4-hydroxylase, matrix metallopeptidase MMP2, and tissue inhibitor of matrix metallopeptidase TIMP1), hence enhancing angiogenesis (Table 11.29).

Table 11.29

Regulation by HIF1α subunit of genes of factors involved in successive steps of angiogenesis (Source: [1544]). Growth factor VEGF is upregulated by HIF1α as well as its VEGFR1 receptor, thereby further increasing VEGF activity.

Vasodilation	NOS
Vascular permeability	VEGF
Extracellular matrix remodeling	MMP, uPAR
Endothelial sprouting	Ang2
Cell proliferation	VEGF, FGF, PDGF, SDF1, CXCR4
Inhibitors	Ang1, Tsp1

The transcriptional response to hypoxia is primarily mediated by HIF1α and HIF2α subunits. Both HIF1α and HIF2α have distinct functions with a functional overlap on targeted genes, which varies according to the cell type. Genes of vascular endothelial growth factor, adrenomedullin, and glucose transporter GluT1 are stimulated by both HIF1α and HIF2α.

Subunit HIF1α uniquely upregulates the expression of glycolytic enzymes. Subunit HIF2α targets genes involved in erythropoiesis, angiogenesis, and cell proliferation, such as those that encode embryonic transcription factor Oct4, cyclin-D1, transforming growth factor-α, and erythropoietin. Subunit HIF2α cooperates with ETS1 transcription factor to synergistically activate VEGFR2 expression and with ELk1 transcription factor for erythropoietin, insulin-like growth factor-binding protein IGFBP3, and plasminogen activator inhibitor PAI1. Subunit HIF2α specifically interacts with NFκB essential modulator NEMo (IKKγ). It also regulates the activity of transcription factors, such as Notch and Myc, thus enhancing expression of cyclin-D2.

Activity of HIF1 depends on the availability of HIF1α subunit. In normoxia, continuously synthesized HIFα subunits have a very short half-life. Subunit HIF1α is only detectable during hypoxia. The concentration and activity of HIF1α is controlled by oxygen-dependent prolyl-hydroxylating domain-containing proteins (PHD) and asparaginyl factor-inhibiting HIF1α hydroxylases (FIH1). Under normoxia, prolyl hydroxylase domain-containing protein⁸⁰ leads to interaction of newly synthesized, oxygen-sensitive HIF1α subunit with von Hippel-Lindau protein for polyubiquitination and proteasomal degradation.⁸¹ In addition, asparaginyl hydroxylase⁸² FIH prevents HIF interactions with CBP and P300 coactivators.

Hypoxia activates HIF1α by suppressing hydroxylation of proline and asparagine residues, thereby preventing HIF1α degradation and favoring its accumulation and dimerization with HIF1β in the nucleus. The dimer then recruits coactivators such as P300 and binds to hypoxia-response elements of target genes.

Many environmental and intracellular factors modulate the activity of prolyl hydroxylase. Nitric oxide at relatively high concentration and ROS inhibit PHD activity [1547].⁸³ Different PHD isoforms differentially contribute to physiological and pathophysiological processes, such as growth, differentiation and survival at the cell level, and angiogenesis, erythropoiesis, and tumorigenesis at the tissue level. These enzymes can function as tumor suppressors. Among the 4 PHD isoforms (PHD1–PHD3 and transmembrane (endoplasmic reticulum) prolyl 4-hydroxylase [_TMP4Hor PH4tm]), HIF1α is more efficiently hydroxylated by PHD2 and HIF2α by PHD1 and PHD3 isozymes. These 3 PHDs are widely distributed among different organs, but at different levels (PHD3 is the highest subtype in the heart). They mostly localize to the cytoplasm. They undergo proteasomal degradation. Both PHD2 and PHD3 bear feedback upregulation by HIFs to suppresses further accumulation of HIFα and ensure rapid removal of HIF after reoxygenation.⁸⁴ Factor TGFβ1 that is also stimulated by hypoxia inhibits PHD2 transcription via SMAD signaling, thereby counteracting the effect of HIF-induced upregulation of PHD2 enzyme. Prolyl hydroxylases also hydroxylate non-HIF substrates and use hydroxylase-independent mechanisms to modify HIF activity [1547]. They can inhibit HIF1α transcriptional activity without decreasing HIF1α amount. Both PHD1 and PHD2 regulate the transcriptional activity of NFκB, hydroxylating IKKβ (Fig. 11.7).

Fig. 11.7

Schematic illustration of HIF activity and regulation by prolyl hydroxylase domain-containing protein (PHD). These enzymes are controlled by NO and ROS agents. Isozyme IKKβ hydroxylated by PHD1 or PHD2 fails to phosphorylate inhibitory factor IκBα, thus being unable to dissociate IκBα from NFκB and to activate NFκB. Hypoxia response element (HRE) are upregulated by HIF factor.

Factor HIF1α is able to complex with [1545]: (1) HIF1β and P300 coactivator that work in transcription for metabolic adaption to hypoxia on the one hand, and angiogenesis and erythropoiesis to increase O₂ delivery on the other hand; (2) FIH1 to inhibit the interaction with P300; (3) heat shock protein HSP90 to stabilize HIF1α; (4) endoplasmic reticulum lectin ERLec2⁸⁵ and prolyl hydroxylase domain-containing protein PHD2 to promote hydroxylation and subsequent degradation; and either (5) spermidine/spermidine acetyltransferase SAT2,⁸⁶ von Hippel-Lindau protein and elongin-C ubiquitin-ligase, or RACK1 and elongin-C for ubiquitination and degradation by 26S proteasome.

11.6.1.1 HIF1 and ROS

Hypoxia-induced production of reactive oxygen species in mitochondria by complex-3 of the electron transport chain under hypoxia is necessary and sufficient for HIF1α accumulation [1548]. Mitochondria acting as oxygen sensors thus regulate HIF1 activity.

11.6.1.2 HIF1 and FoxO3a

Loss in phosphatase and tensin homolog deleted on chromosome-10 (PTen) and subsequent inactivation of Forkhead box factor FoxO3a⁸⁷ relieve HIF1 inhibition [1549]. Overexpression of P300 reverses FoxO3a-mediated repression of HIF1 activity. Factor FoxO3a interferes with transcriptional coactivator P300, thereby impeding HIF1 transcriptional activity.

11.6.1.3 HIF–Notch Coupling

Hypoxia-inducible factor-1α also interacts with the intracellular domain of Notch (Notch^ICD), in addition to the canonical hypoxia-primed HIF pathway. Released Notch^ICD interacts with HIF1α that is recruited to Notch-responsive promoters during hypoxia. Consequently, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations via Notch signaling [1550].

Factor HIF can also enhance expression of Notch target genes, such as HES1 and related HRT2 transcriptional regulators. Moreover, Notch^ICD potentiates the recruitment of HIF1α to HIF-responsive promoters [1551].

Factor-inhibiting HIF1 hydroxylase (FIH1) hydroxylates not only HIF, but also Notch intracellular domain. Enzyme FIH1 has a higher affinity for Notch^ICD than for HIF1αfactor. Intracellular domain of Notch causes FIH1 to deviate away from HIF1α in the absence of excessive FIH1 amount during hypoxia.

11.6.1.4 HIF and RTK-Based Signaling

Heterodimer HIF lowers clathrin-mediated endocytosis at the early endosome sorting stage, thereby prolonging signaling from receptor protein Tyr kinase at the plasma membrane to promote cell survival under hypoxia. In addition, HIF1 and HIF2, during hypoxia, promotes the formation of caveolae, flask-shaped invaginations involved in endocytosis and signaling [1552]. They indeed foster the production of caveolin-1 that binds to EGFR receptors. Caveolin-1 provokes EGFR dimerization within caveolae and subsequent phosphorylation (activation) in the absence of ligand. Caveolin-1 also causes the activation of other RTKs, such as PDGFR and IGF1R receptors.

11.6.2 Hypoxia-Inducible Factor-Independent Axis

11.6.2.1 Target of Rapamycin

Under hypoxia, target of rapamycin activity is impeded to save energy. Stress-response, hypoxia- and DNA damage-inducible transcript-4 (DDIT4)⁸⁸ regulates TOR activity via the TSC1–TSC2 inhibitory complex. In addition, promyelocytic leukemia protein (PML) interacts with TOR and sequesters it into nuclear bodies [1362].

11.6.2.2 Hypoxia-Regulated MicroRNAs

Hypoxia during tumorigenesis upregulates a specific group of microRNAs, the hypoxia-regulated microRNAs (HRM).⁸⁹ They could affect cell apoptosis and proliferation, hence angiogenesis in cancers. Certain microRNAs can be controled by transcription factors in response to endogenous and exogenous stimuli. Transcription factors cMyc and E2F activate the oncogenic miR17–miR92 cluster. Transcription factors P53 and NFκB also affect the expression of microRNAs. Hypoxia-inducible factor activation triggers production of several HRMs [1553].

11.6.2.3 Reactive Oxygen Species

Acute hypoxia and hyperoxia yield excessive amounts of reactive oxygen species. These molecules act not only as toxics, but also as signaling molecules (Sect. 10.6.1). Signaling primed by ROS must be protected, whereas ROS toxicity must be prevented. Intracellular ROS concentration must thus be adjusted. Main sources of ROS are plasmalemmal NADPH oxidases and mitochondria. Nuclear factor erythroid-derived-2-like transcription factor NRF2 is involved in oxidative and environmental stress tolerance and oxidant elimination.

Global long-lasting differentiation pathways regulated by P53, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), MyC transcription factor, and forkhead box-O family proteins yield either long-lasting oxidant-protective responses or damaged cell death [1554]. Reactive oxygen species modulate various redox-sensitive signaling pathways, such as cascades triggered by growth factors, according to the metabolic state of the cell. They target protein Tyr and MAPK phosphatases. Receptors of ROS include peroxidases.

Peroxiredoxin-1 is required in P38MAPK activation by H₂O₂ agent. Peroxiredoxin-2 controls H₂O₂ concentration produced during growth factor signaling. For example, peroxiredoxin-2 interacts with activated platelet-derived growth factor receptor. Peroxidase-3 regulates apoptosis by H₂O₂.

11.6.2.4 Chromatin Remodeling

Endothelial cells respond to hypoxia and reduce abundance of endothelial nitric oxide synthase to cause hypoxia-induced vasoconstriction. Vascular endothelium is characterized by synthesis of labeling proteins, such as NOS3, von Willebrand factor, vascular-endothelial (VE)-cadherin (Cdh5), intercellular adhesion molecule ICAM2, angiopoietin TIE1 and TIE2 receptors, vascular endothelial growth factor receptors VEGFR1 and VEGFR2; Notch-4, and EPHb4 receptor. Hypoxia reduces expression of endothelial nitric oxide synthase.

Short-term hypoxia triggers an acute, transient decrease in acetylation and methylation of histones at Nos3 promoter, hence rapid histone eviction from the Nos3 proximal promoter, especially endothelium-specific histone H2A variant, H2Az, which is not detected in vascular smooth muscle cells [1555]. Therefore, hypoxia represses NOS3 transcription.

Nucleosome accessibility depends on hypoxia duration. The shorter the hypoxia, the higher the nucleosome accessibility, the smaller the chromatin remodeling extent owing to chromatin (and nucleosome) remodeler and transcription activator SMARCa4 (switch–sucrose non-fermentable [Swi–SNF]-related, matrix-associated, actin-dependent regulator of chromatin).

After longer durations of hypoxia, histones are reincorporated at the Nos3 promoter, but they lack substantial histone acetylation [1555]. Chromatin remodeling is reversible upon return to normoxia, as histone acetylation, abundance of RNA polymerase-2 at NOS3 promoter, and NOS3 transcription are restored.

11.7 VEGF Signaling

Intracrine VEGF signaling⁹⁰ in endothelial cells is compulsory for homeostasis of blood vessels in adults, but dispensable for developmental and pathological angiogenesis [1556]. Moreover, paracrine VEGF signaling specific to endothelial cells does not compensate for the absence of endothelial autocrine VEGF signaling (Fig. 11.8).

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Tags: Intracellular Signaling Mediators in the Circulatory and Ventilatory Systems

Jun 3, 2017 | Posted by admin in CARDIOLOGY | Comments Off

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