Vascular smooth muscle cells release: Endothelium-derived NO, the known most powerful vasodilator which activates the soluble guanylate cyclase. Soluble Guanylate cyclase (sGS) enzyme converts GTP to cyclic GMP (cGMP) which activates protein kinase G that leads decreases in the cytosolic calcium concentrations. NO can also affect cellular activity, independently of sGC activation, by the stimulation of the endoplasmic reticulum calcium ATPase, reducing the intracellular calcium concentration and cause relaxation of the smooth muscle. The release of inflammation, vascular cell proliferation, platelet adhesion, and tissue factor are inhibited by NO (Laher 2014).

NO is synthesized from an L-arginine by the enzyme nitric oxide synthases (NOS) as a free radical (Fig. 2). There are three distinct genes encoding NOS isozymes; neuronal NOS (nNOS or NOS-1), cytokine-inducible NOS (iNOS or NOS-2) and endothelial NOS (eNOS or NOS-3) (Melikian et al. 2009) (Table 2). The production of NO from L-arginine by NOS requires the presence of various co-factors including tetrahydrobiopterin, flavin adenine dinucleotide, flavin mononucleotide, calmodulin (calcium binding protein) and iron protoporphyrin (Palmer et al. 1988). nNOS is expressed in the central and peripheral nervous systems, in cardiac and skeletal myocytes, smooth muscles and ECs. NO produced in the nervous systems by nNOS is associated with the regulation of neuronal excitability and synaptic plasticity, memory and learning processes. It has been suggested that the expression of vascular nNOS is also upregulated by stimulation with AT-II and platelet-derived growth factor (Dawson et al. 1991). iNOS is minimal under physiological conditions and is calcium insensitive. When iNOS is stimulated, it continuously produces NO. Induction of iNOS occurs mainly during infection and chronic inflammation. iNOS is expressed in vascular smooth muscle cells following exposure to pro-inflammatory cytokines. It is reported that inflammation-induced iNOS production in the endothelium is related to the vascular dysfunction by limiting the availability of BH₄ for e NOS (Lowenstein and Padalko 2004; Gunnett et al. 2005). eNOS is the major isoform for the regulation of vascular function. The activity of eNOS and the production of NO can be stimulated by shear stress, acetylcholine, bradykinin and histamine by both calcium-dependent and independent ways (Laher 2014). Acetylcholine, bradykinin and histamine bind to specific receptors on the endothelial cell membrane and increase the intracellular concentration of calcium. In a calcium-independent manner, the activation of eNOS is due to the post-translational modification of the enzyme including phosphorylation by NOS kinase and dephosphorylation by phosphatases (Kellogg et al. 2005; Bae et al. 2003). Phosphorylation alters the activity of eNOS, and different sites of phosphorylation can have an opposing effect.The endogenous competitive inhibitor for eNOS is called asymmetric dimethyl arginine (ADMA) (Arora et al. 2013; Zhao et al. 2014). The inhibition of eNOS is correlated with plasma ADMA levels, and plasma ADMA levels are inversely related to endothelium-dependent vasodilation (Vestweber 2012). The acute and chronic rise in the shear stress of blood up-regulates the expression and the activity of eNOS, and thus the release of EDRF/NO (Kolluru et al. 2010; Michel and Vanhoutte 2010). AT-II by binding to its receptor produces bradykinins which stimulate eNOS consequently increases the formation of NO (Yayama et al. 2006).

Additionally, the products of the metabolism of NO are nitrite and nitrate which act as a reservoir of NO. Under certain conditions, several enzymes, such as xanthine oxidoreductase, mitochondrial cytochrome oxidase, aldehyde dehydrogenase 2 and cytochrome P450 reductase, catalyze the reduction of nitrite or nitrate to NO (Weitzberg et al. 2010).

Another vasodilator molecule is prostacyclin (also called prostaglandin I₂ or PGI₂) which is a prostaglandin that belongs to the eicosanoid family of lipid molecules. Prostacyclin is produced in ECs in response to inflammatory mediators, including interleukin (IL)-1 and platelet-derived and epidermal growth factors from prostaglandin H₂ (PGH₂) by the action of the enzyme prostacyclin synthase (Fig. 3). Like NO, it inhibits platelet activation and act as an effective vasodilator (Cahill and Redmond 2016; Siti et al. 2015). PGI₂ is released by healthy ECs and performs its function via paracrine signaling that involves G protein-coupled receptors on both ECs and platelets. PGI₂ binds to endothelial prostacyclin receptors and raise cAMP levels in the cytosol. cAMP activates protein kinase A (PKA) which promotes the dephosphorylation of the myosin light chain kinase. Dephosphorylation of the enzyme results in the inhibition of myosinlight-chainkinase. This leads relaxation of the smooth muscle relaxation and vasodilation (Francis et al. 2010). Prostacyclin has also antiproliferative, antithrombotic, anti-inflammatory and antimitogenic effects on vascular cells. On the other hand, prostanoids, such as PGD₂ and PGF_2, produced in vascular endothelium modulating intracellular Ca²⁺ concentration produce vasoconstriction (Siti et al. 2015).

The term of EDHF represents a mechanism rather than a specific factor (Luksha et al. 2009). Prostacyclin and NO can be considered as an endothelium-derived hyperpolarizing substance. Because most of the available inhibitors of cyclooxygenase abolish the production of prostaglandins in vascular tissues, any endothelium-dependent hyperpolarization observed in the presence of one of these inhibitors is unlikely to involve prostacyclin. NO can also hyperpolarize, or repolarize, vascular smooth muscle cells by activating, in either a c-GMP dependent or –independent pathways through potassium channels such as K-ATP. NO interacts with other ionic channels of the smooth muscle, including chloride and cationic channels and also influences the membrane potential of the smooth muscle cells indirectly in an autocrine fashion (Félétou and Vanhoutte 2006). Members of a class of arachidonic acid derivatives, the epoxyeicosatrien​oic acid, hydrogen peroxide, C-type natriuretic peptide, have been suggested to function as an EDHF in some vascular beds. These molecules are accepted as non-NO–non-PGI₂– EDHFs. EDHF is proposed to be a substance and/or electrical signal that is generated or synthesized in and released from the endothelium. Its function is to hyperpolarize vascular smooth muscle cells, causing these cells to relax. EDHFs are able to activate ion channels, and initiate smooth muscle hyperpolarization and relaxation (Luksha et al. 2009). Calcium-activated K+ channels are opened by EDHFs in vascular smooth muscle cells. The effects of EDHF’s are highest on the small arteries, and are very significant for the regulation of organ blood flow, peripheral vascular resistance and blood pressure, particularly when production of NO is depressed (Michel and Vanhoutte 2010; Luksha et al. 2009; Félétou and Vanhoutte 2006; Khazaei et al. 2008).

Endothelins (ETs) are potent vasoconstrictor molecules having a key role in vascular homeostasis (Fig. 4). Although there are three types of ET, vascular ECs produce only ET-1 which has prominent roles (Wang and Zhao 2010). ET-1 is a 21 amino acid peptide that is synthesized from a 39 amino acid precursor named pre-pro endothelin. Active endothelin molecule is generated by the actions of an endothelin converting enzyme (ECE) found on the endothelial cell membranes. There are two basic types of ET-1 receptors: ET_A and ET_B. Both of these receptors are coupled to a G-protein and to the formation of IP₃ (Barton 2011; Kedzierski and Yanagisawa 2001). In blood vessels, the ET_A receptor is dominant under normal conditions. ET-1 produces vasoconstriction through activation of L-type Ca⁺² channels by binding to ET-A receptors on vascular smooth muscle cells. In addition to the presence of both ET_A and ET_B receptors on the smooth muscle, ET_B receptors are also found on the endothelium, and under the control of vascular tone, considerable cross-talk between ET, NO and prostacyclin occur (Vanhoutte et al. 2009). When ET-1 binds to endothelial ET_B receptors, the formation of NO is stimulated but in the absence of smooth muscle endothelin receptor stimulation, NO causes vasodilation. The other effects of ETs include cell growth, embryonic development, renal functions, neurophysiological functions (such as pain signaling), cardiovascular homeostasis, cancer cell growth, endocrine function, inflammation, pulmonary functions (such as bronchoconstriction) and reproductive system functions (Khazaei et al. 2008). ET-1 production and release are stimulated by AT-II, antidiuretic hormone (ADH), thrombin, cytokines, reactive oxygen species, and shearing forces acting on the vascular endothelium. The ET-1 release is inhibited by prostacyclin, atrial natriuretic peptide and NO (Davenport et al. 2016).

Angiotensin II (AT-II) cause structural changes and vasoconstriction in the arterial wall by affecting many cellular and intracellular events in smooth muscle. Two types of AT-II receptors on ECs are determined. AT-II type-1 receptor is particularly involved in the contraction of vascular smooth muscle cells. AT-II-type-1 receptor blockers increase the release of NO and prostaglandins whereas AT II- type-2 receptors provide the activation of the endothelial relaxation (Masuyer et al. 2014; Jiang et al. 2014).

Thromboxane (TX) A₂ is a member of the eicosanoid lipid family. TXA₂ is generated from prostaglandin H₂ by thromboxane-A synthase. TXA₂ acts by binding to the G-protein-coupled thromboxane receptors. Thromboxane is a vasoconstrictor, and it facilitates platelet aggregation (Fig. 3). Therefore, TXA₂ shows interactions in contrast to prostacyclin (Bauer et al. 2014; Korbecki et al. 2014).

The vascular ECs releases adenosine to produce vaso-relaxation through activation of purinergic (P₂) receptors. Adenosine release is related to local oxygen tension. Also, adenosine metabolites play roles in local vasoregulation and in the physiological control of blood pressure (Ralevic and Dunn 2015).