3.1.1. Sodium–Potassium ATPases

The surface membrane of virtually every animal cell expresses hundreds or even millions of copies of sodium–potassium ATPase (Na⁺/K⁺ ATPases). This “sodium pump” continuously rejects the sodium toward the extracellular space and accumulates potassium in the intracellular medium. This vital process maintains the sodium and potassium ionic gradients and is achieved at the expense of a substantial fraction of the ATP produced by the cell. These ionic gradients drive numerous co- and counter-transporters allowing glucose and amino acids intake, regulation of cell volume, pH and calcium homeostasis and underlie the electrical activity of all excitable cells [82]. Na⁺/K⁺ ATPases are electrogenic as three sodium ions are extruded toward the extracellular medium while two potassium ions are transported. The activity of the pump contributes to the regulation of the cell membrane potential [463].

Na⁺/K⁺ ATPase is composed of a non-covalently linked α and β subunits. Four different isoforms of the α subunit (α1 to α4) and three isoforms of the β subunit (β1 to β3) have been identified in mammalian cells. The enzymatic function has been totally assigned to the multi-spanning membrane α subunit, which also contains the binding sites for ATP and for the inhibitor ouabain. Each combination of α and β subunit produces a functionally active enzyme that possesses distinct affinities for Na⁺ and K⁺ and different ouabain sensitivities [876].

In mammalian arteries both vascular smooth muscle and endothelial cells express the house-keeping form of the Na⁺/K⁺-ATPase which comprises the α1 subunit. This isoform is nearly fully activated at the physiological concentration of extracellular potassium (5 mM). However, depending on the species and/or vascular bed studied, both the endothelial and vascular smooth muscle cells can express the α2 and/or α3 isoforms. These isoforms are activated by increases in extracellular concentration of potassium, in a window compatible with physiological changes in potassium concentrations (between 3 and 15 mM), and therefore can play a role in the regulation of blood flow and blood pressure [602,757,1671].

3.1.2. Potassium Channel Families

Potassium channels are the largest and most diverse sub-group of ion channels. Up to 75 different genes related to potassium channels have been identified in the human genome. The function of all potassium channels is to allow, in a very specific manner, the passage of potassium ions through the plasma membrane. Potassium channels set the resting membrane potential, regulate cell volume and play a key role in many cellular signaling events, including the regulation of smooth muscle tone and therefore blood flow [1408]. Depending on their membrane topology, potassium channels are classified in four subgroups: the voltage-gated (K_V), the calcium-activated (K_Ca), the two-pore-domain (K2P), and the inward rectifier (Kir) potassium channel families [24,466,717,900] (Figure 5).

FIGURE 5

Potassium channel families. The potassium channel family has been classified in four subgroups according to their membrane topology. The first and largest group is the voltage-gated potassium channel subtype family with six trans-membrane domains and (more...)

3.1.2.2. Calcium-Activated Potassium Channels (K_Ca).

The calcium-activated potassium channel family (K_Ca) is divided into two subfamilies, the large conductance (MaxiK or BK_Ca) calcium-activated potassium channels subfamily including the K_Ca1.1 α subunit (also known as Slo1 α), the small conductance calcium-activated potassium channels subfamily (SK_Ca), including K_Ca2.1, K_Ca2.2, K_Ca2.3 subunits (also known as SK1, SK2 and SK3) and the intermediate conductance calcium-activated potassium channels subfamily (IK_Ca) with the K_Ca3.1 (also known as IK1 or SK4) subunit. This IK_Ca is also known as the Gardos channel involved in the regulation of the cell volume of red blood cells [512].

A specific pharmacology for the K_Ca channel family has emerged in the recent years and natural substances as well as synthetic compounds, with potent and specific opening and blocking properties, have been identified (Figure 6).

FIGURE 6

Modulators of calcium-activated potassium channels.

3.1.2.2.1. Large Conductance K_Ca (BK_Ca).

BK_Ca channels are characterized by a high unitary conductance and are both voltage- and calcium-regulated potassium channels, indicating that they play an important role in limiting the entry of calcium and the cell excitability. Unlike SK_Ca and IK_Ca channels, the calcium sensitivity is not linked to an association with calmodulin, but to the presence of two high affinity calcium-sensing regions located on the α-subunit [1368,1723,1782]. Numerous isoforms of the Slo1 α subunit are generated by alternative splicing [884,1026]. In addition, the expression of accessory β subunits (β1 to β4) can lead to channel diversity [1408].

BK_Ca channels are expressed in virtually all vascular smooth muscle cells. However, at membrane potentials close to the resting state, the open-state probability of BK_Ca is very low, suggesting that their contribution in determining the resting membrane potential is modest [1262,1264]. The role of BK_Ca should be seen rather as a physiological brake, a feedback inhibitor of contraction and/or increase in intracellular calcium concentration in response to humoral (e.g., norepinephrine, angiotensin II) or physical stimuli such as an increase in intravascular pressure.

Spontaneous transient outward currents (STOC) are observed in coronary and cerebral arteries as well as in small myogenically active arteries. They are caused by the activation of a group of clustered BK_Ca channels in response to localized, elemental calcium-release events from internal calcium stores, known as calcium sparks [178,1115,1208]. Since these calcium sparks activate BK_Ca channels, they paradoxically lead to a decreased overall intracellular calcium concentration and thus to the relaxation of arterial smooth muscle. In mice with a disrupted gene for the auxiliary β1 subunit, the calcium sparks generated in vascular smooth muscle cells are of normal amplitude and frequency but the frequency of STOCs is reduced. When compared to the wild-type controls, transgenic mice have a higher systemic arterial blood pressure and the contractile responses of isolated aortic rings to agonists and KCl are increased [147,1229], suggesting that STOCs contribute to the general regulation of vascular tone and that the β1 subunit plays an essential role in this process. Furthermore, the deletion of the β1 subunit is associated with the depolarization of the vascular smooth muscle cells and the subsequent increase in NADPH oxidase-dependent production of superoxide anion [1143]. Mice knockout for the Slo 1 α subunit exhibit a moderate increase in blood pressure attributed in part to vascular dysfunctions, such as the absence of STOC and a decrease in the effectiveness of the cGMP/cGMP kinase pathway, but also to primary hyperaldosteronism [1352].

In most endothelial cells, when freshly isolated, BK_Ca channel activity is barely detectable [176,525,831,980]. This can possibly be attributed to the absence in these cells of the regulatory BK_Ca β subunits that enhance Ca²⁺-sensitivity [831,1188,1323].

3.1.2.2.2. Small and Intermediate Conductance K_Ca (SK_Ca and IK_Ca).

SK_Ca and IK_Ca channels are voltage-independent and their calcium sensitivity is ascribed to the association with calmodulin [404,753,829,1722].

In healthy and freshly isolated vascular smooth muscle cells, SK_Ca and IK_Ca channels are generally not or only very poorly expressed [430]. However, in proliferating cells, as seen in culture or after vascular injury, the expression of these channels increases dramatically [833,1117,1542,1543]. Conversely, the deletion of IK1 (K_Ca3.1) reduces vascular smooth muscle cell proliferation [1560]. The selective blockade of IK_Ca channels prevents smooth muscle phenotypic changes and coronary artery neointimal formation in two different models of post-angioplasty restenosis [833,1543]. Coronary arteries from patients with coronary artery disease show elevated levels of IK_Ca channel and pharmacological blockade of this channel reduces the development of atherosclerosis in ApoE-/- mice [1560].

In contrast, the IK_Ca and SK_Ca channels, especially the SK3 α subunit, are constitutively expressed in endothelial cells [161,176,830,831,980]. In endothelial cells, IK_Ca and SK_Ca channels have a specific spatial distribution. In the rat mesenteric artery, SK_Ca are preferentially located at sites of homocellular endothelial gap junctions and caveolin-rich domains and are associated with various connexins, while IK_Ca are preferentially localized at the sites of endothelial projections often associated with myoendothelial gap junctions [4,336,887,1344]. This segregation leads to different functions for each individual channel (see Part 2: EDHF-Mediated Responses “The Classical Pathway”).

In general, an increase in the endothelial [Ca²⁺]_i can activate these two potassium channels and produce the hyperpolarization of the endothelial cells. This in turn favors the entry of calcium by increasing the driving force for this ion [169,246,749,773,953,954] and contributes to the activation of calcium-sensitive enzymes such as eNOS [289,1402,1453]. Additionally, the hyperpolarization of the endothelial cells can evoke the endothelium-dependent hyperpolarization of the vascular smooth muscle cells. Endothelial K_Ca are, therefore, key players in the generation of NO- and EDHF-mediated responses [430].

3.1.3. Chloride Channels

Chloride ions, unlike calcium, are not intracellular messengers. However, chloride channels, which are expressed in both the plasma membrane and intracellular organelles of cells, play an important role in various cell functions, including ion homeostasis, cell volume regulation, transepithelial transport, regulation of electrical excitability and the control of resting membrane potential. Chloride channels are channels that allow the passive diffusion of negatively charged anions along their electrochemical gradient. Some of these channels may conduct other anions (I^-, NO₃^-, Br^-, SCN^-) better than chloride itself but are nevertheless referred to as chloride channels since Cl^- is the most abundant anion in the organism [741]. There is a large variety of chloride channels, expressed on the plasma membrane and/or intracellular organelles, which have been identified according to their biophysical characteristics. However, the molecular structure of the chloride channels is only known for a few of them, suggesting that entire gene families of chloride channels remain to be discovered. Three molecularly distinct chloride channel families are well established, the CLC gene family, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) and the ligand-gated γ-aminobutyric acid and glycine-receptor chloride channels (19 and 4 members in mammals, respectively). However, the genes encoding the Ca²⁺-activated chloride channels (CLCA) and the volume-activated chloride channels (or swelling-activated chloride current or I_Cl,swell) are yet to be properly identified, although the CLC-3 gene may encode the latter [605,741,1521].

In smooth muscle cells, the opening of chloride channels produces an efflux of chloride anions and depolarization [877]. Two major Cl^- currents are recorded in vascular smooth muscle cells: CLCA and I_Cl,swell. I_Cl,swell are activated by low osmotic pressure and by mechanical stress produced, for instance, by vascular distension during a rise in blood pressure. NO tonically down-regulates the activity of I_Cl,swell and this mechanism can contribute to the NO-dependent regulation of smooth muscle cell contractions in various vascular beds including the coronary arteries [387,577,867]. In cerebrovascular smooth muscle cells, ClC-3 chloride channel expression and activity is enhanced along with the severity of cerebrovascular remodeling induced by hypertension [1521], and silencing the ClC-3 genes prevents vascular smooth muscle proliferation [1520,1636]. CLCA are also likely to play a major role in the contractions of vascular smooth muscle cells. Following receptor activation, the release of calcium from intracellular stores opens these channels leading to membrane depolarization, the opening of voltage gated Ca²⁺ channels and the subsequent increase in [Ca²⁺]_i [285,665].

In the endothelial cells, the activation of chloride channels also produces cell depolarization [817,1121]. The volume-regulated anion channel is a multifunctional channel that is expressed constitutively in endothelial cells. Its molecular identity still remains to be determined, but CLC-3 is also highly expressed in endothelial cells [868]. It contributes to the establishment of the resting membrane potential and its blockade provokes the hyperpolarization of the endothelial cells [1126]. I_Cl,swell is not only activated by hypoosmolarity but also by mechanical stimuli, including shear stress, and it contributes to intracellular pH and [Ca²⁺]_i homeostasis [1121]. This channel can therefore be considered to be involved in the paracrine and endocrine activity of the endothelial cells as well as in the proliferation and the differentiation of these cells. CLCA are also expressed in endothelial cells and their activation by an increase in [Ca²⁺]_i depolarizes the cell and counteracts the hyperpolarization evoked by the activation of K_Ca [586,1124].

3.1.4. Voltage-Dependent Calcium Channels

Voltage-gated calcium channels (Ca_V channels) mediate calcium influx in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression. Their activity is essential in the coupling of electrical signals in the cell surface to physiological events in cells. They are members of a gene superfamily of transmembrane ion channel proteins that includes voltage-gated potassium and sodium channels. The Ca_V channels is a superfamily of proteins that is encoded by at least 10 different genes organized into three main subfamilies based on the amino acid sequence of the α1 subunit (Ca_V1 to Ca_V3). According to this nomenclature, the Ca_V1 subfamily includes channels that mediate L-type Ca²⁺ currents, the Ca_V2 subfamily P/Q-, N-, and R-type Ca²⁺ currents and the Ca_V3 subfamily T-type Ca²⁺ currents [198].

In vascular smooth muscle, the Ca_V predominantly expressed is a splice variant of the Ca_V1.2 also expressed in the cardiac muscle (Ca_V1.2a and Ca_V1.2b, for cardiac and smooth muscle, respectively). This channel is the classic dihydropyridine-sensitive calcium channel and is widely expressed in the smooth muscle cells of virtually all vascular beds [723], although some other calcium channels, which are dihydropyridine-insensitive, such as Ca_V3.1 (formerly T-type) or calcium channels with an unknown molecular identity, can be expressed in some vascular smooth muscle cells [1079,1566].

In vascular smooth muscle cells, in response to depolarizing stimuli, the activation of Ca_V1.2 channels is generally the predominant source of calcium entry. In some of these cells, such as those of the longitudinal layers of the portal vein, the activation of L-Type Ca²⁺ currents can trigger the firing of action potentials [411,715]. However, in most of the arteries, the open probability of Ca_V channels is too low to observe the generation of action potentials but sufficient to allow a sustained calcium influx throughout the depolarization. The presence of physiological brakes, such as K_V and BK_Ca channels, prevents the membrane potential from reaching the threshold values required to trigger the action potential [1339], but the inhibition of these potassium channels unmasks the Ca_V channel-dependent action potentials in these normally quiescent smooth muscle cells [428,610]. Additionally, Ca_V1.2 are voltage sensors, which, in the absence of any transmembrane calcium influx, are coupled to the metabolic cascade leading to calcium release from the sarcoplasmic reticulum [303,438].

T-type channels are small-conductance, low voltage-activated, fast-inactivating channels. However, T-type channels display non-inactivating window currents, which may play a role in sustained Ca²⁺ entry. For instance, vascular smooth muscle cells of rat mesenteric arterioles express Ca_V3.1 channels. These channels do not appear to be necessary for the conduction of vasoconstriction, but they are likely to play a role in local electromechanical coupling [740].

Endothelial cells do not express Ca_V1 and, in general, calcium entry does not involve the activation of voltage-gated calcium channels. In some vascular beds, endothelial cells express the T-type channels, Ca_V3.1 and/or Ca_V3.2 [740,1805]. In pulmonary microvascular endothelial cells, Ca_V3.1 channels could be activated by G_q-linked agonist, such as thrombin, and the resulting rise in [Ca²⁺]_i could be associated with the exocytosis of Weibel–Palade bodies leading to the rapid secretion of von Willebrand factor and the membrane expression of P-selectin [1805].

3.1.5. Transient Receptor Potential Channels (TRP)

TRP channels were first identified in the Drosophila where a mutation led to impaired vision caused by the lack of a specific calcium influx pathway into photoreceptors. Homologues of this protein were identified in other species, including more than 30 in mammals. TRP-related proteins are classified into six sub-families: TRPC, TRPV, TRPM and the more distantly related TRPP, TRPML and TRPA. C stands for canonical as this sub-family of protein has the highest homology with the Drosophila TRP channels, V for vanilloid as this sub-family is closely related to the vanilloid receptor TRPV1, M for melastatin as this sub-family has the highest homology with the tumor suppressor melastatin (TRPM1), P for polycystins as these channels were first positionally cloned by linkage analysis of disease genes underlying polycystic kidney disease, the most common form of hereditary kidney disease, ML for mucolipidosis as these channels were also first identified by linkage analysis of a disease gene that mutated in mucolipidosis and A for ankyrin as these channels were characterized by an exceptionally long chain of ankyrin domain repeats. To possibly add to the complexity of this family of ionic channels, TRP channels form homo- and possibly hetero-tetramers [1120,1122,1123,1621]. TRP channels are primary sensors for physical (heat, cold, mechanical stresses) or chemical (e.g., pH, pheromones, capsaicin, bitter and sweet taste) stimuli [1120]. In the TRP channel family, calcium influx channels include all TRPC, TRPV, TRPM1, 2, 3, 6, 7 and 8, TRPP2, 3 and 5, TRPML1, 2 and 3, and TRPA1. These channels can be highly selective for calcium or more generally be merely non-selective cation channels [326,1398].

In both vascular smooth muscle and endothelial cells, some members of this family could be molecular components of several types of calcium-permeable channels, including receptor-operated calcium channels (ROC), activated by agonists acting on their receptors, and possibly contribute in the activation of store-operated calcium channels (SOC) which are activated following the calcium depletion of the internal stores, i.e., the sarcoplasmic reticulum. Their gating is controlled by multiple stimuli, diacylglycerol following receptor activation, arachidonic acid metabolites, kinases, inositol phosphates, calcium, osmolarity, oxidative stress, pH, and physical stimuli such as stretch, pressure, flow, temperature [709,1019].

3.1.6. ORAI Family of Calcium Channels and Store-Operated Calcium Channels (SOC)

The Orai proteins are encoded by a family of three genes and appear totally unrelated to other known channel proteins. They were first identified in human lymphocytes and were named after the Greek mythological characters, the Orai, the three sister keepers of the gates of heaven. Orai is the pore subunit of the long-sought SOC that carry the calcium-release-activated-calcium current, I_crac [1246]. Orai channels are generally highly selective for calcium [1766], but Orai-3 can also act as a cationic channel independently of calcium store depletion [1795].

Store-operated, or capacitive, calcium entry into cells refers to a mechanism that links a decrease in the calcium ion concentration in the sarcoplasmic reticulum to calcium entry into the cell through the plasma membrane [901]. The calcium sensor is a protein that was originally identified as a cell surface protein and named stromal interacting molecule (STIM-1 and STIM-2). However, the STIM proteins are also localized in the membrane of the sarcoplasmic reticulum with the N-terminal region containing the EF-hand calcium-binding motif inside the sarcoplasmic reticulum, while the cytosolic C-terminal region contains the amino acid sequence involved in the protein–protein interaction and activation of Orai [640,1207]. Calcium release from the sarcoplasmic reticulum, following IP₃R activation, decreases the internal calcium concentration and the dissociation of the calcium bound to the low-affinity sites of the EF-hand calcium-binding motif of STIM-1 proteins and promotes their association. The translocation of the oligomers to sites of sarcoplasmic reticulum close to the plasma membrane allows their interaction with Orai, provoking its tetramerization and activation [640,1207]. Furthermore, when CaV are expressed, as, for instance, in vascular smooth muscle cells, STIM-1 can also interact with this calcium channel provoking its immediate inactivation and then its internalization and degradation, highlighting the major role that STIM proteins can play in the regulation of calcium signaling [1189,1654].

Before the recently discovered functions of STIM and Orai, TRPC channels as well as several members of other TRP subfamilies (TRPV6, TRPM3) have at some point been described as SOCs and it remains questionable whether any TRP channel plays such a role [1123]. However, STIM proteins can interact functionally with TRPC channels and the latter physically and functionally interact with Orai-1, indicating that supra-molecular complexes can be formed to generate functional SOC [1653].

3.2.1. Calcium Signaling in Vascular Smooth Muscle Cells

Calcium homeostasis is central to the regulation of vascular smooth muscle functions, including contractility. The contraction of the vascular smooth muscle cells can be elicited by vasoconstrictor agonists, depolarization of the cell membrane or mechanical stimulation, for instance, pressure-induced myogenic tone. It is initiated, and to a lesser extent maintained, by an increase in the intracellular-free calcium concentration ([Ca²⁺]_i). A global increase in [Ca²⁺]_i is achieved through the complex summation of calcium entry and calcium release. In response to physical forces (pressure, stretching), the indirect activation of TRP channels (TRPA1, TRPC1, TRPC6, TRPV2, TRPV4, TRPM4, TRPM7, TRPP1/TRPP2), leading to calcium entry and to depolarization-induced activation of Ca_V, plays a predominant role [326,360,709,1398]. Contractions of vascular smooth muscle activated by neurohumoral mediators usually involve a combination of two major sources of calcium ions, calcium entry from the extracellular space and calcium release from internal stores, although the contribution of each pathway differs markedly depending on the stimulating agonist or the vascular bed studied [119,411].

The sarcoplasmic reticulum is the main organelle capable of taking up, storing and releasing calcium ions. The mitochondrium also plays an important role in calcium homeostasis, especially in situations where the intracellular calcium concentration is elevated [772]. In vascular smooth muscle cells, the sarcoplasmic reticulum is often closely associated with the plasmalemna forming a superficial buffer barrier that allows spatial differences in the intracellular calcium concentration [1585]. Furthermore, some sarcoplasmic reticulum compartments lie just beneath the specialized domain of the plasma membrane containing Na⁺/K⁺-ATPase, Na⁺/Ca²⁺ exchanger, TRP, Ca_V and Orai channels, etc. These microdomains, termed plasmerosomes, often associated with caveolae, form functional units specialized in calcium regulation that have a marked influence on the signaling role in smooth muscle cells [111,181,671,1503]. Specialized calcium pumps (sarcoendoplasmic reticulum Ca²⁺-ATPase, SERCA) generate and maintain the calcium gradient between the inside of the sarcoplasmic reticulum and the surrounding cytoplasm. Three different genes encode the SERCA pumps, and the smooth muscle cells generally express SERCA2 and SERCA3 [1706]. The activity of SERCA is regulated by the expression of an endogenous inhibitor phospholamban, a 52-amino acid phosphoprotein [1154]. Inside the sarcoplasmic reticulum, calcium is stored by binding to specialized proteins such as calsequestrin [1339].

Stimulation of G-protein-coupled receptors activates phospholipase C, leading to the formation of diacylglycerol and inositol trisphosphate (IP₃). Diacylglycerol activates TRP channels (TRPC3, TRPC6) eliciting, again, calcium entry and depolarization-induced activation of Ca_V [709]. IP₃ activates specific receptors/channels situated on the sarcoplasmic reticulum and produces calcium release (Figure 7). Three genes encode the elementary subunits composing the IP₃ receptors [1198]. The activation of IP₃ receptors is regulated by [Ca²⁺]_i. Calcium by itself can activate a sarcoplasmic reticulum receptor/channel, the ryanodine receptor (RyR), to induce calcium release. Three isoforms have been cloned, but smooth muscle cells express preferentially RyR-2 and RyR-3 [1169]. Finally, plasmalemnal Ca_V, in addition to its well-known role as a voltage-dependent selective calcium channel, act as a voltage sensor, which, in the absence of calcium influx, triggers fast G-protein-dependent calcium release from the sarcoplasmic reticulum [303]. The emptying of the calcium stores elicits the refilling of these stores by the opening of store-operated channels (SOC), following the association of STIM and Orai proteins [640,1207].

FIGURE 7

Calcium-dependent and -independent contractions in vascular smooth muscle cells. The stimulation of G-protein-coupled cell membrane receptors evokes a contractile response that, depending on the G-protein involved, implies different coupling mechanisms. (more...)

The [Ca²⁺]_i is regulated differently in the cytosol as a whole and in specialized domains constituted by the portion of the cytosol included between the plasma membrane and closely situated sarcoplasmic reticulum (plasmerosomes). The close spatial relationship of the sarcoplasmic reticulum with the plasma membrane, the direct and indirect activating role of calcium on its own release and the activation of Ca_V and SOC show that calcium entry and calcium release are interdependent phenomena which concur to achieve calcium homeostasis in vascular smooth muscle. For instance, contractile and relaxing agents modulate the incidence of calcium sparks elicited by clustered RyR, activated by calcium entry through Ca_V and/or TRP channels (TRPC1, TRPV4). Calcium sparks can act as a positive feedback to augment the contractility of the smooth muscle directly by increasing [Ca²⁺]_i and indirectly by activating CLCA, giving rise to spontaneous transient inward currents (STIC), and possibly also by activating some calcium-sensitive TRP channels (TRPC1, 4, 6, TRPV4, TRPM4), both leading to further depolarization and calcium entry. Conversely, they also have a relaxing effect by activating BK_Ca giving rise to STOC (TRPV4–ryanodine receptors–BK_Ca complex), leading to repolarization and inhibition of calcium entry [122,709,732,1339].

The general increase in [Ca²⁺]_i activates myosin light chain kinase which in turn phosphorylates the regulatory light chains of myosin II to generate contraction. This calcium-dependent phosphorylation of the light chains of myosin II is modulated in a calcium-independent manner by the constitutively active myosin light chain phosphatase. This enzyme is inhibited by monomeric GTPase Rho and the Rho-associated kinase as well as protein kinase C, and is activated by cyclic-GMP. Furthermore, the myosin light chain kinase activity is also controlled by various kinases (e.g., protein kinase A, protein kinase G, calmodulin-dependent protein kinase II, p21-activated kinase), indicating that even if an increase in [Ca²⁺]_i plays a dominant role in the contraction of smooth muscle, vascular tone is also extensively regulated in both a calcium-dependent and -independent manner, by a complex network of activating and inactivating kinase cascades [501,925,1447,1448] (Figure 7).

The contractions are then terminated when [Ca²⁺]_i returns to control levels. The inactivation of both Ca_V, following cell repolarization, and TRP channels reduce calcium entry. Calcium ions are either pumped out of the cell by a specific plasma membrane, Ca²⁺-ATPase (PMCA), or back into the sarcoplasmic reticulum by SERCA. The PMCA are encoded by at least four genes, PMCA1 and 4 are the most widely expressed and are the two isoforms expressed in vascular smooth muscle cells. This pump is not electrogenic since each calcium ion extruded is exchanged for two protons [193]. However, calcium can also exit via the Na⁺/Ca²⁺ calcium exchanger (NCX1.3 and NCX1.7, predominantly in vascular smooth muscle cells), and the resulting increase in intracellular sodium activates the Na⁺/K⁺-ATPase, which hyperpolarizes the myocytes and reinforces the relaxing process [1154].

3.2.2. Calcium Signaling in Endothelial Cells

Endothelial cells in general do not express fast-activated tetrodotoxin-sensitive Na⁺ channels or Ca_V and are considered to be “non-excitable cells.” Nevertheless, cytoplasmic [Ca²⁺]_i is a key regulator of endothelial function, including the synthesis and release of NO, prostacyclin, endothelium-derived contracting factors, von Willebrand factor and tPA, the generation of EDHF-mediated responses and the control of vascular permeability, cell proliferation and angiogenesis [168,419,1562]. Changes in [Ca²⁺]_i are generated in response to receptor activation and in response to mechanical stimuli, shear stress being a stimulus of utmost importance for endothelial cell physiology. Elevations in [Ca²⁺]_i are generally biphasic, with an initial phase of calcium release from intracellular stores, predominantly the endoplasmic reticulum, followed by calcium entry. For instance, acetylcholine increases [Ca²⁺]_i by activating both calcium release from intracellular stores, which involves IP₃ and ryanodine receptors, and calcium influx from the extracellular space [169,1651]. In addition to IP₃, two other important second messengers, metabolites of pyridine nucleotides, cyclic ADP-ribose, an endogenous activator of the ryanodine receptor, and nicotinic acid dinucleotide phosphate, can trigger the release of calcium [1562].

The increase in [Ca²⁺]_i is associated with the hyperpolarization of endothelial cells due to the activation of calcium-activated potassium channels [169,978,1332]. Agonist-induced hyperpolarization constitutes a positive feedback mechanism for the entry of calcium through receptor-operated channels since the electrical driving force for calcium is enhanced. Depletion of the endoplasmic reticulum calcium stores, following receptor stimulation or by specific inhibitors of the calcium pump (in endothelial cells predominantly SERCA2b and SERCA3), promotes an increase in [Ca²⁺]_i via the activation of SOC [252,1120,1711]. The molecular identity of ROC and SOC in the endothelial cells, as in vascular smooth muscle cells, almost certainly involves TRP channels and the association of STIM and Orai proteins, respectively [640,1207,1562,1791].

In endothelial cells, TRPV4 appears to play a predominant role in flow-mediated endothelium-dependent vasodilatation since these responses are abolished in TRPV4 knockout mice [616]. Shear stress-induced increase in [Ca²⁺]_i involves the formation of EETs, metabolites of arachidonic acid via the cytochrome P450 pathway, which contribute to the activation of TRPV4 [947]. Additionally, TRPC1, TRPV2, TRPP1/2 and TRPM7, which are expressed by the endothelial cells, could be involved in endothelial shear sensing and flow-mediated vasodilatation [1791].

Agonist-induced vasodilatation involves the activation of TRPC4 [475], TRPC6 [461] and again TRPV4 [1792]. Several other TRP channels may also contribute to agonist-induced changes in [Ca²⁺]_i including TRPC1, TRPC3, TRPV1, TRPV3 or TRPA1, and the expression of some of these channels can be altered by pathological processes such as elevated glucose levels or hypertension. The presence of these various TRP channels involved in the regulation of [Ca²⁺]_i may be explained by the formation of heteromeric channels [106,1439,1547,1791].

When the stimulation is terminated, the inactivation of TRP channels reduces calcium entry and calcium ions are either pumped out of the cell by PMCA or back into the endoplasmic reticulum by SERCA. In endothelial cells, PMCA1, 2 and 4 are the isoforms which are likely to play a role in [Ca²⁺]_i homeostasis. In addition, these proteins can directly interact with eNOS and inhibit NO production [667] (Figure 8).

FIGURE 8

Calcium signaling in endothelial cells. Agonists interact with G-protein-coupled receptors (bradykinin, acetylcholine, ATP, etc.) or with tyrosine kinase receptors (growth factors such vascular endothelial growth factor and some inflammatory mediators) (more...)

3.3.1. Connexins and Gap Junctions

Gap junctions are composed of subunit proteins called connexins. Six connexins in one cell are assembled to form hemi-channel or connexon. Two connexons (12 connexins), one on each cell membrane, are linked to form the functional gap junction. In most tissues, these gap junctions are organized as plaques which are aggregates consisting of a few to over a thousand of individual channels. Connexin proteins belong to a highly conserved multigene family with at least 21 identified members in humans (20 in mice) and are classified according to their molecular mass in kDa [752,1679]. The vascular gap junctions are generally assembled from one or more of these four different connexin (Cx) proteins Cx37, Cx40, Cx43 and Cx45. Depending on the species, vascular bed, vessel size and stage of development, the expression of connexins in the vascular wall can be markedly different. Cx45 is expressed exclusively in vascular smooth muscle cells, while both vascular smooth muscle and endothelial cells can express Cx43, Cx37, Cx40; the two latter connexins being preferentially expressed in the endothelium [442,752].

There are three possible assemblies of these connexins to form a gap junction channel. The homotypic type is the assembly of two identical connexons expressed at the membrane surface of each cell type and is therefore a dodecameric structure of identical connexin subunit proteins. A second type is the heterotypic gap junctional channel consisting of two distinct connexins, six in one hemi-channel and six of another type in the other hemi-channel. The third type is a heteromeric gap junction where at least one of the two connexons contains more than one connexin. The number of different gap junction channels that can theoretically be expressed is virtually limitless. Some of these heteromeric channels are expressed in native cells and are likely to have specific characteristics and functions, the extent of which remains to be determined [148,915,1765]. Intercellular channels are sensitive to transmembrane (trans-junctional) voltage differences, as they are able to open or close in response to depolarization or hyperpolarization, and can be regulated by post-translational modifications such as phosphorylations and nitrosylations [752] (Figure 9).

FIGURE 9

Gap junction communication in the vascular wall. (A) Schematic representation of connexins assembling to form gap junctions is shown. Gap peptides such as Gap27, are connexin mimetics which possess conserved sequence homology with the second extracellular (more...)

However, hemi-channels may also remain unpaired and open to release autocrine and/or paracrine signals in the circulation or in the intercellular environment. Since these membrane pores have a large conductance, their opening must be tightly regulated in order to maintain cellular integrity [442]. Additionally, pannexins, closely related to the innexins that form gap junctions in invertebrates also form hemi-channels (pannexons) in vascular smooth muscle and endothelial cells. Pannexin-1 channels are very permeable to ATP and could be involved in the ATP-dependent paracrine calcium wave in the vasculature [752] (Figure 9).

3.3.2. Homocellular and Heterocellular Gap Junctions

Homocellular gap junctions couple vascular smooth muscle cells to vascular smooth muscle cells and endothelial to endothelial cells. In vascular smooth muscle cells of large arteries, Cx43 and Cx45 are predominantly expressed. These junctions play a central role by coordinating changes in membrane potential and [Ca²⁺]_i between adjacent cells and therefore in the contractile response to agonists [148] and could be involved in the maintenance of a non-proliferating state [1798]. In large blood vessels, endothelial cells are well coupled, Cx40, Cx37 being the predominant connexins expressed. Cx43 is expressed in areas of turbulent flow at branching point, possibly regulating endothelial cell proliferation and apoptosis in these areas characterized by elevated shear stress [752].

In resistance vessels, and in contrast to large arteries and veins, the additional formation of heterocellular gap junctions between vascular smooth muscle and endothelial cells can occur via cell projections protruding through holes in the elastin lamina, the myoendothelial gap junctions. This creates a new level of integration in the vascular wall (Figure 9). From a few cells activated by the synaptic release of a transmitter, the passage of a neurohumoral substance in the flowing blood, or an iontophoretically applied agonist in the vicinity of an endothelial or smooth muscle cell, the diffusion of a message by means of gap junctions, allows the synchronized contraction or relaxation of the entire vascular wall [249,581]. In these resistance arteries, Cx40 plays an important role in endothelium-dependent relaxations since the deletion of this gene reduces acetylcholine-induced vasodilatation [441].

Changes in the level of expression of connexins have been correlated with various vascular diseases including hypertension, atherosclerosis and restenosis. Additionally, gap junctions may form between vascular and inflammatory cells and contribute to atherogenesis [149,163].