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Ion channels as effectors of cyclic nucleotide pathways:
functional relevance for arterial tone regulation
Boris Manoury, Sarah Idres, Véronique Leblais, Rodolphe Fischmeister
To cite this version:
Boris Manoury, Sarah Idres, Véronique Leblais, Rodolphe Fischmeister. Ion channels as effectors of cyclic nucleotide pathways: functional relevance for arterial tone regulation. Alimentary Pharmacology & Therapeutics (Suppl), 2020, pp.107499. �10.1016/j.pharmthera.2020.107499�. �hal-02488316�
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P&T #23272
Ion channels as effectors of cyclic nucleotide pathways:
functional relevance for arterial tone regulation
Boris Manoury1, Sarah Idres1, Véronique Leblais1 and Rodolphe Fischmeister1.
1: Université Paris-Saclay, Inserm, Umr-S 1180 - Châtenay-Malabry (France)
Correspondence to:
B Manoury, Université Paris-Saclay, Inserm, Umr-S 1180, 5 rue J-B Clément, 92296 Châtenay-Malabry, France
E-mail: boris.manoury@universite-paris-saclay.fr
Tel: +33-1.46.83.59.06
2 Abstract
Numerous mediators and drugs regulate blood flow or arterial pressure by acting on vascular tone, involving cyclic nucleotide intracellular pathways. These signals lead to regulation of several cellular effectors, including ion channels that tune cell membrane potential, Ca2+ influx and vascular tone. The characterization of these vasocontrictive or vasodilating mechanisms has grown in complexity due to i) the variety of ion channels that are expressed in both vascular endothelial and smooth muscle cells, ii) the heterogeneity of responses among the various vascular beds, and iii) the number of molecular mechanisms involved in cyclic nucleotide signalling in health and disease. This review synthesizes key data from literature that highlight ion channels as physiologically relevant effectors of cyclic nucleotide pathways in the vasculature, including the characterization of the molecular mechanisms involved. In smooth muscle cells, cation influx or chloride efflux through ion channels are associated with vasoconstriction, whereas K+ efflux repolarizes the cell membrane potential and mediates vasodilatation. Both categories of ion currents are under the influence of cAMP and cGMP pathways. Evidence that some ion channels are influenced by CN signalling in endothelial cells will also be presented. Emphasis will also be put on recent data touching a variety of determinants such as phosphodiesterases, EPAC and kinase anchoring, that complicate or even challenge former paradigms.
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Table of contents
1. Introduction ... 6
2. Cyclic nucleotides and vascular tone ... 6
3. Regulation of Ca2+ influx by CNs ... 12
4. cGMP-dependent, Ca2+-activated, Cl- channels in vascular SMCs. ... 27
5. Regulation of K+ channels by CNs in vascular SMCs ... 28
6. Conclusion ... 61
Tables ... 66
Reference list ... 78
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Abbreviations:
2-APB: 2-aminoethoxydiphenyl borate 4-AP: 4-aminopyridine
8-pCPT-AM: 8-pCPT-2’-O-Me-cAMP, acetoxymethyl ester (EPAC-specific cAMP analogue). AC: adenylyl cyclase
AKAP: A-kinase anchoring protein AngII: angiotensin II
ANP, BNP, CNP: atrial -, brain- and c-type natriuretic peptides A1/2/3 R: adenosine receptor type-1 or -2 or -3
ATP: adenosine-tri-phosphate β-AR: -adrenergic receptor
(B/I/S)KCa: (large/intermediate/small conductance,) Ca2+-activated K+ channel [Ca2+]i: intracellular (cytosolic) Ca2+ concentration
cAMP: 3’, 5’-cyclic adenosine monophosphate CBTX: charybdotoxin
cGMP: 3’, 5’-cyclic guanosine monophosphate CGRP: calcitonin gene-related peptide
CN: cyclic nucleotide CLZ: cilostazol
CPA: cyclopiazonic acid DAG: diacylglycerol
DEA-NO: diethylamine NONOate DES: diethylstilbestrol
EC: endothelial cell
EETs: epoxyeicosatrienoic acids
EPAC: exchange protein activated by cAMP Em: cell membrane potential
ER: endoplasmic reticulum FSK: forskolin GPCR: G protein-coupled receptor IBTX: iberiotoxin IP3: inositol-1,4,5–triphosphate IP3R: IP3 receptor ISO: isoprenaline KV: voltage-dependent, K+ channel KATP: ATP-sensitive, K+ channel Kir: inward rectifier, K+ channel
5 LNP: linopirdine
LTCC: voltage-dependent, L-type Ca2+ channel MLCK: myosin light chain kinase
MLCP: myosin light chain phosphatase NCX: Na+, Ca2+ exchanger
NECA: 5′-(N-ethylcarboxamido)adenosine NO: nitric oxide
NP: natriuretic peptides
ODQ: 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one PA: pulmonary artery
PAH: pulmonary arterial hypertension PDE: cyclic nucleotide phosphodiesterase PGI2: prostacyclin
PKA: cAMP-dependent protein kinase PKC: protein kinase C
PKG: cGMP-dependent protein kinase PLB: phospholamban
PLC: phospholipase C
PMCA: plasma membrane Ca2+-ATPase ROC: receptor-operated channel RyR: ryanodine receptor
sGC: soluble guanylyl cyclase
SERCA: sarcoplasmic reticulum Ca2+-ATPase SNP: sodium nitroprusside
SOC: store-operated channels SOCE: store-operated Ca2+ entry SR: sarcoplasmic reticulum
STIM1: stromal interaction molecule-1
STOC: spontaneous transient outward current SUR: sulfonylurea receptor
TEA: tetraethylammonium ion TG: thapsigargin
TRP: transient receptor potential
TTCC: voltage-dependent, T-type Ca2+ channel UTP: uridine triphosphate
VIP: vasoactive intestinal peptide
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1. Introduction
Numerous mediators and drugs regulate blood flow or arterial pressure by acting on vascular tone, and this often involves cyclic nucleotide (CN) intracellular signalling pathways. Ion channels that tune vascular tone are modulated by these CN-driven pathways, making them key effectors of a great, yet complex, diversity of vasodilatory or vasocontrictive mechanisms.
This review will synthesize key data from literature that highlight ion channels as physiologically relevant effectors of CN pathways in the vasculature, including the characterization of the molecular mechanisms involved. Emphasis will be also put on recent data touching a variety of determinants such as CN phosphodiesterases (PDEs), exchange protein activated by cAMP (EPAC) and kinase anchoring, which complicate or even challenge former paradigms. When applicable, data demonstrating the participation of such mechanisms in an integrated vasomotor response will be highlighted. The reader searching for detailed information on general molecular, biophysical and pathophysiological aspects of CN pathways or ion channels will be directed to classic or more recent reviews.
2. Cyclic nucleotides and vascular tone
2.1 Synthesis and elimination of cyclic nucleotides in the vasculature
CNs, namely 3’, 5’-cyclic adenosine monophosphate (cAMP) and 3’, 5’-cyclic guanosine monophosphate (cGMP) are small intracellular molecules acting as ubiquitous second messengers, regulating the function of various systems, including vasculature. Generally speaking, CNs have vasodilating, antiproliferative and platelet inhibitory properties (Francis, et al., 2010; Koyama, et al., 2001; Morgado, et al., 2012; Smolenski, 2012). Cyclic GMP is classically produced in vascular smooth muscle cells (VSMCs) in response to exogenous stimuli originating from the endothelial cell (EC) layer or the blood circulation. Cyclic GMP can be generated from GTP either
7 by the soluble guanylate cyclase (sGC) or the “particulate”, membrane-bound cyclases, namely natriuretic peptide receptors A and B (NPR-A and NPR-B) (Francis, et al., 2010; Kuhn, 2016). These pathways are also functional in ECs where they control endothelial permeability (Kuhn, 2016). While sGC can be activated by endogenous nitric oxide (NO), synthetic NO donors or direct synthetic pharmacological activators such as riociguat, NPR-A and NPR-B are stimulated by circulating natriuretic peptides (NP), namely atrial, brain and C-type natriuretic peptides (ANP, BNP and CNP, respectively). The resulting elevation of intracellular cGMP levels modifies the function of many downstream cellular effectors, mainly via activation of the cGMP- dependent protein kinase (PKG) isoforms PKGI and PKGII and substrate phosphorylation (Francis, et al., 2010). The use of mice deficient for the PKGI highlighted the role of this kinase in the vasodilating properties of the NO-cGMP signalling (Pfeifer, et al., 1998). PKGI comprehends two related isoforms, and , that are equivalently expressed in arterial tissue and display similar affinity for [cGMP] (half activation constant of 0.29 and 0.44 µM, respectively) (Wolfe, et al., 1989). PKGI can also undergo cGMP-independent, constitutive activation following exposure to H2O2, probably mediated by oxidation of critical cysteine residues (Burgoyne, et al., 2007; Sheehe, et al., 2018). Oxidation of PKGI, however, hampers cGMP-dependent activation.
Cyclic AMP is produced by adenylyl cyclases (ACs) which use adenosine-tri-phosphate (ATP) as substrate. Among the eight isoforms described, mainly AC3, AC5 and AC6 have been shown to be expressed and functionally relevant in quiescent VSMCs (Nelson, et al., 2011; Ostrom, et al., 2002), while AC2 and AC8 expression is turned on in dedifferentiated cultured VSMCs (Clement, et al., 2006; Gueguen, et al., 2010; Nelson, et al., 2011). ACs are classically stimulated by Gs protein-coupled receptors, and AC6 was proposed as the most relevant contributor to cAMP production and recruitment of downstream effectors (Nelson, et al., 2011). VSMCs express a great variety of such receptors, including -adrenergic receptors (-AR), adenosine receptor type-2 (A2A-R), eicosanoid receptors (e.g. receptor for prostacyclin, PGI2), calcitonin gene-related peptide (CGRP) receptor. The main documented effector for cAMP is the cAMP-dependent protein kinase (PKA), a serine/threonin protein kinase formed by 2 catalytic “C” subunits that are inhibited by 2
8 regulatory “R” subunits in the absence of cAMP. PKA holoenzymes can exist as 2 isoforms, namely type I and type II PKA. Type I PKA can be composed of RIor RIsubunits whereas type II contains RII or RIIsubunits. R subunits are all expressed in excess and buffer C subunits (Tasken & Aandahl, 2004; Walker-Gray, et al., 2017). Binding of cAMP to the R subunits (Kact = 50-100 nM for type I, 200-400 nM for type II) releases catalytic activity of C (Ercu & Klussmann, 2018). A single report mentions expression of RI and RII proteins in cultured rat aortic smooth muscle cells (Indolfi, et al., 2000) but relative expression of R subunits in vascular cell types remains globally uncharacterized. Binding of a specific region in the N-terminus of the R subunits to A-kinase anchoring proteins (AKAPs) targets PKA activity to specific subcellular membranes and multiprotein complexes (Ercu & Klussmann, 2018).
Of note, the concentration of cAMP in vascular tissues is usually 5 times higher than cGMP (Lugnier, et al., 1986) although relative levels can show great variability. Intracellular levels of cAMP and cGMP are mitigated by the activity of PDEs, a large group of enzymes which in mammals is composed of 11 families that encompass 21 genes and multiple variants (see reviews by (Bobin, et al., 2016; Keravis & Lugnier, 2012; Maurice, et al., 2014)). Main PDE activity in VSMCs and ECs is generated by PDE3 and PDE4 isoforms for cAMP, and PDE5 for cGMP, but activity of PDE1 or PDE2 is also described. Function of a PDE family can be revealed by selective pharmacological inhibition of its activity, resulting in potentiation of CN signalling. Accordingly, PDE3, PDE4 and PDE5 inhibitors have well-described vasorelaxant effects in a variety of vascular beds (see classic review by (Polson & Strada, 1996)) and are used therapeutically to treat pulmonary artery hypertension, intermittent claudication, erectile dysfunction and chronic obstructive pulmonary disease (Maurice, et al., 2014). Development of new molecules that could discriminate between variants within a PDE family, or target other PDE families is ongoing (Maurice, et al., 2014). Progress in this field, especially for cAMP signalling, has been fostered by description of how PDEs delineate discrete CN pools which control molecular targets in their vicinity, creating signalling complexes scaffolded by AKAPs (Ercu & Klussmann, 2018). This would result in compartmentalization of CN signalling in the cell, controlled by specific PDE machinery targeted
9 in subcellular domains. This would allow to create multimodal signals from the production of a single messenger. Of note, cGMP and cAMP can also modulate PDE activities with possible important consequences on autoregulation of the system, and allowing crosstalks between both pathways (Keravis & Lugnier, 2012).
Besides PDE activity, elimination of intracellular CNs can also occur through export by ABC transporter multidrug resistance-associated protein 4 (Krawutschke, et al., 2015; Sassi, et al., 2008).
2.2 Overview of mechanisms by which CNs regulate vascular smooth muscle tone
VSMCs tone is commanded by the phosphorylation state of the 20kDa light chain subunit (MLC20) of the smooth muscle myosin, which is associated with formation of actin-myosin complex responsible for force generation (reviewed by (Cole & Welsh, 2011)). Ser-19 of MLC20 is phosphorylated by myosin-light chain kinase (MLCK) and dephosphorylated by the catalytic (PP1c-δ) subunit of the myosin light chain phosphatase, and the balance of the respective activities of these enzymes determines contractility of VSMCs.
Contraction is initiated by elevation of global intracellular concentration of Ca2+ ([Ca2+]i) which complexes with calmodulin and activates MLCK. Elevation of [Ca2+]i classically occurs upon mobilization of intracellular Ca2+ stores by inositol-1,4,5–trisphosphate (IP3) binding to its Ca2+ channel IP3 receptor (IP3R) following stimulation of Gq protein-coupled receptors. IP3R was shown to be a substrate of PKGI, and inhibition of the channel is proposed as a mechanism of relaxation by cGMP pathways (Figure 1).
Increase in global [Ca2+]i can also result from Ca2+ influx through ion channels in the plasma membrane. Among these, the voltage-dependent, L-type Ca2+ channels (LTCCs) play a key role in regulating blood pressure by allowing Ca2+ influx upon cell membrane potential (Em) depolarization, as occurs for instance during elevation of intraluminal pressure (myogenic tone) or following stimulation of Gq-coupled receptors (Moosmang, et al., 2003). Other Ca2+-entry pathways include voltage-dependent T-type Ca2+ currents (Harraz, et al., 2015b) but also
store-10 operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry, which involve transient receptor potential (TRP) cation channels and ORAi1 channels (reviewed in (Avila-Medina, et al., 2018; Earley & Brayden, 2015)).
[Ca2+]i lowering mechanisms include re-uptake by the sarcoplasmic reticulum Ca2+-ATPase (SERCA) or extrusion via the plasma membrane Ca2+-ATPase (PMCA) and, probably to a lesser extent, the Na+-Ca2+ exchanger (reviewed by (Karaki, et al., 1997)). Action of CNs on these systems is summarized in Figure 1.
In addition, Em depolarizing currents (cationic and chloride fluxes) are counterbalanced by repolarizing currents mainly carried by K+ channels. VSMCs membrane exhibits a variety of K+ channels that contribute to maintaining resting Em close to the K+ equilibrium potential, or to repolarization following a depolarization. This activity impedes LTCCs activation and therefore inhibits contraction. As described in the following sections, regulation of the activity of several K+ channels constitutes the endpoint of CN-elevating pathways and accounts for a substantial part of their VSMCs-relaxant action (Figure 1 and 2).
For a given calcium concentration, several sensitizing mechanisms can potentiate contraction by inhibiting MLCP activity. Conversely, mechanisms that decrease sensitivity to Ca2+-calmodulin, some of which are initiated by CN signalling, lead to relaxation of smooth muscle (Figure 1). PKA can phosphorylate MLCK (Conti & Adelstein, 1981) and this decreases the affinity of the enzyme for Ca2+-calmodulin.
The actual contribution of this particular mechanism to the relaxation evoked by cAMP remains however unclear. Taking advantage of a mouse line expressing a FRET biosensor for MLCK activation, Raina et al. reported that MLCK activation was decreased by forskolin (FSK), a direct AC activator, in mesenteric arteries contracted by high external [K+], a depolarizing condition where intracellular Ca2+ should be kept constantly elevated (Raina, et al., 2009). Kinetics of MLCK inhibition by FSK paralleled tone reduction, suggesting that MLCK inhibition participated in relaxation. Next, authors used isoprenaline (ISO), a -adrenergic receptor (-AR) agonist well
11 known to evoke vasorelaxation and to elevate cAMP (Holman, et al., 1968; Meisheri & van Breemen, 1982; Schoeffter, et al., 1987). ISO did not change force, [Ca2+]i nor MLCK activation under similar high [K+] conditions (although ISO decreased all these parameters when vessels were pre-contracted using phenylephrine, a -adrenergic receptor agonist)(Raina, et al., 2009). This suggests that -AR-mediated cAMP signals may not relax VSMCs via direct MLCK inhibition but rather by regulating Ca2+ handling mechanisms, where ion channels play pivotal roles. This specificity of cAMP signalling may be explained by differential intensity of the cAMP production stimulus, forskolin evoking broader AC stimulation than receptor agonists. Alternatively, this may be a consequence of cAMP being compartmentalized, with limited diffusion around receptors-associated signalosomes.
CN pathways also decrease Ca2+ sensitivity of the contractile apparatus in smooth muscle via various mechanisms involving Rho kinase inhibition or myosin targeting subunit MYPT1 activation (reviewed in (Loirand & Pacaud, 2014; Puetz, et al., 2009), Figure 1). For instance cGMP relaxant effect is not seen in permeabilized, phenylephrine-stimulated arteries if MLCP is inhibited by calyculin A (Nakamura, et al., 2007), highlighting the relevance of these mechanisms in controlling vascular tone.
2.3 Critical overview of pharmacological tools used to study cAMP and cGMP pathways Large amounts of intracellular cAMP can usually be obtained by directly stimulating AC with FSK, or by broadly inhibiting PDE using 3-isobutyl-1-methylxanthine (IBMX) or another non-selective PDE inhibitor. Soluble GC can be stimulated with NO donors (e.g. sodium nitroprusside, SNP, diethylamine NONOate, DEA-NO) to enhance intracellular cGMP (Miller & Megson, 2007), but NO may also have cGMP-independent, direct action on various target proteins via cysteine S-nitrosylation (Lima, et al., 2010). NP receptors are usually stimulated by using recombinant NPs, ANP and CNP being more selective to NPR-A and NPR-B, respectively (Alexander, et al., 2017a; Kuhn, 2016). Abundant data highlighting the respective roles of molecular intermediates in the CN pathways were obtained by using cell permeant, non-hydrolysable CN analogues (e.g.
8-Br-12 cGMP or 8-Br-cAMP), pharmacological inhibitors of kinases, cyclases and other proteins. Alternatively, patch-clamp experiments offer the possibility to add cAMP or cGMP in the patch pipette filling solution to allow CN dialysis into the intracellular medium. Exposure of the excised cell membrane patches to recombinant catalytic subunit of PKA (PKAc) or PKG was used to demonstrate direct effects of these kinases on single channel activity. However, due to limited selectivity of CN analogues and pharmacological modulators, unexpected effects on alternative targets may occur, e.g. PDE inhibition (see for instance (Lochner & Moolman, 2006; Poppe, et al., 2008), which complicates the interpretation of the results obtained with these molecules. As always, conjunction of data obtained with several modulators with different chemical structures or mechanisms of action (e.g., for PKA inhibitors, a cAMP-analogue vs. an inhibitor of the catalytic site) would provide more robust evidence of the involvement of a specific pathway. With respect to PKA and PKG, the use of PKI (Cheng, et al., 1986) or DT-2 (Taylor, et al., 2004), respectively, are recommended selective peptide inhibitors to be used when studying the role of these two kinases.
3. Regulation of Ca
2+influx by CNs
3.1 Regulation of voltage-gated, L-type Ca2+ channels by CN in VSMCs
Voltage-gated, Ca2+ influx in VSMCs mainly reflects the activity of LTCCs, identified as CaV1.2. These channels are composed of a pore forming -subunit encoded by the gene CACNA1C (formerly designated as 1C), associated with auxiliary and 2-δ subunits (reviewed in (Hofmann, et al., 2014)). Native L-type Ca2+ current features high activation threshold, slow inactivation and sensitivity to dihydropyridines. Vascular smooth muscle expresses mainly the CaV1.2b variant (often referred to as the “smooth muscle” variant). Alternative promoter variant CaV1.3c was described in cerebral arteries, and the CaV1.2a, “cardiac”, variant was recently shown to be promoted by the mineralocorticoid receptor signalling in VSMCs (Mesquita, et al., 2018). The exon 8b variant present in the smooth muscle isoform makes the channel more sensitive to
13 dihydropyridines (Welling, et al., 1997). Smooth muscle specific deletion of CaV1.2 channels highlighted their key role the in myogenic tone and blood pressure regulation (Moosmang, et al., 2003). Being of therapeutic relevance, CaV1.2 channels are the target of Ca2+ influx inhibitors with vasodilating properties such as dihydropyridines, verapamil and diltiazem, which are indicated in various cardiovascular disorders.
3.1.1 Regulation of CaV1.2 by cGMP in vascular myocytes
Several studies performed using rodent and human VSMCs isolated from various vascular beds, used either freshly or in culture, consistently reported that native L-type Ca2+ current is reversibly inhibited by NO donors (Clapp & Gurney, 1991; Quignard, et al., 1997; Tewari & Simard, 1997), 8-Br-cGMP (Blatter & Wier, 1994; Quignard, et al., 1997; Taguchi, et al., 1997; Tewari & Simard, 1997; Xiong, et al., 1994b) or PKG. These data were obtained by using Ba2+ as the charge carrier. Percentage of inhibition in whole-cell recordings ranged from <50% inhibition (Quignard, et al., 1997; Tewari & Simard, 1997) to almost complete abolition of the current (Blatter & Wier, 1994). Cell-attached recordings allowing to study unitary conductances displayed a concentration-dependent inhibition of NPo (N x single channel opening probability) with SNP concentrations
200 nM (Tewari & Simard, 1997). Paradoxically, channel activity in the presence of higher SNP concentration (1 or 10 µM) was not different from control. Inhibition of channel activity by 100 µM 8-Br-cGMP or SNP did not change channel conductance, voltage dependence or open time characteristics (Tewari & Simard, 1997). PKG inhibitors (Rp 8-Br PET cGMPs, 10 nM) reduced whole-cell basal current Velasco, et al., 1998) and reversed the action of 8-Br-cGMP (Ruiz-Velasco, et al., 1998). Likewise, H-8, a weakly selective PKG inhibitor (Hidaka, et al., 1984), reversed the effect of nitroprusside (Tewari & Simard, 1997). So far, no biochemical evidence that the smooth muscle CaV1.2 channel is a substrate of PKG has been provided. While it was inferred from mutagenesis experiments in oocytes that PKG phosphorylates the “cardiac” CaV1.2a 1C subunit at Ser-533 (rabbit sequence)(Jiang, et al., 2000), this was not confirmed by others who studied the channel expressed in human HEK-293 cells (Yang, et al., 2007). Rather, the latter group
14 demonstrated that PKG phosphorylates 1C subunit at Ser-1928, a site also proposed as a substrate for PKA and PKC (Yang, et al., 2007). Interestingly, Ser-496 of the 2a subunit was also shown to be a substrate of PKG in HEK-293 cells and rat cardiomyocytes, and appeared actually to play a key role in mediating the inhibition of Ba2+ current by 8-Br-cGMP in HEK-293 cells (Yang, et al., 2007). In vascular tissue 2 and 3 subunits are expressed (Murakami, et al., 2003) and whether their phosphorylation by PKG is relevant in modulating CaV1.2 activity remains to be addressed.
Of note, S-nitrosylation, i.e. the covalent modification of a cysteine thiol by NO, may also decrease activity of CaV1.2 channel and many other ion channels in the vasculature in a cGMP-independent manner (see review by (Olschewski & Weir, 2015)).
Among the diverse mechanisms that could mediate cGMP-induced vasorelaxation, the particular contribution of CaV1.2 channel inhibition is still unclear. Contribution of CaV1.2 channels may be inferred from the effect produced by selective blockers on the responses to cGMP. Nevertheless, because CaV1.2 is necessary for myogenic tone and most agonist-evoked vasoconstrictions, this approach may introduce a bias, as control and inhibitor conditions will generally not be studied at the same contractile tone. Indeed, it is usually considered that the higher the precontraction is, the smaller is the subsequent vasorelaxant response (see for instance Eckly et al., 1994). Still, examples exist were comparison may be made: Liu et al. (Liu, et al., 2016) for instance observed that responses to S-nitrosothiols (s-NO) and cGMP were higher in sheep mesenteric arteries than femoral arteries pre-contracted with serotonin. Nifedipine (10 µM, high concentration) inhibited contractile response in a similar manner (24%) in both vascular beds and, interestingly, inhibited response to S-NO only in mesenteric arteries, bringing it to the level of that in femoral. These data suggest that inhibition of the CaV1.2 channel by S-NO may be responsible for the more robust vasorelaxant response to S-NO observed in the mesenteric bed. In a recent study in isolated rat tail artery, NO-cGMP anti-contractile effects appeared to be due to inhibition of Ca2+ influx rather than intracellular Ca2+ store release during methoxamine-induced contraction (Schmid, et al., 2018). Taken together, these data suggest that CaV1.2 inhibition participates in the vasorelaxation
15 evoked by NO and cGMP. However, results obtained with nifedipine at high concentrations (>1 µM) may be considered cautiously since this drug can also affect vascular T-type Ca2+ current (Harraz & Welsh, 2013), which is also a target of CN signalling (see section 3.2).
Intriguingly, other data obtained in pressurized rat cerebral arteries show that the contribution of LTCCs to myogenic tone (defined as the vasoconstriction sensitive to 1 µM nifedipine) was decreased in the presence of L-NAME (10 µM) (Howitt, et al., 2013). These observations are at odds with the notion that endogenous NO represses myogenic tone via CaV1.2 inhibition. Thus, the exact influence of endogenous NO release on the contribution of CaV1.2 to tone regulation remains to be further delineated.
3.1.2 Regulation of CaV1.2 by cAMP in vascular myocytes
In the heart, it is widely accepted that CaV1.2 activity is stimulated by the cAMP-PKA axis and that this regulation plays key roles in Gs-coupled receptor-mediated responses to neurohormones such as catecholamines. Still, the exact molecular mechanisms involved in the regulation of CaV1.2 activity by PKA have remained uncertain despite extensive research that has been reviewed by others (Catterall, 2015; Keef, et al., 2001; Weiss, et al., 2013). In brief, proposed mechanisms include phosphorylation of 1C (mostly at sites located at the distal C-terminus region) or
subunits, requirement of the distal C-terminus domain that can undergo proteolytic truncation and requirement of AKAP that may hold together the channel subunits and the kinase, thus facilitating the phosphorylation. Recently, PKA-phosphorylation and subsequent removal of the inhibitory Rad protein from the calcium channel vicinity was demonstrated as another credible mechanism to explain activation of CaV1.2 channel by -adrenergic stimulation (Liu, et al., 2020).
The regulation of CaV1.2 in isolated SMCs by -adrenergic stimulation, FSK and cAMP analogues was addressed by several groups, yielding to non-unanimous conclusions which are reviewed elsewhere (Keef, et al., 2001). Manoeuvres that would activate the cAMP pathway (e.g. 8-Br-cAMP
10-100 µM, ISO 1 µM, FSK 10 µM, broad PDE inhibitor papaverine) generally induced a modest (<50%), still significant increase in Ba2+ currents carried by the CaV1.2 channel in myocytes
16 isolated from rabbit portal vein (Ishikawa, et al., 1993; Ruiz-Velasco, et al., 1998; Shi & Cox, 1995), porcine coronary artery (Fukumitsu, et al., 1990), rat mesenteric (Yokoshiki, et al., 1997) or tail (Fusi, et al., 2016) arteries or in A7r5 cells (Kimura, et al., 2000; Marks, et al., 1990). Recordings from cell-attached patches in cultured rat mesenteric artery SMCs (Taguchi, et al., 1997) or guinea-pig freshly isolated basilar artery myocytes (Tewari & Simard, 1994) provided consistent results with a 1.5- to 2.7-fold increase in channel activity in response to cAMP stimulation. Still, it was noted that only 50% of cells responded to stimulation in the latter study (Tewari & Simard, 1994). Only minor modification in the voltage-dependency of channel activity was observed (Kimura, et al., 2000; Tewari & Simard, 1994). Importantly, stimulation by cAMP was reported to be blocked by PKA inhibition using Rp-cAMPS (10 µM) (Kimura, et al., 2000), KT-5720 (0.2 µM) (Zhong, et al., 1999b) or PKI (1 µM) (Yokoshiki, et al., 1997). Direct intracellular exposition to Gs protein also stimulated IBa (Xiong & Sperelakis, 1995; Zhong, et al., 1999a) and Rp-8-Br-cAMPS inhibited this effect (Zhong, et al., 1999a). Also, FSK (10 µM) increased density of Ca2+ sparklets, i.e. optically-recorded Ca2+ influx events via LTCCs occurring at physiological membrane potential and Ca2+ concentration (Navedo, et al., 2010).
Since CaV1.2 channels form the major route for Ca2+ entry in SMCs, and an increase in Ca2+ generally leads to vasoconstriction, it is odd that elevation of channel activity by the cAMP/PKA pathway leads to vasorelaxation. A mechanism was proposed to explain this discrepancy (Keef, et al., 2001): it involves an interplay between CaV1.2 channels and neighbouring Ca2+-activated K+ (KCa) channels (see section 5.4), with subsarcolemmal rise of [Ca2+] near CaV1.2 activating nearby KCa channels without affecting global intracellular [Ca2+], resulting in membrane hyperpolarisation and relaxation (Guia, et al., 1999). However, the actual relevance of this mechanism on tone regulation remains unclear.
In a recent study, Nystoriak et al. (Nystoriak, et al., 2017) demonstrated that PKA activated CaV1.2 channels in mouse and human VSMCs in the context of exposure to high glucose. Phosphorylation of ser-1928 of the 1C subunit was necessary for high glucose to increase Ba2+ current and [Ca2+]i, and to evoke vasoconstriction of mouse cerebral arteries. Moreover, the effects of glucose were
17 abolished if the A-kinase anchoring protein 150 (AKAP150), a scaffolding protein important for targeted PKA activity (Ercu & Klussmann, 2018), was absent or ablated for its PKA-interacting domain. Using super resolution microscopy a subpopulation of 1C subunits was localized in the vicinity of PKA. These data suggest that a particular signalosome held by AKAP150 and involving phosphorylation of a single CaV1.2 residue by PKA, is sensitive to high glucose exposure and potentiates vasoconstriction. This finding may have clinical relevance, since PKA-mediated stimulation of Ca2+ influx was increased in diabetic mice fed with a high-fat-diet, and in VSMCs from diabetic patients (Nystoriak, et al., 2017). However, the mechanisms linking high glucose with stimulation of this specific PKA activity remain to be elucidated.
PKA was also proposed to potentiate CaV1.2 channel activity in VSMCs by activation of 51 integrin, a transmembrane cell adhesion molecule involved in the interaction with extracellular matrix and in mechanotransduction (Chao, et al., 2011). This regulation involved the dual phosphorylation of the 1C protein at ser-1901 and tyr-2122, which are PKA and Src sites, respectively. This phosphorylation seems to favour association of the 1C with the 1-integrin, but other molecular partners such as focal adhesion kinase may also be involved.
Alternative, CN-independent, regulatory mechanisms have also been described to modulate LTCCs activity upon GPCR stimulation. For instance, during -adrenergic receptor stimulation, the G-PI3K-PKC axis was shown to stimulate the CaV1.2 channel in portal vein myocytes (Viard, et al., 2001; Zhong, et al., 2001). This is consistent with the enhancement of channel activity evoked by PKC activation (Keef, et al., 2001), e.g. as occurs in response to angiotensin II (AngII) (Nystoriak, et al., 2017). Also, stimulation of the adenosine A2A receptor, known to be coupled via Gs to cAMP production, was reported to decrease the Ca2+ channel current via a tyrosine phosphatase activity (Murphy, et al., 2003).
At variance with the above studies, a number of studies showed that cAMP produces either biphasic effect or steady state inhibition of CaV1.2 current (Ishikawa, et al., 1993; Ruiz-Velasco, et al., 1998; Shi & Cox, 1995; Xiong, et al., 1994a). However, these experiments were often performed
18 with very high concentrations of cAMP (e.g. 3 mM 8-Br-cAMP) (Xiong, et al., 1994b), which could stimulate not only PKA but also PKG by “cross-activation” (Dhanakoti, et al., 2000), leading to a PKG-mediated inhibition of CaV1.2 channels (Ruiz-Velasco, et al., 1998) and vasorelaxation (Jiang, et al., 1992).
3.2 Regulation of voltage-gated, T-type Ca2+ channels (TTCC) by cyclic nucleotides in
vascular SMCs
TTCC expression was more recently characterized in rodent and human vascular SMCs (Abd El-Rahman, et al., 2013; Braunstein, et al., 2009; Harraz, et al., 2014; Harraz, et al., 2015b; Howitt, et al., 2013; Navarro-Gonzalez, et al., 2009). T-type Ca2+ (or Ba2+) current can be isolated from L-type current on the basis of its biophysical properties (low voltage activation threshold and fast activation/inactivation kinetics), low sensitivity to dihydropyridines and sensitivity to a diversity of molecules including mibefradil and NNC 55–0396. CaV3.1 and CaV3.2 subunits were first detected in rodent VSMCs. In human, however, CaV3.3 (CACNA1I) subunit substitutes for CaV3.1 (CACNA1G) while the CaV3.2 (CACNA1H) homologue was also expressed. By taking advantage of a high sensitivity of CaV3.2 channels to Ni2+ compared to other nifedipine-insensitive currents, it was possible to dissect the TTCC current (Harraz, et al., 2015b; Harraz & Welsh, 2013). Intriguingly, inhibition or invalidation of the CaV3.2 channel led to a paradoxical increase in myogenic tone in rodent and human arteries (Harraz, et al., 2014; Harraz, et al., 2015a; Harraz, et al., 2015b). Further studies demonstrated that sarcolemmal Ca2+ influx through this channel could serve as a trigger for the opening of adjacent RyR channels on the SR membrane (Harraz, et al., 2014; Harraz, et al., 2015a). Ca2+ locally and transiently released by the RyR (Ca2+ sparks) would in turn activate sarcolemmal BKCa channels that would relax VSMCs via Em repolarization (see section 3.4, similar coupling with TRPV4, and 5.4 for introduction of RyR – BKCa channel coupling). Therefore, CaV3.2 channels may promote a negative feedback mechanism acting against vasoconstriction. Other CaV3.x channels were shown to rather facilitate myogenic tone at low intraluminal pressure (Abd El-Rahman, et al., 2013; Bjorling, et al., 2013; Harraz, et al., 2015b).
19 The effects of CN signalling on T-type current were mainly studied in rat cerebral artery by Harraz and Welsh in a couple of reports (Harraz, et al., 2013; Harraz & Welsh, 2013). -AR stimulation with ISO (1 µM), direct AC activation with FSK (1µM), or cell-permeable cAMP derivative db-cAMP (200 µM) all reduced T-type Ba2+ current up to 50-70% inhibition. The effect of FSK was inhibited by PKI 14-22 (1 µM) and KT-5720 (1 µM), pointing to a PKA-mediated effect. Also, Ht31, a peptide that disrupts PKA-AKAP interaction, also prevented current suppression by FSK. Because no further inhibition was observed when adding 50 µM Ni2+ on top of FSK or db-cAMP, authors suggest that the CaV3.2 may be the main target responsible for the inhibitory effect of PKA on T-type current. This is actually not consistent with the notion that CaV3.2 channels globally facilitate vasodilation by activating the above-mentioned Ca2+ sparks and BKCa signalling. These observations are also at odds with the stimulatory action of cAMP signalling on T-type current in neurons and cardiac myocytes. A deeper biochemical characterization of the phosphorylation sites present in the CaV3.2 subunit expressed in the vasculature may help to solve this discrepancy.
Although the above studies convincingly demonstrated that cAMP-PKA signalling inhibits T-type channel, relevance to vascular tone has not been explored and remains elusive. In contrast, experimental observations suggested that TTCCs inhibition by NO-cGMP translates into vasodilation. A first study assessed the influence of endogenous NO on the TTCC-dependent component of myogenic tone in isolated cerebral arteries and cremaster muscle arterioles in vivo (Howitt, et al., 2013). TTCC-mediated tone was defined as the component sensitive to TTCC inhibitor NNC 55–0396 (NNC, 3 µM), in the presence of 1 µM nifedipine. In this study, it was found that abrogating NO production in arterioles by acute incubation with L-NAME (10 µM) enhanced TTCCs contribution to myogenic tone to a similar level as LTCCs contribution, suggesting that endogenous NO is an important repressor of TTCCs that limits vascular tone. Experiments performed in phenylephrine-constricted mesenteric arteries from CaV3.1-knockout or CaV 3.2-knockout mice indicated that both channels participate in this regulation. Likewise, Welsh’s group reported that TTCCs contribution to tone development (defined as sensitivity to 1 µM NNC on top of 200 nM nifedipine) was strongly repressed in the presence of NO donor SNAP (from 12% to
20 5%, at 50 mmHg). Importantly, these authors further demonstrated that NO donors inhibited nifedipine-resistant T-type current. This effect was mediated by PKG since it was mimicked by the cGMP analogue db-cGMP and prevented by the PKG inhibitor KT-5823 (1 µM). Overall these data suggest that the cGMP/PKG cascade inhibits TTCCs, resulting in attenuation of arterial tone.
The molecular mechanism underlying this regulation remains unknown. In both PKA- and PKG-induced inhibitions of vascular TTCCs, a rightward shift of the curve for voltage-dependence of inactivation was observed (Harraz, et al., 2013; Harraz & Welsh, 2013). It is likely that TTCC subunits are directly phosphorylated by these kinases, as TTCC subunits display multiple phosphorylation sites for various kinases which can modulate its gating properties (Blesneac, et al., 2015; Harraz & Welsh, 2013). Alternatively, the regulation may also involve trafficking of channel subunits to membrane or specific subcellular compartments. This was suggested by the observation that 30 min incubation with L-NAME increased signal yield by an anti-CaV3.1 antibody at sarcolemma of isolated rat cerebral artery SMCs, and enhanced signal for both anti-CaV3.1 and anti-CaV3.2 in rat arterioles (Howitt, et al., 2013).
3.3 Regulation of store-operated Ca2+ entry by CNs
Store-operated Ca2+ entry (SOCE) is one mechanism of [Ca2+]i elevationtriggered by depletion of the endoplasmic reticulum (ER) Ca2+ store and mainly involving Ca2+ influx through cation channels different from voltage-gated Ca2+ channels (recently reviewed by (Avila-Medina, et al., 2018)). Store depletion can occur upon inhibition of SERCA (e.g. using thapsigargin, TG, or cyclopiazonic acid, CPA) or upon stimulation with vasoconstricting agonists which lead to activation of IP3R and sarcoplasmic reticulum (SR) Ca2+ release. SOCE can promote contraction in a dihydropyridine-independent manner (Dominguez-Rodriguez, et al., 2012; Ng & Gurney, 2001).
Located at the ER membrane, stromal interaction molecule-1 (STIM1) protein has been established as the main sensor for ER Ca2+-depletion (Avila-Medina, et al., 2018; Liou, et al., 2005; Zhang, et al., 2005). Upon stimulation, STIM1 migrates into clusters located at ER-plasmalemmal junctions where it launches Ca2+ influx through 2 main types of store-operated Ca2+ (SOC)
21 channels, a Ca2+ selective one, the Ca2+ release-activated Ca2+ channel (CRAC) and non-selective SOC (reviewed in (Albert & Large, 2006; Ambudkar, et al., 2017; Avila-Medina, et al., 2018)). The Ca2+ selective channel Orai1 was identified as a key molecular correlate of ICRAC (Ambudkar, et al., 2017; Prakriya, et al., 2006) and contributes to SOCE in VSMCs (Beech, 2012; Dominguez-Rodriguez, et al., 2012; Potier, et al., 2009). Expression of Orai1 is more robust in proliferating, non-contractile SMCs in comparison to freshly isolated vascular tissue or SMCs (Beech, 2012; Shi, et al., 2017a). Transient receptor channels, TRPC1 in particular (Ambudkar, et al., 2017; Xu & Beech, 2001),are considered to constitute SOC channels, with higher conductance and lower Ca2+ selectivity than CRAC (Avila-Medina, et al., 2018; Earley & Brayden, 2015). Depolarization evoked by cation influx carried by TRPC1 and Orai1 activates LTCCs (Park, et al., 2008a) which were also demonstrated to participate in SOCE in VSMCs (Avila-Medina, et al., 2018). Many intermediate mechanisms have been proposed as activators of Orai1 and SOCs, including direct molecular interaction with STIM1, interaction with additional partner proteins and activity of Ca2+ -independent-phospholipase A2 (iPLA2) (Avila-Medina, et al., 2018; Smani, et al., 2004). A combination of STIM1, PLC1 and PKC was also proposed to activate SOCs composed of TRPC1 in VSMCs (Shi, et al., 2017a). Interestingly, whether Orai1 is necessary or not for TRPC1 to function as SOC is controversial (Ambudkar, et al., 2017; Shi, et al., 2017b). Nevertheless, several evidence suggest that STIM1, Orai1, TRPC1 channels and LTCCs act within a macromolecular signalling complex, which may be relevant for vascular tone regulation (Ambudkar, et al., 2017; Avila-Medina, et al., 2016; Avila-Avila-Medina, et al., 2018).
Few studies from different groups account for an inhibition of SOCE and SOC channels by cAMP pathways in various SMCs, a mechanism that may contribute to relaxing effects of cAMP-elevating agents. Permeant cAMP analogues can nearly abolish TG-evoked SOCE in cultured or freshly isolated rat SMCs isolated from coronary or mesenteric arteries (Smani, et al., 2007; Wang, et al., 2009). This effect is inhibited by 1 µM KT-5720, suggesting that PKA is involved. This mechanism appears to mediate the relaxant effects of adenosine and its Gs-coupled A2A receptor in mesenteric artery (Wang, et al., 2009) and of urocortin, a 40-amino acid peptide, agonist of
corticotropin-22 releasing factor receptor-2 in coronary artery (Smani, et al., 2007). Indeed, adenosine and urocortin were demonstrated to inhibit contraction produced by TG or phenylephrine, the latter contractile response being much sensitive to SOC channels inhibitors (diethylstilbestrol, DES, 2-aminoethoxydiphenyl borate, 2-APB), while displaying a large nifedipine-resistant component in these studies. Smani et al. also reported that db-cAMP and urocortin effects were associated with inhibition of iPLA2 by cAMP (Smani, et al., 2007). Urocortin did not exert its effects on pathways downstream iPLA2 activity (i.e. COX-sensitive or stimulated by lysophosphatidylinositol or lysophosphatidylcholine), highlighting this enzyme as a key target of cAMP-PKA for reducing SOCE and associated tone in the vasculature. The fact that iPLA2 activity was downregulated by exposure to urocortin for as little as 10 min also highlights this pathway as mediator of a robust tone-inhibiting process.
In addition, electrophysiological evidence for a cAMP-PKA inhibition of SOC was provided in isolated SMCs from rabbit portal vein (Liu, et al., 2005). Using various configurations of the patch clamp technique, bath application of CPA or BAPTA-AM (a Ca2+ chelator) evoked a cation current with a positive reversal potential compatible with the activation of a store-operated, non-selective, cationic channel (Albert & Large, 2006). This current was reduced by 85-95% by ISO, FSK or 8-Br-cAMP. A similar current could be directly activated (independently from store-depletion) by a diacylglycerol (DAG) analogue and PKC activator phorbol-12,13-dibutyrate (PDBu), that was still sensitive to FSK and 8-Br-cAMP, suggesting that cAMP-pathways act directly on the channel to inhibit its activity. This is supported by the observation that bath application of PKAc to inside-out patches was able to blunt the stimulation of the current evoked by the PKC activator. Interestingly, pharmacological inhibition of PKA evoked a current in cell-attached but also in inside-out patches, while application of 8-Br-cAMP also blunted the PDBu-induced current in the latter configuration. This suggests that the PKA enzyme is membrane-bound and exerts a tonic action on the channel. In line with these observations, another study (Chen, et al., 2011) reported that a CPA-evoked, non-selective, cationic current was increased by relatively high concentration (10 µM) of H-89 (an inhibitor acting on many kinases, including PKA) but also by
23 the PKG inhibitor KT-5823 (3 µM) in SMCs isolated from rat pulmonary artery (PA). This current seemed to be carried by a TRPC channel, as the non-selective TRPC blocker SKF-96365 (10 µM) blunted these effects on the whole-cell current. Nevertheless some caution is needed to interpret these results because of the poor selectivity of the inhibitor (Singh, et al., 2010). Overall, substantial evidence supports that cAMP, generated by multiple stimuli, can blunt SOCE in freshly isolated VSMCs and hamper associated contraction.
In addition, regulation of SOCE by the NO-cGMP-PKG pathway was demonstrated in cultured ECs where it was proposed to act as a negative feedback following activation of NO synthase by Ca2+ influx (Dedkova & Blatter, 2002; Kwan, et al., 2000).
3.4 Regulation of transient receptor potential (TRP) channels by CNs
The notion of SOC channel inhibition by CN pathways can actually be extended to a more general regulation of members of the TRP channels family by CNs and associated effectors. TRP channels constitute a large group of non-selective, cationic channels (for a more comprehensive review see (Earley & Brayden, 2015)). TRP channel proteins are encoded by 28 genes in mammals, yielding subunits which can assemble in homo- or heterotetramers to form a functional channel. Members of TRP channels can be found in most cell types where they are involved in sensory or signal transduction. In ECs and SMCs, TRP channels can be activated by a variety of physical (membrane stretch, temperature) or pharmacological (fatty acids, receptor-activated signalling) stimuli. TRP channel families display a large array of permeability for cations, with some being permeant for only monovalent ions (TRPM5-6), some being selective for Ca2+ (TRPV5-6), and most being permeant for both monovalent and divalent cations with various preferences. Thus, activity of TRP channels can participate in Em regulation and/or Ca2+ influx in SMCs and ECs (Table 1). Evidence demonstrating the contribution of a given TRP channel family in regulating a particular function are often based on the use of weakly selective ligands (Ni2+, SKF-96365, La3+), but also more convincingly with antisense/silencing RNA approaches (Takahashi, et al., 2008) or addition of interfering antibodies dialyzed intracellularly via the patch pipette (Chen, et al., 2009). In
24 addition to their role in SOCE, several members of TRP channels participate in the establishment of arterial tone subsequent to Gq-coupled receptor activation (receptor-operated channel: TRPC3, TRPC6, and TRPC7, activated by DAG) or elevation in intraluminal pressure (myogenic tone) (Earley & Brayden, 2015). Elementary Ca2+ signals generated by Ca2+ influx from single TRPV4 or TRPA1 channels (sparklets) were shown to mediate endothelial and SMC vasodilatory mechanisms (Mercado, et al., 2014; Sonkusare, et al., 2012; Sullivan, et al., 2015).
Most data on TRP regulation by CNs were provided for members of the TRPC family, often obtained in HEK-293 cells used as an expression system. Evidence for direct phosphorylation of serine or threonine residues is generally available, using alanine scan or in vitro phosphorylation assays. Nevertheless, actual relevance of these mechanisms in regulating vascular tone remains unclear, since data on native vascular beds or even primary cells with contractile phenotype are scarcely reported.
Several reports have shown phosphorylation of TRPC1, TRPC3 and TRPC6 channels by PKG, resulting in inhibition of activity (Chen, et al., 2009; Kwan, et al., 2004, 2006; Takahashi, et al., 2008) (see Table 1). Takahashi et al. demonstrated that native TRPC6-like current evoked by Arg8 -vasopressin was inhibited by cGMP-PKG in A7r5 rat embryonic aortic cell line (Takahashi, et al., 2008). Conversely, heterologously expressed TRPC6 was not sensitive to cAMP (Sung, et al., 2011), suggesting here that the two CN pathways do not necessarily share common targets. More physiologically relevant was the demonstration that TRPC1/TRPC3-like currents were inhibited by a NO donor in rat carotid artery SMCs (Chen, et al., 2009). A direct nitrosylation of channels by the NO donor was unlikely since a cGMP analogue evoked a similar response and both effects were sensitive to PKG inhibition (using KT-5823). Interestingly, only TRPC1 and TRPC3 proteins could be detected in this tissue while TRPC6 displayed only RNA expression and TRPC7 was not detected. Currents evoked by uridine triphosphate (UTP) were inhibited by both TRPC1 or TRPC3 antibodies, and TRPC1 co-immunoprecipitated with TRPC3, leading to the hypothesis that a TRPC1/TRPC3 heteromeric channel was blunted by cGMP in these cells. A hint towards a possible contribution of these channels to vascular tone was the finding that the vasodilating response to
25 SNP of carotid artery segments precontracted with UTP was partially inhibited by 100 µM La3+. However, caution must be taken in drawing definitive conclusion from data relying on the use of this poorly selective inhibitor.
In parallel, an extensive study was performed showing inhibitory effects of cAMP-elevating agents on DAG-sensitive TRPC channels, and TRPC6 in particular (Nishioka, et al., 2011). The authors used cilostazol (CLZ), a PDE3 inhibitor with vasodilatory and anti-platelet aggregation properties. Although this was not demonstrated in the study, CLZ should inhibit cAMP breakdown and thus increase intracellular levels of the CN. CLZ potently reduced contractile response to AngII in rat aorta, and this was abolished by inhibition of SKF-96365-sensitive Ca2+ entry, indicating a role for receptor-operated channels. Ca2+-influx carried by DAG-activated TRPC channels, namely TRPC3, 6 and 7, expressed in HEK-293 cells, were all dose dependently-inhibited by CLZ. The authors robustly demonstrated that CLZ effect on TRPC6 was mediated by phosphorylation of thr-69, a substrate shared by PKA and PKG. Although high concentrations of CLZ (> 1 µM) may also inhibit PDE5 (Sudo, et al., 2000), and thus activate the cGMP-PKG pathway, here CLZ action was abolished by 0.1 µM KT-5720, but not 0.3 µM KT-5823, indicating a role of PKA in this regulation. In native rat aortic SMCs, gene silencing indicated that inhibition of AngII-evoked Ca2+ influx by CLZ depends on TRPC6, but not on TRPC3 or TRPC7. Cells expressing TRPC6 channels harbouring mutation at thr-69 displayed AngII-induced Ca2+ influx with reduced sensitivity to CLZ. The mutation also rendered the contraction of rat aortic SMCs less sensitive to the PDE inhibitor, consistent with a role of TRPC6 thr-69 in mediating cAMP-PKA vasorelaxation.
The cAMP-PKA axis was also reported to act on TRPC4 and TRPC5 channels, both being close homologues, insensitive to DAG and shown to participate in SOCE (Earley & Brayden, 2015). Data are, however, somewhat contradictory. When expressed in HEK-293 cells, TRPC5 mediated a Ca2+ influx that remained unaffected by CLZ (see above)(Nishioka, et al., 2011). By contrast, in another report (Sung, et al., 2011), the inward current carried by human TRPC5 channel expressed in HEK-293 and stimulated by GTPs was inhibited by FSK, 8-Br-cAMP or expression of a constitutively active GsQ227L protein. Similarly, TRPC4 ( and splicing variants) was sensitive to GsQ227L, but,
26 surprisingly, not TRPC6. Contradictory results may have emerged from different protocols and readouts to determine channel activity (intracellular Ca2+ level versus current) or the use of channels from different species. Another discrepancy emerged from the study by Wie et al. who reported that udenafil (“Zydena”), a PDE5 inhibitor, but also cilostamide (PDE3 inhibitor) and EHNA (PDE2 inhibitor), but not rolipram (PDE4 inhibitor) actually enhanced TRPC4 currents (Wie, et al., 2017). Because the actual concentration of inhibitors used are not indicated, it makes it difficult to conclude on the actual PDE isoforms that were inhibited in these experiments. Nevertheless, the authors reported that cGMP enhanced a Cs+-activated, TRPC4-like native current in human prostate SMCs in culture, suggesting that this regulation may be relevant for function visceral tissues.
In blood vessels, different vasorelaxant pathways coexist and may interfere with each other. An example is given by Zhang et al. who described a mechanism by which NO-cGMP signalling inhibits vascular relaxation by 11, 12-epoxyecosatrienoic acid, an endothelium-derived hyperpolarizing factor (EDHF) (Zhang, et al., 2014b). Epoxyeicosatrienoic acids (EETs) are derived from arachidonic acid and are involved in the vasodilating responses to various endothelium-acting agonists (Bellien, et al., 2011). EETs are reported to activate TRPV4 in both ECs and SMCs and to mediate vasodilation. In SMCs, a well-documented paradigm proposes that TRPV4 channels generate local Ca2+ increase that would activate Ca2+ sparks from neighbouring RyR, which in turn would activate BKCa channels, causing eventually hyperpolarization and vasorelaxation (Earley & Brayden, 2015; Filosa, et al., 2013). Also, TRPV4 was reported to form heteromeric channels with other TRP subunits, including TRPC1. Interestingly, data obtained by Zhang et al. demonstrated that TRPC1 is a target of PKG at ser-172 and/or thr-313 in HEK-293 cells (Zhang, et al., 2014b). Moreover, by using a small permeant competing peptide to inhibit PKG phosphorylation of TRPC1, the authors demonstrated that this regulation is also relevant in endothelium-denuded porcine coronary artery and is crucial for the NO-mediated inhibition of EETs-induced relaxation. Functional and biochemical data support the existence of a ternary complex including TRPC1, TRPV4 and BKCa (KCa1.1) channels in these arteries which would then be tamed by the
NO-cGMP-27 PKG axis. It is proposed that this intriguing regulation may allow EDHF mechanisms to act as a “second line” vasodilating mechanism in situation of loss of NO bioavailability. Consistent with these data is the observation that inhibitors of NO-cGMP-PKG axis potentiate vasodilatory response to carvacrol, a TRPV3 activator, in rat uterine radial arteries (Murphy, et al., 2016). Response to carvacrol was endothelium-independent and sensitive to the KCa3.1 blocker Tram-34, suggesting that multiple coupling combinations associating TRP and K+ channels may occur in vascular SMCs and are subjected to regulation by the cGMP pathway.
TRPV4 channels are also expressed in the endothelium and are thought to activate Ca2+-dependent K+ channels which contribute to EDHF signalling (Earley & Brayden, 2015). Arachidonic acid can activate endothelial TRPV4 and this was shown to be inhibited by the PKA inhibitory peptide PKI (Zheng, et al., 2013). Recent data further explored this mechanism by showing that TRPV4 is actually phosphorylated by PKA at ser-824, a modification associated with increased Ca2+ influx (Cao, et al., 2018). This mechanism was proposed to mediate the vasodilating properties of arachidonic acid in human coronary artery.
Besides direct post-translational modifications affecting their gating, TRP channel activity may also be regulated by modifications of their membrane trafficking (Earley & Brayden, 2015). Although this hypothesis was addressed in some of the above-cited studies, no evidence of alteration of the localization of TRP channel subunits by CN signalling was reported (Cao, et al., 2018; Kwan, et al., 2004; Sung, et al., 2011).
4. cGMP-dependent, Ca
2+-activated, Cl
-channels in vascular SMCs.
Two distinct Ca2+-activated, Cl- currents (ICl(Ca))have been identified in vascular SMCs (see(Dam, et al., 2014) for review): one “classical” current with small (1-4 pS) conductance, displaying voltage-dependent outward rectification and sensitive to niflumic acid (100 µM); one cGMP-activated current, with higher conductance (15-55 pS), displaying linear conductance-voltage
28 relationship and highly sensitive to Zn2+(Matchkov, et al., 2004; Piper & Large, 2004). It is now accepted that the former is carried by the TMEM16A protein which promotes cell contraction and myogenic tone in different vascular beds (Bulley, et al., 2012; Dam, et al., 2013; Heinze, et al., 2014; Manoury, et al., 2010). However, the molecular correlate of the cGMP-dependent current remains uncertain. By siRNA-approach, it was shown that bestrophin-3 (vitelliform macular dystrophy 2-like 3 protein) was necessary to observe the current (Matchkov, et al., 2008). This is however challenged by the fact that downregulation of TMEM16A abrogates both currents. The role of TMEM16A may actually span beyond simple Cl- conductance, and it was proposed that TMEM16A knock-down may also influence other ion channel expression, including LTCCs (Dam, et al., 2014).
Expression of the cGMP-dependent current is present in various vascular SMCs, although absent in PA, where both bestrophin-3 protein and current are not observed (Dam, et al., 2014; Matchkov, et al., 2008). Importantly, cGMP-dependence of the current takes place via PKG activity, and it was suggested that PKGII isoform, that is membrane-bound, could be involved. Partial silencing of bestrophins does not result in major alteration of vascular tone, but it abrogates rhythmic contractions (Broegger, et al., 2011).
5. Regulation of K
+channels by CNs in vascular SMCs
As presented above, K+ channels are pivotal in regulating vascular tone by their role in balancing Em of SMCs toward more polarized values, therefore limiting Ca2+ influx through LTCCs. CN pathways generally increase K+ channel activity, and thus oppose VSMCs depolarization and contraction. This notion is supported by the fact that cAMP and NO donors hyperpolarize VSMCs and this largely depends on K+ conductance (Somlyo, et al., 1970) (Tables 2-4). Moreover, CN-elevating agents added on isolated vessels rings studied in a myograph and contracted using high K+ solution (which abolishes K+ gradient and therefore K+ fluxes) often produces a much weaker relaxant effect than in vessels contracted with a Gq-coupled receptor agonist (e.g. (Bracamonte,
29 et al., 1999; Raina, et al., 2009; Tanaka, et al., 2000)). Thus K+ conductances generally participate in the vasorelaxant properties of CN pathways. Nevertheless, because a myriad of K+ channel subtypes are expressed in VSMCs, dissecting the contribution of each possible pathways has been the focus of abundant research effort.
Virtually all families of K+ channels are present in SMCs, namely inward rectifiers K+ channels (Kir), ATP-sensitive K+ channels (KATP), Ca2+-activated K+ channels (typically, large conductance, BKCa, in SMCs, and small conductance, SKCa, in ECs), voltage-gated K+ channels (KV), and two-pore-domain K+ channels. Expression of a great diversity of channel subunits, within the KV family in particular, can be observed. Since K+ channels are created from the assembly of several pore subunits (generally forming tetramers, except for K2P which assemble in dimers), additional diversity can be created by assembly of different subunits (e.g. KV7.4 and KV7.5) or different subunit variants (e.g. for BKCa channel) into heteromers. Regulation of K+ channels by cGMP and cAMP has been intensively scrutinized for 3 decades. KATP and BKCa channels were the most extensively studied, probably because they displayed singular biophysical properties and selective pharmacological tools were available and allowed to isolate their activity more readily. Nevertheless, emergence of new molecular, genetic and pharmacological tools has helped to highlight the role of other K+ channel families, KV7 channels in particular.
5.1 Inward rectifier K+ (Kir) channels
The Kir channel family includes pore-forming subunits with 2 transmembrane domains that assemble in tetramers. Kir2.1 (KCNJ2) and Kir2.2 (KCNJ12) expression, but not Kir2.3 (KCNJ4) nor 2.4 (KCNJ14) (Alexander, et al., 2017b), can be found in vascular SMCs and ECs (Sancho, et al., 2017; Schubert, et al., 2004). Channel activity can be isolated by using small Ba2+ concentration (micromolar range, mostly selective at <50 µM) (Nelson & Quayle, 1995; Park, et al., 2008b) and is characterized by stronger conductance in the inward direction (i.e. at potentials negative to EK) than when K+ ions flow in the outward direction. Moderate increase in extracellular [K+] enhances channel activity and this may be involved in mediating EDHF-evoked vasodilation due to local
30 release of K+ (Park, et al., 2008b). Kir channels were proposed to participate in global cellular K+ conductance when Em is close to the resting potential. Kir channel activity was shown to mediate cicaprost-evoked hyperpolarization and relaxation (Orie, et al., 2006), an effect, however, unlikely to be mediated by a cAMP-related mechanism. In rabbit coronary artery myocytes Kir activity was reported to be increased by 30% by adenosine, via the A3 receptor (Son, et al., 2005). Sensitivity of this response to PKA inhibitors and the AC inhibitor SQ22536 and comparable effect obtained with FSK pointed to a cAMP-PKA-dependent signalling. This regulation may have physiological relevance as 50 µM Ba2+ hampered the vasodilatory response to adenosine in perfused heart. Although sequence of Kir2 subunits displays PKA consensus sites (Park, et al., 2008b), experimental exploration of this regulation gave contradictory results. Still, a study performed in a heterologous expression system demonstrated that enhancement of Kir2.1 activity by cAMP stimuli was facilitated by AKAP79 protein, provided PKC inhibitors were present (Dart & Leyland, 2001). Co-immunoprecipitation studies in the same expression model showed that AKAP79 interacts with Kir2.1, suggesting the possibility of tight regulation of the channel within a PKA-AKAP signalling complex. Kir channel participation in NO-evoked vasodilation has been suggested in pressurized rat tail small arteries (Schubert, et al., 2004): 10 µM Ba2+ attenuated dilation to SNP, which was abolished by a NO-scavenger, ascribing the vasodilation to a NO-related mechanism. SNP (100 µM) increased a Ba2+-sensitive current by 80%. Overall, these data suggest that Kir2.1 in particular is regulated by CN pathways and participates in vascular tone regulation.
5.2 KATP channels
5.2.1 General properties of KATP channels expressed in the vasculature
ATP-sensitive K+ (KATP) channels have been extensively documented in VSMCs (for reviews see (Cole & Clement-Chomienne, 2003; Foster & Coetzee, 2016; Quayle, et al., 1997)). KATP channels are K+ selective, voltage independent. A variety of KATP conductances with differential sensitivities to intracellular ATP, Mg2+, pharmacological modulators, and nucleosides di- and tri-phosphate has been reported. KATP channels regulate Em in many cell types, including VSMCs, and, being generally
31 inhibited by intracellular ATP, are considered to transduce changes of the metabolic state of the cell into changes of membrane excitability and cellular activity. In blood vessels, activation of these channels is observed in situations of hypoxia, metabolic inhibition, acidosis. KATP activation produces hyperpolarisation and contributes to subsequent vasodilation as observed in response to the above stimuli. Therefore, KATP channel activity is thought to be pivotal for adapting local blood flow and nutrient supply to organs relative to demand (Cole & Clement-Chomienne, 2003; Foster & Coetzee, 2016). KATP channels are hetero-octamers made of four Kir6 subunits and four SUR (for sulfonylurea receptor) subunits (Cole & Clement-Chomienne, 2003), with the variety of Kir/SUR combinations accounting for diversity of function and regulation of the channel. Two different types of KATP-like conductances have been characterized in VSMCs: (i) a predominant small conductance (e.g. 20 pS at physiological [K+] (Nelson, et al., 1990), 35pS in symmetrical [K+] (Dart & Standen, 1993)). The presence of intracellular nucleotide diphosphates and Mg2+ is crucial for this channel activation while inhibition by intracellular ATP occurs at higher concentrations (>1 mM) than in cardiac or -pancreatic cells (Cole & Clement-Chomienne, 2003; Foster & Coetzee, 2016). These channels are probably mostly composed of Kir6.1 subunits and SUR2B subunits (Cole & Clement-Chomienne, 2003); (ii) a medium conductance (50-70 pS), less frequently observed and displaying higher sensitivity to ATP, spontaneous activity in the absence of dinucleotides and reduced sensitivity to KATP activators has also been characterized in rat portal vein myocytes (Zhang & Bolton, 1996).
The role of KATP channels in regulating vasomotor tone has been highlighted by phenotyping knock-out models: Kir6.1 knockout mice do not show vasodilation to pinacidil, a KATP activator, and are more prone to coronary vasospasm (Miki, et al., 2002). Conversely, transgenic mice expressing an ATP-insensitive Kir6.1 mutant (gain of function mutation) display lower blood pressure and a slight impairment of mesenteric artery vasoconstriction in response to phenylephrine (Li, et al., 2013). Smooth-muscle-specific deletion in Kir6.1 leads to a mild hypertensive phenotype, yet without higher sensitivity to vasospasms (Aziz, et al., 2014). SUR2 knockout mice display hypertension and vasospastic episodes which were rescued by treatment
