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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

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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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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: [email protected]

Tel: +33-1.46.83.59.06

Fax: +33-1.46.83.54.75

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.

Keywords: cAMP, cGMP, PKA, PKG, ion channels, vascular tone

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 Figure legends: ...... 99

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

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 (V)SMCs: (vascular) smooth muscle cells

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

6 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

7 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 RIor RIsubunits whereas type II contains RII or RIIsubunits. 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

8 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-

9 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

10 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-

11 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 Ca2+ 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

12 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 (Ruiz-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

13 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

14 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

15 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 Gs 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

16 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 51 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

17 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).

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 CaV3.2- 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

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 elevation triggered 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)

20 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, PLC1 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-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-

21 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

22 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

23 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

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 GTPs 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,

25 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-

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, Ca2+-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

27 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,

28 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

29 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

30 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

31 with the Ca2+ channel blocker nifedipine. Surprisingly, however, re-expression of SUR2 under the control of a SMC-targeting promoter failed to restore a normal phenotype, thus questioning the role of VSMC KATP channels in controlling vasomotor tone, at least in coronary circulation (Kakkar, et al., 2006).

KATP channels have also been characterized in ECs (Aziz, et al., 2017; Katnik & Adams, 1997;

Norton & Segal, 2018), where Kir6.1 appeared to be the subunit constitutive of the channel. A recent study characterized genetically-modified mice with endothelial-targeted deletion (eKO) of

Kir6.1 subunits using expression of a Cre recombinase under the Tie2 promoter (Aziz, et al., 2017).

In perfused hearts, vasodilatory responses to pinacidil or hypoxia were reduced in Kir6.1 eKO mice. These animals displayed also higher susceptibility to global ischemic injury. Endothelial KATP channel activation raised cytosolic Ca2+ (probably by increasing driving force for Ca2+) which may serve as a stimulus for endothelial mediator release such as NO. Therefore both smooth muscle and endothelial KATP channels seem to participate in the regulation of local blood flow and arterial pressure (Aziz, et al., 2017).

KATP channels are also located in intracellular organelles such as mitochondria (Foster & Coetzee,

2016). Although their molecular composition is still uncertain, cardioprotective properties of such channels have been proposed (reviewed and discussed in (Foster & Coetzee, 2016)).

Functional characterization of KATP channels in VSMCs has also been largely enabled by the use of inhibitors such as sulfonylurea antidiabetic drug glibenclamide usually used at 1-10 µM (Foster &

Coetzee, 2016; Quayle, et al., 1997). Conversely, channel activators such as pinacidil and levcromakalim produce both ECs and VSMCs hyperpolarization, vasorelaxation (Norton & Segal,

2018; Quayle, et al., 1997; Standen, et al., 1989) and some are used clinically as vasodilators indicated against angina (nicorandil, with a dual KATP and NO donor-like mechanism (Holzmann, et al., 1992)) or hypertension (minoxidil). Noteworthy, glibenclamide can inhibit contractile response to several agonists, including methoxamine (Randall & McCulloch, 1995) and U46619

(Huang, et al., 2003; Wareing, et al., 2006), and this may have interfered with subsequent studies of relaxing mechanisms.

5.2.2 Regulation of KATP channels by cAMP pathways

Numerous studies performed in the 1990s and 2000s provided wide description of the upregulation of vascular KATP channel activity by agonists of Gs-coupled receptors (Table 2) and other cAMP stimuli.

Calcitonin gene-related peptide (CGRP) is a peptide released at sensory nerve terminals and exhibits potent vasodilating action. CGRP acts on the CGRP receptor, a GPCR associated with partner single transmembrane domain protein, RAMP1 (Poyner, et al., 2002). Stimulation of CGRP receptor leads to an increase in intracellular cAMP (Poyner, et al., 2002). Patch-clamp studies in freshly isolated myocytes from rabbit mesenteric artery (Nelson, et al., 1990; Quayle, et al., 1994) or porcine coronary artery (Wellman, et al., 1998) demonstrated that a glibenclamide-sensitive conductance was dramatically increased by CGRP. This was also found in cultured porcine coronary artery myocytes (Miyoshi & Nakaya, 1995). This response was shown to be abolished by PKA inhibition (PKI, H-8, Rp-cAMPS, H-89), but not by PKG inhibitor (KT-5823) (Quayle, et al.,

1994; Wellman, et al., 1998), and was potentiated by PDE inhibition (Wellman, et al., 1998).

Consistent with a response mediated by cAMP-PKA, FSK application or intracellular dialysis with cAMP or PKAc from the pipette solution also activated a glibenclamide-sensitive current

(Wellman, et al., 1998). Also, CGRP hyperpolarized rabbit mesenteric and mouse pulmonary arteries, and potently dilated rabbit mesenteric artery, mouse PA, rat basilar and pial artery in a glibenclamide-sensitive manner (Kitazono, et al., 1993; Nelson, et al., 1990; Norton & Segal, 2018)

(Table 2). Accordingly, hyperpolarizing response, obtained by using the sharp microelectrode technique, was only minor in PAs from Kir6.1 knockout mice, supporting a key role for KATP channels in mediating this response (Norton & Segal, 2018). Nevertheless, other data obtained in porcine coronary artery and rabbit basilar artery account for a CGRP-evoked vasodilation that

33 was glibenclamide-insensitive, despite being potentiated by PDE inhibition (Kageyama, et al.,

1993; Prieto, et al., 1991; Sutter, et al., 1995).

In addition to SMCs, the CGRP-cAMP-PKA-KATP channel axis was also demonstrated in endothelial tubes preparations isolated from mouse PA (Norton & Segal, 2018). CGRP induced hyperpolarization and rise in [Ca2+]i in ECs, in a glibenclamide- and PKI-sensitive manner.

Accordingly, CGRP receptor proteins were detected in both ECs and SMCs. Interestingly, endothelial-dependent component appeared to account for 20% of the vasodilatory response to

CGRP in this model.

Stimulation of a glibenclamide-sensitive current was obtained by using ISO (Miyoshi & Nakaya,

1993; Shi, et al., 2007; Wellman, et al., 1998). Besides, ISO was shown to evoke hyperpolarization of canine saphenous vein (Nakashima & Vanhoutte, 1995) or rat mesenteric arteries (Goto, et al.,

2000). This was blunted by glibenclamide, suggesting that -AR stimulation can activate KATP channel, resulting in SMCs hyperpolarization. In rat mesenteric artery, hyperpolarization induced by ISO was more sensitive to 1- than 2-AR antagonism (Fujii, et al., 1999). The response was also blunted in 24-month-old animals, but this was suggested to be due to altered AC capacity and coupling to -AR rather than to a decrease in KATP channel density (Fujii, et al., 1999). Regarding vasodilatory responses, glibenclamide moderately right-shifted concentration-response curves to

-AR agonists in pressurized rat mesenteric artery (Randall & McCulloch, 1995) but had no effect in other studies (Garland, et al., 2011; Hong, et al., 1996; Huang & Kwok, 1997; Satake, et al.,

1996b). Nevertheless, KATP channel contribution may be revealed under specific metabolic stress condition, as potentiation of ISO vasorelaxation by hypoxia was glibenclamide-sensitive in isolated porcine coronary artery (Fukuda, et al., 1999).

Adenosine is an autacoid mediator liberated upon metabolic imbalance, with potent vasodilating properties, especially in coronary circulation (reviewed in (Headrick, et al., 2013; Mustafa, et al.,

2009)). Adenosine can interact with four receptor types namely A1, A2A, A2B, and A3 receptors

(AxR). A1R and A3R are mainly coupled to Gi, while A2AR and A2BR are coupled to Gs and cAMP production. A1R, A2AR, A2BR are expressed in coronary SMCs and ECs and some reports mention pharmacological evidence of A3R expression. Both A2AR and A2BR participate in mediating vasodilation of coronary bed in various species, and both endothelium-dependent and - independent actions were reported (Headrick, et al., 2013; Mustafa, et al., 2009; Teng, et al., 2016).

Whether A1R and A3R rather mediate coronary vasoconstriction or vasodilation is unclear

(Merkel, et al., 1992; Mustafa, et al., 2009; Son, et al., 2005). Still; both A1R and A3R may be less prominent than A2R in mediating the vasoactive response to adenosine in vivo (Teng, et al., 2016).

Adenosine activates a glibenclamide-sensitive current in freshly isolated rabbit mesenteric

(Kleppisch & Nelson, 1995a) and basilar (Kleppisch & Nelson, 1995b) arteries, and in porcine and cat coronary myocytes (Dart & Standen, 1993; Xu & Lee, 1994). Response to 5 µM adenosine in rabbit mesenteric artery was shown to be sensitive to PKA inhibition (Rp-cAMPS, PKI[5-24]amide,

H-89) and was mimicked by cAMP analogues and a A2AR agonist (CGS-21680, 200-500 nM), but not by a A1R agonist (CCPA, 100-250 nM). In anesthetized dog, vasodilatory response to non- selective agonist NECA (5′-(N-ethylcarboxamido)adenosine) in the presence of A1R antagonist

(DPCPX) was inhibited by glibenclamide, suggesting a A2R-mediated action of adenosine on KATP channels. By comparing the effects of several adenosine analogues with various selectivity for

A2AR and A2BR, it was suggested that glibenclamide-sensitive hyperpolarization of guinea pig coronary artery was mediated by A2BR (Mutafova-Yambolieva & Keef, 1997).

By contrast, adenosine effect on KATP channel activity in porcine coronary artery has the feature of an A1R-mediated response, as CCPA (5 µM) mimicked the effect of adenosine, but CGS 21680

(50-100 µM) had no effect. Besides, activation of the glibenclamide-sensitive current by adenosine in coronary myocytes was observed in the presence of CGS 15943A (100 nM), an antagonist more potent against the A2R subtype, but not in presence of the A1R antagonist DPCPX (1 µM), indicating a role for A1R, but not A2R. This was somewhat supported by a study in perfused rabbit heart

(Nakhostine & Lamontagne, 1993) and by the study by Merkel et al. in isolated porcine coronary

35 artery rings (Merkel, et al., 1992). In the latter work, relaxation to a rather A1R-selective agonist

(N6-cyclopentyladenosine, CPA), but not to an A2R-selective agonist (DPMA), was right-shifted by

0.3 µM glyburide, a KATP inhibitor. However, relaxation to the A1R agonist was not modified by an

A1R antagonist (DPCPX, 20 nM), making it difficult to draw definitive conclusion on these data.

The actual relevance of this paradoxical vasodilatory role of A1R-mediated activation of KATP in

VSMCs, in regard to the rather vasoconstricting influence of A1R, remains unclear.

As stated above, vasodilatory effects of adenosine were also endothelium-dependent in some studies, and were associated with an increase in NO synthesis in a cAMP-independent manner

(Hein & Kuo, 1999; Kuo & Chancellor, 1995). Some data were compatible with a role for KATP channels in the endothelium (Kuo & Chancellor, 1995) that could present substantial contribution to vasodilation at small adenosine concentrations (<0.1 µM). As already mentioned, endothelial

KATP activity may facilitate NOS activation by increasing driving force for Ca2+ influx. This notion was recently robustly supported by Aziz et al., whose study on endothelial-specific Kir6.1-/- arterial rings provided evidence that endothelial KATP channels are also activated by NECA, and participate in coronary vasodilation (30%) (Aziz, et al., 2017) in response to this adenosine receptor agonist.

In addition, KATP activity appeared to initiate endothelium-dependent, longitudinally spreading vasodilation following focal application of ISO in rat mesenteric artery (Garland, et al., 2011), and this may be important for local regulation of blood flow.

Activation of glibenclamide-sensitive currents and vasorelaxation induced by additional signalling molecules coupled to cAMP pathway (vasoactive intestinal polypeptide, VIP, PGI2 …) are also presented in Table 2.

In addition to GPCR stimulation (adenosine, ISO), cAMP enhancers can evoke glibenclamide- sensitive hyperpolarization (Mutafova-Yambolieva & Keef, 1997; Nakashima & Vanhoutte, 1995) and K+ current (Quayle, et al., 1994; Wellman, et al., 1998) or 86Rb+ efflux (Kessler, et al., 1997) in

VSMCs. Intracellular dialysis with PKAc also activated the current (Hayabuchi, et al., 2001a;

Quayle, et al., 1994; Wellman, et al., 1998). PKA inhibition with Rp-cAMPS diminished KATP current

36 evoked by pinacidil, suggesting that activation of the channel by PKA occurs also in the absence of cAMP stimulation (Hayabuchi, et al., 2001a). This action was blunted by dialysis with the AKAP-

PKA interaction disruptor peptide, Ht-31, suggesting that regulation of the KATP channel by PKA is integrated within a complex stabilized by an AKAP (Hayabuchi, et al., 2001a). This signalling platform may include other kinases or phosphatases, as PKC and phosphatase 2B (calcineurin) were shown to inhibit vascular KATP channels (Cole & Clement-Chomienne, 2003) via mechanisms possibly involving interaction with PKA (Hayabuchi, et al., 2001b; Orie, et al., 2009). Besides, both

CNs and PKC were shown to be targeted to sarcolemmal domains by AKAP proteins in VSMCs, participating in regulation of other ion channels (e.g. CaV1.2) (Navedo & Santana, 2013). Possible participation of KATP channels to such a macromolecular complexes remains to be further characterized.

Regarding vascular tone regulation, while GPCR activation often produces glibenclamide- sensitive vasodilation (Table 2), the response to direct cAMP enhancers (FSK, db-cAMP, Sp-5,6-

DCI-cBIMPS) is sometimes (Schubert, et al., 1997), but not always, sensitive to glibenclamide

(Jackson, 1993; Omar, et al., 2000; Randall & McCulloch, 1995). Also, agonist-evoked hyperpolarization does not necessarily translate into vasodilation (e.g. in rat mesenteric artery,

(Garland, et al., 2011)) probably because of the occurrence of additional vasodilating mechanisms.

Alternatively, cAMP-independent mechanisms may also mediate GPCR-activation of KATP, as it was suggested in another system (Suga, et al., 2000). Because KATP activity is enhanced by moderate hypoxia and inhibited by vasoconstrictors promoting PKC activation (Quayle, et al., 1997), it cannot be ruled out that apparent contribution of these channels to tone regulation is highly sensitive to experimental settings (such as P(O2)) and basal or stimulated kinase activity. It was also reported that relaxation to ISO was glibenclamide-sensitive in a 2-kidney-one clip hypertension model in rat, while it was not in control animals. This suggests that KATP contribution may be enhanced under pathological conditions (Callera, et al., 2004).

Although compelling data support the notion that KATP channel is activated by PKA, actual molecular characterization of this regulation has been controversial. Phosphorylation sites were identified in both Kir6.2 and SUR1 subunits which form the pancreatic KATP channel (Beguin, et al.,

1999; Lin, et al., 2000). Likewise, ser-385 in Kir6.1, or thr-633 and ser-1465 in SUR2B were proposed as the phosphorylation targets responsible for the increase of the vascular-like KATP channel activity by PKA in HEK-293 cells (Quinn, et al., 2004; Tanaka, et al., 2007). These data, however, could not be confirmed by another group, which instead demonstrated that ser-1387 in

SUR2 is necessary for ISO or FSK to stimulate channel activity and that this residue was phosphorylated by PKA in vitro (Shi, et al., 2007). This critical residue is located in the second nucleotide binding domain (NBD2) of the SUR2B protein and was shown to interact with other neighbouring residues at the interface with transmembrane domain 1 of SUR2 and facilitate channel activation (Shi, et al., 2008).

Besides PKA, exchange protein activated by cAMP (EPAC) may also mediate some of the cAMP effects on KATP channels function (reviewed in (Lezoualc'h, et al., 2016)). Cyclic AMP can bind a high affinity domain in EPAC1 or EPAC2 proteins and this activates downstream Rap GTPase signalling cascade. Growing literature on EPAC contribution in the cardiovascular system highlighted its role in several functions, including regulation of cardiomyocyte hypertrophy, intracellular Ca2+ handling, excitation-transcription coupling and vascular tone. Of the two EPAC isoforms, only EPAC1 seems clearly to be expressed in vasculature, in both SMCs and ECs (Davies, et al., 2010; Roberts, et al., 2013). Stimulation of EPAC using the EPAC-selective cAMP analogue 8- pCPT-2’-O-Me-cAMP (5 µM) was shown to inhibit pinacidil-evoked KATP current in rat aortic SMCs

(Purves, et al., 2009). In the presence of PKA inhibitors (KT-5720, 1 µM or Rp-cAMPS, 100 µM),

FSK induced similar effect. EPAC agonist also induced transient raise in [Ca2+]i, and its action on

KATP current was proposed to be mediated by the previously described calcineurin-mediated inhibitory pathway (Cole & Clement-Chomienne, 2003). This action, however, is not consistent with a hyperpolarizing/vasodilating action for EPAC via KATP regulation. The authors speculate that this may serve as a negative feedback on KATP channel activity at high cAMP levels, because

EPAC activation by the nucleotide occurs at higher concentrations than PKA activation (EC50 are

30 µM and 1 µM, respectively). Nevertheless, other actions via Rho kinase inhibition or activation of other K+ channels, BKCa channels in particular, likely mediate EPAC-mediated vasorelaxant effects of cAMP (see following sections).

Being an endpoint effector of cAMP pathway, some studies examined the subcellular localization of sarcolemmal KATP channels with respect to other key signalling proteins. By immunoprecipitating the Kir6.1 subunit, Dart and colleagues found that KATP channel can be pulled down with AC isoforms and PKA RII subunits in cultured rat aortic SMCs (Sampson, et al., 2004).

The former were located in caveolae, cholesterol-enriched, caveolin-coated domain compartments which serve as sarcolemmal signalling platforms by gathering receptors and associated signalling molecules (Hardin & Vallejo, 2006; Razani, et al., 2002; Sampson, et al.,

2004). Interestingly, integrity of these caveolae was necessary for proper tonic activation of the

KATP current by PKA in rat mesenteric artery myocytes, suggesting that proximity of the channel with signalling partners is mandatory to ensure adequate coupling. Likewise, the same group demonstrated that immunoprecipitated EPAC1 protein was found in a complex with the SUR2B subunit of the KATP channel (Purves, et al., 2009). The actual organization of a possible scaffolding structure involving both PKA and EPAC that would fine-tune KATP channel activity remains to be clarified.

5.2.3 Regulation of KATP channels by cGMP pathways

Vascular KATP channel regulation by the cGMP-PKG pathway is controversial (Table 2). Evidence exists for a stimulatory effect of NO donors on the vascular KATP channel in cultured vascular myocytes (Kubo, et al., 1994; Miyoshi, et al., 1994) and whole arteries (Armstead, 1996; Hein, et al., 2006; Murphy & Brayden, 1995; Wu, et al., 2004). For instance, a NO donor, 3- morpholinosydnonimine (SIN-1, 3 µM), induced a hyperpolarization in rabbit mesenteric artery that was inhibited by glibenclamide, supporting a stimulatory action on the KATP current (Murphy

& Brayden, 1995). SIN-1 effect was observed only if tonic hyperpolarizing influence of endothelial

NO was suppressed by endothelium-denudation or NOS inhibition. In keeping with a role for cGMP in these responses, the effects of NO donors were potentiated by zaprinast, a non-selective cGMP-

PDE inhibitor, in some studies (Hein, et al., 2006; Murphy & Brayden, 1995), and 8-Br-cGMP produced similar effects on KATP channel activity and tone (Armstead, 1996; Kubo, et al., 1994).

ANP was also reported to activate a glibenclamide-sensitive current, suggesting that activating both soluble and particulate guanylate cyclases can lead to stimulation of KATP channels in VSMCs.

Some reports accounted for a prominent KATP activation by NOS activity after stimulation with bacterial lipopolysaccharide, suggesting that inducible-NOS activation may stimulate KATP channels (Miyoshi, et al., 1994; Wu, et al., 2004). In agreement with these data, NO signalling on

KATP and on other channels appears to mediate vascular hyporeactivity to vasoconstrictors in a rat model of endotoxic shock (Chen, et al., 1999; Wu, et al., 2004).

Yet, a number of other studies reported the absence of KATP channels activation by NO-cGMP pathway in different vascular territories (reported in Table 2). For instance, despite being able to hyperpolarize Em in arterial rings in a glibenclamide-sensitive manner (Murphy & Brayden, 1995),

SIN-1 had no effect on the KATP current in myocytes isolated from the same vessels (Quayle, et al.,

1994). Besides the contribution of KATP channels to the vasorelaxant effect of NO donors appears to be limited, regardless of the vascular bed studied (Table 2). These differences may be due to experimental conditions, as patch-clamp experiments do not necessarily recapitulate the situation of a whole organ or in vivo. Therefore, the exact influence and contribution of a cGMP-PKG-KATP channel pathway on VSMC electrical and mechanical activities remain uncertain and may vary depending on the preparation used (isolated VSMC) versus whole artery or in vivo models, and on the concentration and/or selectivity of the pharmacological agents used. Interestingly, the mechanism for activation of the cardiac KATP channel (Kir6.2/SUR2A) by NO stimulation may involve reactive oxygen species, ERK1/2 and calmodulin-dependent protein kinase II (Zhang, et al., 2014a). Whether this pathway exists in VSMCs remains to be shown.

5.3 KV channels

The denomination “KV” refers to K+ channels formed by an assembly of 4 pore-forming subunits, each having 6 membrane-spanning domains (Alexander, et al., 2017b). Classification of KV channels subunits comprehends 12 families, each including several isoforms coded by specific genes (Alexander, et al., 2017b). In VSMCs, KV1.x (products of KCNAx genes), KV2.x (KCNBx), and

KV7.x (KCNQx) are the most well represented families (Jackson, 2018). Channels activated by intracellular Ca2+ will be the object of section 5.4. Extensive information on physiology and pharmacology of KV channels can be found in classic (Coetzee, et al., 1999; Nelson & Quayle, 1995) and recent (Jackson, 2018) reviews. KV channels are essentially voltage-dependent, meaning that depolarization of cell Em promotes channel opening.

In VSMCs, it is classically considered that concentrations up to 1 mM of 4-amino pyridine (4-AP) inhibit rather selectively KV channels relative to KCa and Kir channels (Nelson & Quayle, 1995).

Also, sensitivity of KV channels to tetraethylammonium ion (TEA) occurs usually at higher concentrations (> 5mM) than for KCa (Coetzee, et al., 1999; Nelson & Quayle, 1995). Still, the relative affinity of 4-AP varies among various KV members (Coetzee, et al., 1999), and some channels, such as the KV2 and KV7 family, show relative resistance to this blocker (Coetzee, et al.,

1999; Jackson, 2018). More selective pharmacological modulators are now available to study more precisely the function of the KV channels (Jackson, 2018). In vascular SMCs, KV-like currents exhibit a variety of biophysical and pharmacological properties, reflecting the high diversity in molecular expression of the KV subunits in the cell, stoichiometry of KV subunits within a tetramer

(homo- or hetero-tetramer) and their association with ancillary, regulating proteins (e.g. KCNE for KV7 channels). KV channels have been involved in the regulation of resting Em of SMCs in some vascular beds and in the negative feedback upon depolarization of Em (Jackson, 2018; Joshi, et al.,

2009). Some of them were shown to be involved as functional endpoints of CN signalling and to mediate associated vasorelaxant effects (Table 3).

5.3.1 KV1 channels

Many studies reported a stimulatory effect of cAMP and PKA on KV current in VSMCs, mainly based on sensitivity to 4-AP (1-5 mM) (Aiello, et al., 1998; Aiello, et al., 1995) (for review see (Jackson,

2018)). Likewise, some vasodilatory responses are also sensitive to 4-AP at similar or even smaller concentrations (< 1 mM) (Berwick, et al., 2010; Dong, et al., 1998; Heaps & Bowles, 2002;

Li, et al., 2003; Satake, et al., 1996b), likely indicating a participation of KV1 channels. Other drugs described as more selective blockers of some KV1 channels (correolide, dendrotoxin, Psora4)

(Jackson, 2018) were able to blunt some responses to Gs-coupled vasodilators (Table 3).

Accordingly, the KV1.2 channel was shown to be a substrate of PKA at ser-449 (rabbit) (Johnson, et al., 2009). In rat cerebral arteries, KV1.2 phosphorylation by PKA requires the binding to postsynaptic density-95 (PSD95)-AKAP scaffolding protein (Moore, et al., 2015; Moore, et al.,

2014). Selectively targeting this association using a disrupting peptide induced KV1 inhibition, cell

Em depolarization and vasoconstriction. Possible interaction between the 1-AR and PSD95 may favour stimulation of KV1 current by ISO (Moore, et al., 2015). Other indirect actions of PKA or

PKG, mediated by RhoA inhibition, have also been suggested (Luykenaar & Welsh, 2007).

5.3.2 KV7 channels

In the vasculature, KV7 channels are mainly formed by KV7.1, KV7.4 and KV7.5 subunits, with KV7.4 and KV7.5 probably building active heteromers (Chadha, et al., 2014). During the last decade, the use of relatively selective blockers (mainly linopirdine, LNP, and XE991) allowed to dissect the contribution of KV7 to global outward, voltage-dependent current and vasorelaxation (Chadha, et al., 2012a; Haick & Byron, 2016; Jepps, et al., 2011; Joshi, et al., 2009; Yeung & Greenwood, 2005).

The interesting therapeutic potential of these drugs to improve some vascular disorders such as hypertension or hypotension, cerebral vasospasm and coronary artery disease has been explored in preclinical models and is reviewed elsewhere (Haick & Byron, 2016).

There is now substantial evidence that cAMP “signals” to KV7 channels as terminal effectors in

VSMCs (Table 3). KV7 blockers inhibit the relaxant response to A2AR or -AR stimulation (Chadha, et al., 2012b; Khanamiri, et al., 2013), cAMP analogues (Khanamiri, et al., 2013) and FSK (Chadha,

42 et al., 2012b). In the presence of blockers of KV1 (correolide), KV2 (stromatoxin-1) or KCa (penitrem

A) channels, ISO (1 µM) slightly, but significantly increased the outward current in renal artery myocytes, and this was prevented in the presence of LNP. Partial silencing (by 40-60%) of the

Kcnq4 gene expression in intact arteries (Chadha, et al., 2014; Chadha, et al., 2012b) led to a much less potent vasorelaxant response to ISO or CGRP, thus emphasizing the role of the KV7.4 pore- forming subunit in these responses. Expression of this protein was found to be decreased in renal and coronary arteries from spontaneously hypertensive rats, but not in cerebral arteries (Chadha, et al., 2014; Chadha, et al., 2012b; Khanamiri, et al., 2013). This was associated with a dramatic reduction in the vasorelaxant response to cAMP stimulation in renal and coronary arteries, but not in cerebral arteries, pointing to a strong link between depression of the cAMP-KV7.4 axis and loss of vasorelaxant response in this pathological context. In another study in rat left coronary artery, high glucose reduced the response to FSK, which was concomitant with a loss of KV7 contribution to tone regulation, suggesting that these channels may contribute to coronary vascular dysfunction occurring in diabetes (Morales-Cano, et al., 2015).

Interestingly, studies exploring how cAMP signal is conveyed to KV7 channels may conclude on either mediation by PKA (Khanamiri, et al., 2013) or EPAC (Stott, et al., 2016), depending on which vascular bed is studied. Stott et al. (Stott, et al., 2016) showed that activation of EPAC with 8-pCPT-

AM (5 µM) elicited a vasorelaxation that could be substantially inhibited by pretreatment with

LNP (1 & 10 µM) in both rat mesenteric and renal arteries. The selective KV7.1 selective blocker

HMR 1556 (10 µM) or endothelial denudation had no effect on the response to 8-pCPT-AM.

Moreover, 8-pCPT-AM (5 µM) enhanced LNP-sensitive outward current in isolated myocytes, as well as KV7.4 current heterologously expressed in HEK-293 cells. Thus KV7.4, and possibly KV7.5 channels, appear to be a target of EPAC activation in vascular SMCs, with a relevance in vascular tone regulation. Interestingly, the authors looked further at the respective contributions of EPAC and PKA pathways to the vasorelaxant response to -adrenergic stimulation. Surprisingly, the relaxant response to ISO in mesenteric artery was shifted to the right by the EPAC1/2 inhibitor

ESI-09 (used at 100 nM), whereas it was not altered by PKA inhibition with 1 µM KT-5720 or [14-

22]amide PKI. LNP effect on ISO response was lost in the presence of EPAC inhibition but not with

KT-5720. The relative contribution of the two EPAC isoforms remains unclear, since a selective inhibition of EPAC1 (with CE3F4, 1 µM) or EPAC2 (with HJC0350, 1 µM) had no effect when used separately, but significantly inhibited the ISO response when used in combination, suggesting a redundant role for the two proteins (Stott, et al., 2016). These results demonstrated the functional relevance of EPAC pathway targeting KV7 channels in a vasorelaxant response initiated by - adrenergic stimulation, a physiological stimulus, in mesenteric artery. By contrast, ISO response in renal artery was not modified by EPAC inhibition whereas it was clearly reduced by KT-5720 and PKI and a disruptor of PKA anchoring, the Ht31 peptide, pointing to a mechanism relying on

PKA rather than EPAC in this vessels (Stott, et al., 2016). This functional dichotomy between both vascular beds was further supported using proximity ligation assay which showed a physical proximity between KV7.4 channels and Rap1 or Rap2 proteins (downstream partners of EPAC) in mesenteric arteries, and between KV7.4 channels and AKAP (a downstream partner of PKA) in renal arteries (Stott, et al., 2016). Also, KV7.4 localization close to Rap1 and Rap2 was increased by ISO in mesenteric artery myocytes, while it was unchanged in renal artery myocytes.

Conversely, ISO decreased the proximity of KV7.4 with AKAP-150 in mesenteric arteries, but increased it in renal VSMCs. This study provides a clear example on how an apparently equivalent response to a stimulus in two vascular beds can actually be underlain by separate mechanisms, namely EPAC and PKA signalling in mesenteric and renal artery, respectively.

The exact molecular determinants of stimulation of KV7 channels by either EPAC or PKA in vasculature remain unclear. PKA regulation of KV7.1 has been shown to involve interaction with the Yotiao protein (AKAP9) which binds to the channel and is key for phosphorylation at ser-27 and enhanced channel activity (Marx, et al., 2002). However, while KV7.1 is mainly expressed in cardiomyocytes where it co-assembles with KCNE1 to give rise to the IKs current, it is probably not the major KV7 isoform that is relevant for tone regulation (Chadha, et al., 2014; Chadha, et al.,

2012a; Khanamiri, et al., 2013; Tsvetkov, et al., 2017). By studying native or exogenously expressed KV7 channels, Mani et al. (Mani, et al., 2016) reported that endogenous KV7.5 in A7r5

44 cells are highly responsive to cAMP stimulation while KV7.5/7.4 channels exhibited poor response, and KV7.4 were non responding at all. This suggests that the relatively modest increase in current intensity that was described in native VSMCs (Chadha, et al., 2014) may reflect stimulation of channels that include the KV7.4 and KV7.5 subunit.

Activation of -AR or CGRP-R have also been reported to stimulate KV7 channels through G proteins in some vascular beds (Stott, et al., 2018).

It was shown recently that microtubular disrupting agents, namely colchicine or nocodazole, potentiate the vasorelaxant response to ISO in isolated rat renal and mesenteric arteries

(Lindman, et al., 2017). Indeed, in mesenteric arteries depolarized/contracted with methoxamine, colchicine potentiated Em hyperpolarization and relaxation evoked by a small concentration of ISO

(30 nM), and this potentiation was abolished in the presence of the KV7 inhibitor XE991 (3 µM).

Conversely, colchicine potentiated relaxation produced by KV7 activators and increased localization of the KV7.4 subunit at the cell membrane, indicating that microtubule disruption may also increase availability and vasorelaxant contribution of KV7 channels. By contrast, the inhibiting effect of the BKCa blocker IBTX on -adrenergic vasorelaxation (see following section) was abolished by colchicine, suggesting that contribution of BKCa channels to the ISO response becomes redundant. Further work is needed to decipher the molecular mechanisms involved in the regulation of both KV7 and BKCa channels by cytoskeleton.

Cyclic GMP signalling also appears to target KV7 channels, as vasorelaxant responses of isolated rat aorta to ANP, CNP and SNP were all blunted by LNP (10 µM) (Stott, et al., 2015), (Table 3). The

KV7.1 channel blocker HMR 1556 did not mimic this effect, again supporting a prevalent role for

KV7.4 and/or KV7.5 subunits. Similarly, based on pharmacological evidence, activation of KV7 channels by NO donors was recently demonstrated to mediate part of their relaxant action in rat

PA (Mondejar-Parreno, et al., 2019).

Since cAMP- or cGMP-elevating agents exert a relatively modest effect on the whole-cell KV7 current at physiological Em in isolated VSMC, it is not clear why LNP or XE991 exert such strong inhibition on the relaxant responses of isolated vessels to these CNs (Chadha, et al., 2012b;

Khanamiri, et al., 2013; Mondejar-Parreno, et al., 2019). One possible explanation is that VSMCs usually display a large input resistance, so that a small effect on current may translate into a substantial repolarization, sufficient to hamper voltage-gated Ca2+ channel activation (Nelson, et al., 1995). One may thus assume that KV7 channels are tightly coupled to Em-controlled contractile mechanisms, making this channel family an important effector of vasodilatory stimuli.

5.4 BKCa channels

5.4.1 General properties of BKCa channels

Ca2+-activated, K+ channels are functionally important channels in blood vessels. KCa1.1 ( subunit, product of the KCNMA1 gene), often referred to as large conductance, Ca2+-activated, K+

(BKCa) channel, is abundant in smooth muscle (Dopico, et al., 2018; Latorre, et al., 2017).

Comprehensive description of channel structure, biophysics, gating models and regulation has been the object of a number of recent reviews (Dopico, et al., 2018; Latorre, et al., 2017). Other members of the KCa channels family, namely small (KCa2.x, KCNN1-3, SKCa) and intermediate

(KCa3.x, KCNN4, IKCa) channels, are mostly characterized in the endothelium, where they participate in hyperpolarizing signals (Alexander, et al., 2017b; Bellien, et al., 2011).

Among other K+ channels expressed in vascular smooth muscle, BKCa channels are important contributors to the vasorelaxing mechanisms: their activity repolarizes Em, which opposes contraction. BKCa channels activity in SMCs has been abundantly documented. They display large unitary conductance, ranging from 60-100 pS in physiological [K+] to values around 200 pS or above in high, symmetrical [K+] (e.g. (Benham, et al., 1986; Latorre, et al., 2017; Pallotta, et al.,

1981)). This high conductance, coupled with high channel density in SMCs, makes it possible to generate polarizing current even if the open channel probability Po is low (Nelson, et al., 1995;

Nelson & Quayle, 1995). Exploration of BKCa function has been facilitated by the use of relatively selective inhibitors such as TEA at small concentration (apparent Kd  0.2 µM) (Brayden & Nelson,

1992), paxilline (Zhou & Lingle, 2014) and scorpion toxins charybdotoxin (Miller, et al., 1985) and highly selective iberiotoxin (IBTX) (Galvez, et al., 1990).

The channel () subunit shares general similarities with the voltage-gated K+ (KV) channel family, displaying six hydrophobic  helixes (S1 to S6) and assembling in tetramers. However, the presence of a supplementary S0 membrane spanning domain makes the N-terminal domain extracellular. The C-terminal forms a massive cytosolic region, containing Ca2+ sensors (Latorre, et al., 2017). Gating of the channel is favoured by membrane depolarization or binding of intracellular Ca2+ with a relatively low (micromolar) affinity. Open probability of the channel is very low at resting [Ca2+]i (10-7 M), while exposure to micromolar [Ca2+]i concentrations dramatically increases channel activity (Latorre, et al., 2017; Tanaka, et al., 1997). Such high [Ca2+]i can be attained in response to pharmacological agents such as caffeine, or, more physiologically, by the occurrence of sporadic, transient, SR release of Ca2+ via the RyR, i.e. Ca2+ sparks (Bolton,

2006; Jaggar, et al., 2000; Nelson, et al., 1995). The latter is facilitated by the formation of clusters of BKCa channels located close to the SR/ER which can be stimulated by Ca2+ sparks, locally raising

[Ca2+] above 10 µM. Activated BKCa channels coupled to Ca2+ sparks generate a macroscopic current, identified as spontaneous transient outward current (STOC) (Bolton, 2006; Bolton &

Imaizumi, 1996; Lifshitz, et al., 2011). BKCa activation can also occur via sarcolemmal Ca2+ influx through LTCCs (Guia, et al., 1999; Liu, et al., 2004) or other cationic channels (Kwan, et al., 2009).

In smooth muscle, most  subunits assemble with an auxiliary 1 subunit (KCNMB1) (Knaus, et al., 1994), which confers a higher sensitivity to internal Ca2+ (Tanaka, et al., 1997) and plays a key role in vasomotricity regulation (Brenner, et al., 2000; Pluger, et al., 2000; Tanaka, et al., 1997).

An elegant study by Jaggar’s group (Leo, et al., 2014) demonstrated that anterograde trafficking of 1 to the membrane is dynamically regulated by both cGMP and cAMP stimuli (Leo, et al., 2014).

This occurs by rapid increase in the transport of 1 subunits in Rab11A-positve recycling

47 endosome to the sarcolemma, independently from the -BKCa subunit. It was shown that this process participates in vasodilating action of endothelial NO or SNP by increasing Ca2+ sensitivity of the channel.

It is generally accepted that Ca2+-activated K+ channel activity acts as a negative feedback (or

“brake”) against Ca2+ increase, by “pushing” the Em toward resting levels and restoring relaxed state (Brayden & Nelson, 1992; Wu & Marx, 2010). Accordingly, pharmacological inhibitors of

BKCa produce depolarization and steady vasoconstriction when applied on isolated vessels submitted to increased parietal tension (Brayden & Nelson, 1992; Brenner, et al., 2000; Cabell, et al., 1994; Taniguchi, et al., 1993). Tibial arteries from Kcnma1-/- mice display globally a higher myogenic tone, but surprisingly an unaltered contractile response to rapid changes of pressure steps (Sausbier, et al., 2005). Vascular myocytes from Kcnma1-/- mice display depolarized resting

Em (Sausbier, et al., 2005), but the contribution of the channel to resting tone and Em appeared, however, limited in other studies (Taniguchi, et al., 1993). Hyperaldosteronism is observed in models with genetic disruption of BKCa channel  or  subunits, contributing to higher blood pressure (Grimm, et al., 2009; Sausbier, et al., 2005).

BKCa channels have been shown to be involved in the vasorelaxant responses to a variety a hormones, autacoïds and signalling peptides, most of them involving CN signalling. This is highlighted by a myriad of studies demonstrating sensitivity of vasodilatory mechanism to specific

BKCa inhibitors (see Table 4 and (Kotlikoff & Kamm, 1996; Nelson & Quayle, 1995) for classic reviews).

Vasodilatory signalling involving BKCa channels as an endpoint effector may directly involve biochemical modification of channel subunit (e.g. phosphorylation by PKA, PKG). However, other pathways that modify cellular Ca2+ homeostasis are also involved in BKCa channel regulation. The influence of cAMP and cGMP signalling on BKCa channel activity was documented in electrophysiological studies, by recording single channel activity, steady state whole-cell current or calcium-mediated STOCs.

By stimulating vascular smooth-muscle relaxation, BKCa channel openers provide an interesting therapeutic potential for the treatment of a number of disorders, such as hypertension and stroke

(Kaczorowski & Garcia, 2016). However, small molecule candidates have failed until now to reach clinical application because of limited potency and selectivity. Since BKCa channels are activated by CN, acting on CN signalling pathways may provide an alternative avenue to increase BKCa channel functional benefits.

5.4.2 Regulation of BKCa channels by cAMP pathways

The activation of BKCa channels by cAMP signalling has been extensively studied. The channel is activated by agonists of Gs-coupled receptors (see Table 4), cAMP analogues (Sadoshima, et al.,

1988; Schubert, et al., 1996; Song & Simard, 1995) or direct cyclase activation using FSK (Minami, et al., 1993; Sadoshima, et al., 1988; Song & Simard, 1995) in VSMCs, as it is in other SMC types

(e.g.(Kotlikoff & Kamm, 1996)). The effect of cAMP on BKCa current is potentiated by intracellular

Ca2+ (Murphy, et al., 2003). Stimulation of the cAMP pathway induces a hyperpolarization of SMCs

Em which is partially inhibited by BKCa blockers (Von der Weid & Van Helden, 1996).

Pharmacological inhibition of BKCa also antagonizes the response to GPCR agonists (Table 4), cAMP mimetics (Armstead, 1997a; Barman, 1997; Paterno, et al., 1996; Taguchi, et al., 1995;

Tanaka, et al., 1999), or FSK (Taguchi, et al., 1995), which shows that the cAMP-BKCa channel axis is an important contributing mechanism to vasodilatory response evoked by the cAMP pathway.

This was not observed, however, in some reports (Khan, et al., 1993).

PKA appears to convey the stimulation of BKCa channel activity by cAMP, since exposure of the channel to PKA(c) in the presence of ATP increases channel open probability (Minami, et al., 1993;

Sadoshima, et al., 1988; Schubert, et al., 1996) and this involves a tight interaction between the kinase and the channel (Dai, et al., 2009). Conversely, PKA inhibition (Rp-8-CPT-cAMPs) was shown to abolish the effect of a cAMP analogue on the current (Schubert, et al., 1996). Basal application of PKA inhibitors (Rp-CPT-cAMPS or H-89) to pressurized small tail arteries produce a dose-dependent increase in myogenic tone, indicating that tonic activity of PKA is sufficient to

49 activate the channel in the absence of exogenous stimulation (Schubert, et al., 1999). This effect was mimicked by, and not additive to, IBTX, pointing to a specific contribution of BKCa channels in this effect.

In rat aorta rings, IBTX-sensitive component of ISO response was attributed to the stimulation of

2-AR, but not -AR, and was sensitive to PKA inhibition by KT-5720 (0.5 µM) (Satake, et al.,

1996b). Liu et al. (Liu, et al., 2004) demonstrated that BKCa channels immunoprecipitated with the

β2-AR in rat aorta and human primary cultured VSMCs. Association of the β2-AR with additional partners such as AKAP79/150 (AKAP5) and the CaV1.2 channel may provide a scaffold to integrate efficient adrenergic/PKA signalling toward activation of BKCa channel by phosphorylation and

Ca2+ (Dai, et al., 2009; Liu, et al., 2004).

Phosphorylation of the -BKCa channel subunit by PKA, PKG and also PKC in the C-terminus has been well characterized (reviewed in (Kyle & Braun, 2014). There are functional and biochemical evidence that PKA targets a conserved, weak consensus PKA phosphorylation site at ser-869

(human) at the C-terminal part of the channel (Nara, et al., 1998; Tian, et al., 2004; Tian, et al.,

2001). This site was demonstrated to mediate the responses to 2-AR activation and to FSK in reconstituted channels expressed in oocytes (Nara, et al., 1998). Further investigations demonstrated that regulation of various channel variants by either PKA or PKG actually obeys to interesting, although complex, modalities. The STREX variant of -BKCa, which exhibits a 59-amino acid exon inserted in the tail region (Saito, et al., 1997; Xie & McCobb, 1998), was actually shown to be inhibited by PKA phosphorylation within the STREX insert (Tian, et al., 2001), as opposed to the “ZERO” variant, which lacks the insert. Tian et al. (Tian, et al., 2004) further demonstrated that the presence of a single STREX subunit in the BKCa tetramer is sufficient to confer this PKA- inhibited property to the channel. Conversely, all four “ZERO” -subunits need to be phosphorylated by PKA at ser-899 to ensure proper stimulation by PKA (Tian, et al., 2004).

Complex crosstalk between PKA/PKG phosphorylation and PKC phosphorylation adds further complexity which was unravelled by a series of studies by Zhou et al. (Zhou, et al., 2012; Zhou, et

50 al., 2001; Zhou, et al., 2010). PKC phosphorylates the channel at key PKC consensus sites, namely ser-1151 and ser-695, and this inhibits the “ZERO“ channel (Zhou, et al., 2010). These modifications are necessary for cGMP-PKG to potentiate channel activation, whereas PKAc is ineffective. Conversely, invalidating the PKC sites, or even inhibition of PKC, renders the channel insensitive to PKG and turns it to a PKA-sensitive form. The presence of the STREX insert promotes palmitoylation of conserved cysteine residues that associate the C-terminal with the membrane.

This abolishes above-mentioned inhibition of the channel by PKC. PKA suppresses this membrane anchoring and makes the channel strongly PKC-inhibitable (Tian, et al., 2008; Zhou, et al., 2012).

Interestingly, expression studies reported that the STREX variant is expressed in vasculature

(Nourian, et al., 2014), however relevance of these important regulatory systems in the VSMCs remain unclear (Nystoriak, et al., 2017).

EETs constitute an important component of endothelium-derived hyperpolarising factors and, as such, evoke vasodilation by modulating ion channels, including activation of BKCa channels

(Bellien, et al., 2011). Proposed mechanism involves Gs protein activation by EETS via stimulation of a putative receptor, or via direct ADP-ribosylation (Li, et al., 1999). Contribution of cAMP-PKA axis to this pathway was demonstrated in small renal arteries (Carroll, et al., 2006) but was not verified in HEK-293 cells expressing the -BKCa channel.

The possibility of a direct activation of the channel by cAMP has also been raised in some reports

(Minami, et al., 1993), but not verified by others (Schubert, et al., 1996). On the other hand, a cAMP-independent action of Gs-coupled receptor agonists such as ISO in SMCs was also suggested

(Kume, et al., 1994; Scornik, et al., 1993).

Because BKCa channel opening is dependent on intracellular calcium, modification of Ca2+ handling by CNs can indirectly regulate their activity. Stimulating cAMP synthesis with FSK increased Ca2+ sparks frequency by 2-3-fold in cerebral and coronary myocytes in rat or mice, and this was antagonized by PKA inhibitors (Porter, et al., 1998; Wellman, et al., 2001). Consistent with a coupling of BKCa with underlying Ca2+ sparks, FSK increased the frequency of STOCs to the same

51 extent. Other stimuli increased STOC activity, namely cAMP analogues, adenosine, and also NO donors (Bychkov, et al., 1998; Clapp & Gurney, 1991; Hayoz, et al., 2007; Porter, et al., 1998).

Vascular reactivity experiments in pressurized artery showed that suppression of Ca2+ sparks with a high concentration of ryanodine (10 µM) inhibited vasodilation evoked by FSK by 79%, suggesting that a “cAMP-PKA/Ca2+ sparks/STOC” axis accounts for a great proportion of the cAMP-evoked vasodilation in such conditions. This attractive paradigm was further explored by studies in mice lacking the phospholamban (PLB) protein, a negative regulator of SERCA pump and SR refilling, which is a classic target of PKA at ser-16. This phosphorylation relieves SERCA from inhibition by PLB and elevates SR Ca2+ load. Basal sparks and STOCs frequencies, as well as

SR Ca2+ load were actually higher in PLB-knockout myocytes and were not increased by FSK.

Sparks reserve did not appear to be exhausted in PLB-knockout myocytes since STOC activity could be increased by a small concentration of caffeine. Overall these data suggest that SERCA activation may be a key effector of cAMP-PKA for increasing Ca2+ sparks. Nevertheless, because sparks activity also generally depends on RyR activity, Ca2+ influx and Em (Jaggar, et al., 2000), the participation of other targets related to Ca2+ handling (RyR, LTCC) cannot be ruled out.

STOCs activity show heterogeneity among vascular beds, with myocytes from resistant arteries being more likely to display STOCs than conduit (e.g. aorta) arteries (Hayoz, et al., 2007). It was suggested that cAMP-PKA stimulation may trigger STOC activity in these so called “silent” myocytes (Hayoz, et al., 2007).

Several studies account for responses to -AR stimulation that was relatively resistant to inhibitors of PKA (e.g. (Matsumoto, et al., 2012)). As mentioned in a preceding section, the possibility of a cross activation of PKG by cAMP has been suggested in vascular smooth muscle

(Barman, et al., 2003; Eckly-Michel, et al., 1997; Jiang, et al., 1992). This may provide a plausible hypothesis for resolving PKA-independent effects of cAMP on BKCa channel. Indeed, data from

White et al. showed that the increase in BKCa channel opening elicited by various cAMP-elevating agents (used at 10 µM) could be inhibited by PKG inhibitors KT-5823 and Rp-8-pCPT-cGMPs, but not by the PKA inhibitor KT-5720. Interestingly, however, cGMP levels were not increased by

52 these stimulations, and activation by a cAMP analogue was inhibited by KT-5823. These data and others (Barman, et al., 2003; Natarajan, et al., 2010; Zhu, et al., 2002) support the notion that cAMP signal can activate BKCa channel and evoke vasorelaxation via phosphorylation by PKG, and not necessarily by PKA.

More recently, EPAC protein was also highlighted as a possible mediator of cAMP pathways able to modulate BKCa channel function. In rat mesenteric arteries, acetoxymethyl ester form of the

EPAC agonist 8-pCPT-2’-O-Me-cAMP (8-pCPT-AM, 5 µM) induced a vasorelaxation that was reduced when using high [K+] to contract the vessel, suggesting the involvement of K+ channels in this response (Roberts, et al., 2013). Moreover, it was shown that 100 nM IBTX decreased by half the vasorelaxation evoked by 8-pCPT-AM, pointing to a participation of BKCa channels. Similar results were obtained by another group using paxilline (1 µM) in rat mesenteric and renal arteries

(Stott, et al., 2016). In keeping with the paradigm of a coupling between RyR activity and BKCa presented above, 8-pCPT-AM also promoted Ca2+ sparks and associated STOC activity. CaMKII

(probably activated via PLC and IP3-mobilized Ca2+) appears to be a key mediator of this regulation promoted by the EPAC agonist (Humphries, et al., 2017). Indeed, EPAC pathway is known to activate CaMKII in other systems, including cardiomyocytes (Lezoualc'h, et al., 2016), and here 8- pCPT-AM induced phosphorylation of variants of CaMKII, more abundant than CaMKIIδ in smooth muscle. Mechanisms downstream of CaMKII activation remain unclear, but do not involve the extent of Ca2+ store content. Although the above-presented data convincingly demonstrate that activating EPAC using a permeant agonist promotes activation of the Ca2+-sparks-BKCa- vasodilation axis, it remains unclear whether this route is relevant in the context of cAMP stimulation by using a GPCR agonist or FSK. Current development of selective EPAC inhibitors or knockout animals (Lezoualc'h, et al., 2016) may offer methodological opportunities to address this issue. Furthermore, Roberts et al. observed that the vasorelaxant response to 8-pCPT-AM was partially endothelium-dependent, and inhibited by blockade of EDHF-driving SKCa channels (by using apamin and TRAM-34), or by the NO synthase inhibitor L-NAME. Consistent with a role of

EPAC in regulating endothelial function, this adds up more complexity to the understanding of the regulation of vascular tone by various cAMP pathways.

5.4.3 Regulation of BKCa channels by cGMP pathways

A number of studies demonstrated the stimulation of BKCa channel by NO donors, ANP, cGMP and associated PKG signalling (Table 4). Inhibition of sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-

1-one (ODQ) or methylene blue abolished channel activation by a NO donor (Li, et al., 1998; Peng, et al., 1996).

Membrane permeant cGMP analogues or internal cGMP tested at high concentration (0.4 µM – 2 mM) increase BKCa open state probability or macroscopic current (Archer, et al., 1994; Fujino, et al., 1991; Peng, et al., 1996; Robertson, et al., 1993; White, et al., 1995; Williams, et al., 1988). In isolated myocytes, ODQ decreased basal activity in the absence of any stimulation, suggesting that tonic activity of the enzyme is sufficient to stimulate the channel (Li, et al., 1998).

In various vascular reactivity models, manoeuvres that enhanced or mimicked cGMP evoked vasorelaxation which was often partially inhibited by BKCa inhibitors, such as IBTX, charybdotoxin

(CBTX, 50-100 nM) or small concentrations of TEA (<1 mM) (Bracamonte, et al., 1999; Carrier, et al., 1997; Jiang, et al., 1998; Paterno, et al., 1996; Price & Hellermann, 1997; Tanaka, et al., 1998;

Taniguchi, et al., 1993). In some studies, however, the effect of cGMP stimulators was also mediated by channels sensitive to 4-AP or glibenclamide rather than BKCa blockers (Table 4), suggesting that Em-dependent relaxant responses rely on several K+ channel families.

Consistent with PKG being the main effector of this pathway, kinase inhibition with drug of variable selectivity (KT-5823, Rp-cGMPs, H-8) or ablation of PKGI gene in mice blunted the effects of cGMP analogues or NO (Carrier, et al., 1997; Gerzanich, et al., 2003; Peng, et al., 1996; Sausbier, et al., 2000). Also, application of PKG to the cytosolic side of excised membrane patches from isolated myocytes potentiated channel activity in the presence of ATP and cGMP, suggesting that kinase promotes channel activity (Gerzanich, et al., 2003; Robertson, et al., 1993; Taniguchi, et al.,

1993; Wellman, et al., 1996). In one report, a cGMP analogue was able to stimulate channel activity

54 in excised patch, suggesting that PKG was present in the membrane (Peng, et al., 1996). Also, inhibition of dephosphorylation in the same patch-clamp configuration enhanced channel activity, which suggests the presence of phosphatases in the vicinity of the channel. Biochemical evidence of a direct phosphorylation of the BKCa channel by PKG was demonstrated by several groups

(Alioua, et al., 1995; Alioua, et al., 1998; Swayze & Braun, 2001). However, the exact substrate targeted by the kinase has not yet been fully characterized. Substituting ser-1072 by alanine in cloned canine KCNMA1 (Slo) gene suggested that this residue is critical for channel activation by

PKG in HEK-293 cells (Fukao, et al., 1999). Another group studying ANP and NO donor responses of mutants of the human channel expressed in oocytes found no effect of alanine substitution of ser-1072, but found that combined mutations of ser-855/869 abolished the whole response

(Nara, et al., 2000). Further investigations by another group (Kyle, et al., 2013) using the murine sequence demonstrated that several serines were actually phosphorylated by PKGI in vitro, namely ser-691, ser-873, and probably either of 3 serines at positions 1111-1113 (murine form ser-1112 being homologous to the above-cited ser-1072). Alanine substitution of 691 or 873 residues, and of all three serine 1111-1113 together, reduced the response to a NO donor or a cGMP analogue when expressed in A7r5 VSMCs cell line, suggesting that these residues are targeted by PKG in a living system. Other serine residues, namely ser-418 and ser-446, although not phosphorylated in vitro, appeared to be important for activation for the response to PKG stimulation.

In a rat model of hypertension induced by chronic infusion of AngII, BKCa channel activity in basilar artery myocytes displayed a reduced response to stimulation by SNP and 8-Br-cGMP (Gerzanich, et al., 2003). It was also observed that expression of the PKGI splicing variant was increased in basilar arteries from hypertensive rats, while PKGI was slightly decreased. Because the

PKGIhas slightly less affinity to cGMP than PKGIand displays differences in its N-terminal domain (Sheehe, et al., 2018), this switch may account for the weaker response of the channel to

NO-cGMP pathway in this pathophysiological model.

Constitutive activation of PKGI by oxidation (H2O2) can also lead to subsequent BKCa stimulation

(Burgoyne, et al., 2007; Sheehe, et al., 2018; Zhang, et al., 2012). This would be due to translocation of the kinase to the membrane and increase in BKCa channel activity (Zhang, et al., 2012). It was recently proposed that activation of PKG by oxidation may also account for BKCa channel stimulation in response to high intraluminal pressure (Khavandi, et al., 2016). This was inferred from the observation of arteries isolated from knock-in mice expressing a PKGI mutant cys-42- serCys-42 residue was considered as the key element for redox sensing and enzyme dimerization. Cys-42-ser arteries displayed reduced engagement of the BKCa channel during myogenic tone. The data point to an indirect mechanism involving constitutive activation of PKGI by endogenous oxidants upon elevation of intraluminal pressure, resulting in higher Ca2+ sparks and STOC activity, enhancing BKCa ability to oppose myogenic tone (Khavandi, et al., 2016). Still, results obtained by another group suggest that another cysteine (cys-117) may be responsible for sensitivity to oxidation and related disruption of sensitivity to cGMP (Sheehe, et al., 2018).

However, the exact role of cys-42 remains unclear, as [cys-42-ser] PKG mutant retains its sensitivity to oxidation, displays reduced activation by cGMP and may alter PKG action on the kinetics of the BKCa channel.

Because CNs influence both BKCa channel activity and intracellular Ca2+ movements that contribute to contractions, dissection of the exact contribution of each particular effector is complex, and yields to results that may appear paradoxical at first sight. An interesting example of such paradox was recently provided by Schmid et al. (Schmid, et al., 2018): by comparing shifts of concentration-response curves to the vasoconstrictor methoxamine, they found that the anti- contractile response to NO donors was actually increased by IBTX. The authors provided data supporting the following hypothesis: at relatively small intensity of contractile response (low concentration of methoxamine), NO anti-contractile effects would originate mainly from inhibition of Ca2+ influx through voltage-operated Ca2+ channels (see section 3.); by inhibiting Ca2+ influx, BKCa channels would reduce the contribution of this mechanism by limiting the number of open Ca2+ channels, which would explain why IBTX exerts an apparent anti-contractile effect.

Conversely, at high concentration of methoxamine, Ca2+ influx appeared to be equivalent in the presence or absence of NO. Here, cGMP-induced inhibition of BKCa channels translates into relaxation, so that IBTX produces and anti-relaxant effect.

5.4.4 cGMP-independent stimulation of BKCa channels by NO or NO donors.

A number of other reports suggested that the vasorelaxant effects of NO or NOS-dependent signalling might be mediated by cGMP-independent activation of BKCa channels (Mughal, et al.,

2018; Plane, et al., 1998; Sampson, et al., 2001; Satake, et al., 1996a; Sun, et al., 2000; Zhang, et al.,

2006). Cross activation of PKA by high concentrations of cGMP may participate in vasodilating effect of NO, but this does not seem to activate BKCa channel (Sausbier, et al., 2000). Direct activation of BKCa channels by NO has been demonstrated, both at the current and vasorelaxant response levels (Table 4) (Bolotina, et al., 1994; Mistry & Garland, 1998). Making the sulfhydryl groups biochemically unavailable for nitrosylation abolished the direct effect of NO on the channel, suggesting that nitrosylation steps might be involved (Bolotina, et al., 1994). Surprisingly, in a study conducted on canine middle cerebral artery, BKCa contribution to DEA-NO-evoked relaxation was actually stronger when cGMP synthesis by sGC was inhibited by ODQ (Onoue &

Katusic, 1998). This suggests that direct activation of the channel by high [NO] may contribute to vasodilation.

However, it may be somewhat hazardous to draw any definitive conclusion from such studies, since the results may be influenced by several experimental factors: for instance endothelium- dependent mechanisms may influence contribution of CBTX-sensitive channels in response to NO

(Plane, et al., 1996), and the action of physiological NO may differ from that of NO donors (Plane, et al., 1998). Moreover, working at different precontraction levels or vasoconstrictor concentrations may lead to opposite results in terms of subsequent vasorelaxant responses, as illustrated in (Plane, et al., 1998). The choice of K+ blocker may also lead to confounding results: for instance, CBTX is less selective than IBTX and its use may actually reveal the contribution of other K+ channels (Plane, et al., 1998).

Intriguingly, in retinal arteries and choroid, NO donors were shown to increase PGI2 production

(sensitive to PGI2 synthase inhibitor trans-2-phenylcyclopropylamine) via a BKCa-dependent and cGMP-independent mechanism (Hardy, et al., 1998). This process must take place in ECs as i) it is absent following endothelium denudation, and ii) SNP stimulates PGI2 synthesis in cultured retinal ECs. Moreover, this process is inhibited by IBTX or CBTX, while it is mimicked by the BKCa channel openers NS1619 or NS004, which is somewhat surprising since BKCa channels are generally considered to be absent from the ECs layer (Gauthier, et al., 2002). The study of Hardy et al. thus raises a possible relevance of BKCa channels located in the endothelium, which are able to evoke relaxation of VSMCs upon activation by NO, possibly through stimulation of cAMP production by PGI2 via a paracrine route. The latter mechanisms, however, have not been further investigated, and other studies suggest the presence of endothelial BKCa that could be activated by the cGMP pathway (Begg, et al., 2003; Luedders, et al., 2006). Vasodilatory action may thus occur by several mechanisms, including increase in driving force for Ca2+ in ECs and stimulation of the release of endothelial vasodilatory autacoids as suggested above, or participation of an endothelium-derived hyperpolarizing component.

5.4.5 Specific association of BKCa channels with PDEs

The possibility that ion channels, BKCa channels in particular, are tightly regulated by distinct PDEs that are pivotal for vasomotion control has been addressed by several studies. Contribution of

BKCa channel was probed by the effect of IBTX or paxilline on the vasorelaxant effects of PDE3,

PDE4 and PDE5 inhibitors (Bardou, et al., 2002; Idres, et al., 2018; Kaneda, et al., 2010; Kyle, et al.,

2017; Li, et al., 2015; Rieg, et al., 2013; Taylor & Benoit, 1999). Still, relative contribution of BKCa channel varies according to the nature and concentration of the inhibitor used, the vascular territory and the animal species (Bardou, et al., 2002; Idres, et al., 2018; Kaneda, et al., 2010; Kyle, et al., 2017; Li, et al., 2015; Rieg, et al., 2013; Taylor & Benoit, 1999). For instance, BKCa channel activity appears to be important for PDE3 inhibition in aorta and coronary artery (Idres, et al.,

2018; Li, et al., 2015), but is less evident in PA or cerebral arterioles (Bardou, et al., 2002;

Nakamura, et al., 2006; Rieg, et al., 2013). BKCa channels may share this participation with KV7 channels, which inhibition can also hamper relaxation to PDE3/4 inhibition in rat coronary artery

(Idres, et al., 2018). Interestingly, such contribution may be altered in pathological situation. In a rat model of heart failure, inhibition of BKCa channels was no longer able to hamper relaxation to

PDE3/4 inhibition in coronary artery, a result that may be explained by a decrease in channel expression and remodelling of the expression of some PDE isoforms (Idres, et al., 2018).

The extent of BKCa contribution to the response to a vasorelaxant agonist can be inferred from shifts in the concentration-response relationships in the presence or absence of BKCa blockers.

Using such approach, it was shown that under BKCa inhibition, PDE3 and PDE4 inhibition was no longer able to potentiate vasorelaxant responses to -AR stimulation, whereas potentiation was still observed in responses to FSK (Idres, et al., 2018). This supports the notion that vasoactive -

AR signalling is mainly hampered by PDEs that control BKCa channels. BKCa channel is known to interact with the 2-AR in VSMCs. Proximity ligation assay data showed that the BKCa channel - subunit localizes close to PDE3 and PDE4 (Idres, et al., 2018) or PDE5 (Kyle, et al., 2017) isoforms.

Thus, the control of -AR-stimulated BKCa channel by PDEs may be facilitated by proximity, if not direct protein-protein interactions, between receptors, PDEs and channels.

5.5 TASK-1/KCNK3 channel

Belonging to the 2-pore-domain K+ (K2P) channel family, the TASK-1/KCNK3 channel carries acid-sensitive K+ background currents in some VSMCs and was demonstrated to participate in the resting membrane potential of PA SMCs while loss of function of the channel was associated with the development of pulmonary arterial hypertension (PAH) (reviewed by (Olschewski, et al.,

2017)). In human PA SMCs, non-inactivating current, thought to be mainly carried by TASK-1 channel, can be enhanced by treprostinil, a PGI2 analogue (EC50  1 µM)(Olschewski, et al., 2006).

This was mimicked by 8-Br-cAMP (100 µM), inhibited by KT-5720 (300 nM), and associated with an increased ser/thr phosphorylation of TASK-1 protein, suggesting a cAMP-PKA-mediated effect.

The sequences of KCNK3 and other K2P channels display PKG-consensus sites, suggesting

59 possible regulation by the NO-cGMP pathway. The NO donor SNP (100 µM) or 8-Br-cGMP (10 µM) did not seem, however, to have any effect on global TEA (10 mM)-resistant outward current

(putatively including KCNK3-current) in rat middle cerebral arteries (Lloyd, et al., 2009). In contrast, the sGC activator, riociguat, was recently shown to activate recombinant wild type

KCNK3 channels, but not mutants that were associated with PAH (Cunningham, et al., 2018).

Recently, it was shown that relaxation produced by sildenafil in rat PA was attenuated by a pharmacological inhibitor of KCNK3 or in transgenic rats with non-functional KCNK3 channel compared with wild type (Lambert, et al., 2019). While KCNK3 was already shown to be stimulated by cGMP in other cell types, these data interestingly link the KCNK3 channel to cGMP pathway, which is therapeutically used to alleviate the symptoms of PAH.

6. Conclusion

6.1 Basic knowledge that has been gained in the field

Studies on ion channel regulation by cAMP and cGMP and associated pathways span over more than three decades, and substantial basic knowledge has been accumulated regarding the main channels that were characterized in VSMCs, namely LTCCs (CaV1.2), KATP channels and BKCa channels (summarized in Figure 2). Classic data obtained using relatively selective drugs have been useful to demonstrate the mechanisms underlying these signalisations, mainly at the cellular levels and in whole blood vessels. While most studies used isolated conduit or resistance arteries, in situ preparations allow to study small sized-vessels, providing closer insight on mechanisms occurring in vivo and in microvasculature. Biochemical investigations also provided important insights on how cAMP- and cGMP-activated kinases impact ion channel activity at the molecular level.

Also, a more recent wave of pharmacological, molecular and silencing techniques fostered the investigation of ion channels such as TRP channels family, KV7 channels and endothelial KATP channels. Moreover, novel, “non-canonical” signalling molecules such as EPAC, or regulators of channel subunits trafficking have been increasingly well explored and gave birth to more complex paradigms (some of which are summarized in Figure 2). New approaches combining genetic models, pharmacology with high resolution imaging and protein-protein interaction studies have started to further delineate the molecular organization of these pathways. One may logically predict that the use of ever improving techniques will allow further important discoveries.

Nevertheless, studying the molecular mechanisms responsible for tuning vascular tone faces challenges and complexity, some of which may be specific of the field of vascular research.

6.2 Remaining challenges for research in the field

6.2.1 Characterization of signal heterogeneity among vascular beds and in disease

Classic and more recent studies highlighted the heterogeneity of signalling routes between different vascular beds. For instance, Stott et al. (Stott, et al., 2016)) observed that the response to a -AR stimulation in mesenteric or renal arteries follows distinct signalling pathways, either

EPAC or PKA-dependent, respectively. The ability of -AR stimulation to generate Ca2+ sparks- related STOC-activity in myocytes also differs depending on the vessel of origin, e.g. aorta or resistive artery (Hayoz, et al., 2007). The contribution of a particular ion channel to vasorelaxant responses may also vary among models, including the type of artery investigated (see Table 2 &

4). For instance, CGRP response is glibenclamide-sensitive in mesenteric artery (Nelson, et al.,

1990), but not in rabbit basilar artery (Sutter, et al., 1995). Likewise, modalities of the experimental procedures used to investigate vascular reactivity may influence the final conclusion on the engagement of an ion channel: for instance, IBTX had opposite effects on vasorelaxant response to ISO in two studies conducted on rat 3rd order mesenteric arteries, but using slightly different protocols: in (Garland, et al., 2011), 70 mmHg was used as a reference to set basal tension

(Nyborg, et al., 1987), phenylephrine was used for precontraction, and IBTX did not change the response. In (White, et al., 2001), however, when basal tension was higher (100 mmHg was used) and another vasoconstrictor agonist (methoxamine) was used, IBTX significantly attenuated ISO response. These observations make even more obvious the notion that each conclusion should be restricted only to the model and experimental procedures that have been used. The localization of the vascular bed, the basal tension and the precontraction level are important variables to consider before driving a general conclusion from a single observation.

An additional complexity arises when studying cells and tissues in disease conditions. In particular, the coupling of various ion channels with CN signalling appears to be altered in pathologies such as hypertension, heart failure or diabetes (Callera, et al., 2004; Chadha, et al.,

2014; Chadha, et al., 2012b; Gerzanich, et al., 2003; Idres, et al., 2019; Khanamiri, et al., 2013; Li, et al., 2003; Morales-Cano, et al., 2015; Nystoriak, et al., 2017). Knowledge of such alterations is still limited which provides interesting research opportunities.

Yet, to dissect the causes of regional heterogeneity or pathological alterations of a given signalling pathway may represent a great challenge due to the multiplicity of possible underlying mechanisms, such as: 1) changes in the relative expression of receptors, kinases, signalling proteins, ion channel subunits and their variants; 2) the dynamics of the coupling within each signal transduction step, that may vary according posttranslational modifications of proteins, abundance of their regulatory subunits or architecture of multiprotein signalling domains; 3) the prevalence of the inhibitory processes that inhibit the signalling, e.g. PDE and phosphatase activities.

One may assume that this field will benefit from the combination of relevant in vivo models with accurate ex vivo and molecular approaches. For instance, the exploration of dynamics of CNs in native vascular beds can be facilitated by using sophisticated genetic models expressing fluorescent probes reporting intracellular signalling (Raina, et al., 2009; Thunemann, et al., 2013).

This may be coupled with vessel diameter or tone measurement, allowing to investigate signal dynamics with associated variations of tone in intact resistance arteries or superficial or microcirculatory beds (Spiranec, et al., 2018).

6.2.2 Exploring the impact of pre-translational molecular diversity and regulation

Various molecular actors mentioned in this review potentially display important molecular diversity via the production of several transcript variants that may behave differently regarding

CN signalling. This is illustrated by the STREX/ZERO variants of the -BKCa subunit (see section

5.4.2). While other examples may arise from future research work, physiological significance of this diversity is still unclear and will deserve deeper examination. The systematic characterization of various transcript variants expression in normal and pathological conditions may help to correlate the transcriptome dynamics with altered signalling and organ function. Emerging single cell transcriptomics technologies and bioinformatics are likely to provide an important support to address these questions.

6.2.3 Mapping subcellular dynamics of CN signalling

Correct targeting of proteins to membranes or subcellular niches is an important level of posttranslational regulation of signalling platforms. Some mechanisms related to CN signalling have been described, involving trafficking of channel regulatory subunits to the membrane (Leo, et al., 2014), or AKAP-dependent targeting of PKA to the CaV1.2 channel in diabetes (Nystoriak, et al., 2017). In addition, exploration of the interaction of ion channels subunits with AKAP proteins,

PDEs and phosphatases may help to delineate a more comprehensive scheme of CN compartmentalization within SMCs and ECs. Current and cutting edge techniques used to demonstrate protein-protein interactions (co-immunoprecipitation, FRET, BRET interaction studies, protein-fragment complementation assay (Schramm, et al., 2018)) or to quantify proximity of different partners (super resolution microscopy (Nystoriak, et al., 2017)) will be valuable approaches to address these questions. This field is only emerging in vascular research and probably deserves further effort in order to improve our understanding of subcellular processes in health and disease.

6.2.4 Ion channel contribution to the integrated vasomotor response

Although the existence of a given signalling pathway may be demonstrated at the molecular or cellular level, it may be functionally redundant due to the multiplicity of effectors that mediate integrated vasorelaxant mechanisms. For instance, an ion channel may not contribute significantly to a vasomotor response despite being involved in Em hyperpolarisation (Garland, et al., 2011). Furthermore, the respective contribution of a given pathway may be enhanced, decreased or unchanged in a pathological context (Idres, et al., 2019). Further research may be needed to define more accurately the signalling axes that are relevant to arterial tone regulation, and to detect key alterations associated with pathological situations.

A deeper understanding of such mechanisms may be useful for drug discovery, when developing a vasoactive action is sought as primary pharmacodynamics of a new pharmaceutical.

Alternatively, regarding drugs developed for other indications, this would improve the prediction of potential secondary pharmacological effects on blood flow regulation and arterial blood

64 pressure. Also, because most ion channel families are ubiquitously expressed, this would help to define more targeted strategies and safer mechanisms of action.

Conflict of Interest Statement: The authors declare that there are no conflicts of interest.

Tables

Table 1: Studies testing the regulation of TRP channels by cAMP and cGMP pathways in vasculature, SMCs and expression systems.

Channel Model Observation / Mechanism References NO-cGMP inhibited 11, 12-EETs-induced hyperpolarization and vasorelaxation via phosphorylation of TRPC1 pCorA; HEK-293 expr. channels (Zhang, et al., 2014b) TRPC1 at ser-172 and thr-313 by PKG. TRPC1, Native currents carried by TRPC1 and TRPC3 inhibited by NO donor and cGMP via PKG activity. rCarA ( fresh SMCs, ring) (Chen, et al., 2009) TRPC3 La3+ (100 µM) partially inhibited SNP-relaxation of UTP-contracted arteries. TRPC3 HEK-293, expr. PKG and TRPC3 cGMP reduced SOCE via phosphorylation of TRPC3 at thr-11 and ser-263 by PKGI. (Kwan, et al., 2004) HEK-293, expr. TRPC4 or TRPC5; TRPC5 current inhibited by cAMP, FSK, ISO or co-expression of a constitutively active GsQ227L protein. TRPC5, Responses to cAMP were blunted by PKA inhibitors or when using S796A and S794A mutants. TRPC4 (Sung, et al., 2011) TRPC4 m small intestine SMCs (fresh) and TRPC4, but not TRPC6 or TRPV6 were also sensitive to GsQ227L. In native SMCs, inward cation current carried by Cs+ were inhibited by cGMP or ISO. HEK-293 expr. channels; Activation of TRPC4 current by PDE inhibitors (cilostamide, PDE3, EHNA and zydena, PDE5) and cGMP. TRPC4 (Wie, et al., 2017) h prostate SMCs Effect was absent with channels carrying a mutated ser-688. TRPC6 HEK-293, expr. TRPC6; A7r5 NO donor or cGMP inhibited receptor-stimulated TRPC6 current via phosphorylation at thr-69 by PKG. (Takahashi, et al., 2008) rAo Cilostazol (CZL, PDE3 inhibitor) inhibited AngII-induced contraction via reduction of Ca2+ influx. HEK-293, expr. channels; CZL inhibited TRPC3, TRPC6 and TRPC7 (but not TRPC5)-mediated Ca2+ influx. TRPC6 CLZ effect on TRPC6 was mediated by phosphorylation of thr-69 by PKA. (Nishioka, et al., 2011) rAo SMCs; CLZ effect was dependent on endogenous TRPC6, not TRPC3/7. VSMCs-reconstituted ring Valid thr-69 of TRPC6 was necessary for CLZ relaxation. Inhibitions of NOS (L-NAME), sGC (ODQ) or PKG (KT-5823) potentiated endothelium-independent TRPV3 rUtRadA (Murphy, et al., 2016) vasodilation to carvacrol, a TRPV3 activator. AA evoked TRPV4 activity via PKA. PKA phosphorylated TRPV4 at ser-824, a modification associated (Cao, et al., 2018; Zheng, TRPV4 hCorA ECs, expr. TRPV4; hCorA with increased Ca2+ influx and proposed to participate in the vasodilation to AA. et al., 2013) Abbreviations: for species: h: human; m: mouse; p: porcine; r: rat; ; for vascular beds: Ao: aorta; CarA: carotid artery; CorA: coronary artery; UtRadA: uterine radial artery; other abbreviations: AA: arachidonic acid; CZL: cilostazol; ECs: endothelial cells; expr.: heterologously expressing; ISO:

66 isoprenaline; NO: nitric oxide; PKA: cAMP-dependent protein kinase; PKG: cGMP-dependent protein kinase; SMCs: smooth muscle cells; UTP: uridine triphosphate.

Table 2: Studies testing the regulation of KATP channels by some cAMP and cGMP –associated pathways in vasculature.

Stimulus Model Response studied* Observation / Mechanism Reference channel activation(w-c; c-att.) activation of a glib-sensitive current via PKA; rabMA hyperpolarization, (Nelson, et al., 1990; Quayle, et al., 1994) glib-sensitive hyperpolarization and vasorelaxation vasorelaxation(NA) pCorA channel activation(w-c; c-att.) activation of a glib-sensitive current via PKA (Wellman, et al., 1998) calcitonin cultd SMCs (pCorA) channel activation(c-att.; i-o)) activation of a glib-sensitive current via PKA (Miyoshi & Nakaya, 1995) gene- rCorA vasorelaxation(PGF2) NOT glib-sensitive (Prieto, et al., 1991) related rBA vasodilation(in vivo tone) glib-sensitive (Kitazono, et al., 1993) protein (CGRP) pCorAendo(-) vasorelaxation(ET) cAMP-dependent; NOT glib- sensitive (Kageyama, et al., 1993) rabBA vasorelaxation(PKC activation) NOT glib-sensitive, but induced an increase in cAMP (Sutter, et al., 1995) rPialA vasodilation(in vivo tone) glib-sensitive and CBTX-sensitive (Hong, et al., 1996) hyperpolarization, mPA glib-sensitive, PKA-inhibited and abolished in Kir6.1 knockout mice (Norton & Segal, 2018) vasodilation(UTP) (pp) pCorA channel activation activation of a glib-sensitive current via A1A receptors (Dart & Standen, 1993) (w-c) rabMA channel activation activation of a glib-sensitive current via A2 receptors and PKA (Kleppisch & Nelson, 1995a)

gpCorA hyperpolarization glib-sensitive, participation of A2B receptor suggested (Mutafova-Yambolieva & Keef, 1997) pCorA vasorelaxation(PGF2) glyburide-sensitive responses to adenosine R agonists (Merkel, et al., 1992; Merkel, et al., 1993) vasodilation(perfused, hypoxic adenosine rabCorA glib-sensitive response to A1 R agonist (Nakhostine & Lamontagne, 1993) heart) rabCorA vasodilation(perfused heart + PE) glib-sensitive (Jackson, et al., 1993) (in vivo tone) canCorA vasodilation glib-sensitive, probably via A2 receptors (Akatsuka, et al., 1994) pRetA vasodilation(in vivo) glib-sensitive (Gidday, et al., 1996) pPialA vasodilation (in vivo) glib-sensitive (Armstead, 1997b)

Stimulus Model Response studied* Observation / Mechanism Reference glib-sensitive, partly endothelial-dependent response; cAMP- pCorA vasodilation(m.t.) (Hein & Kuo, 1999) independent cultd SMCs (pCorA) channel activation(c-att.; i-o)) activation of a glib-sensitive current (Miyoshi & Nakaya, 1993) pCorA channel activation(w-c) activation of a glib-sensitive current (Wellman, et al., 1998) rMA hyperpolarization glib-sensitive response to ISO (Fujii, et al., 1999) canSV hyperpolarization glib-sensitive response to ISO (Nakashima & Vanhoutte, 1995) rMA vasodilation(perfused + meth.) glib-sensitive responses to -AR agonists (Randall & McCulloch, 1995) rMA hyperpolarization glib-sensitive response to NA (under -AR blockade) (Goto, et al., 2000) rAo(2-k-1clip HT vs control) vasorelaxation(PE) glib-sensitive response to ISO reduced in 2-k-1clip HT, not in control (Callera, et al., 2004) hyperpolarization, rMA glib inhibited ISO-evoked hyperpolarization, not vasodilation (Garland, et al., 2011) vasorelaxation(PE) rAo vasorelaxation(PE) response to ISO was NOT glib-sensitive (Satake, et al., 1996b) rPialA vasodilation (in vivo) response to ISO was NOT glib-sensitive (Hong, et al., 1996) rMA vasorelaxation(PE) response to ISO unaltered by glib, but was by CBTX (Huang & Kwok, 1997) rMA vasorelaxation(meth.) ISO potentiated response to levcromakalim via PKA (White, et al., 2001) channel activation(w-c); activation of a glib-sensitive current; rMA, rPA (Yang, et al., 2008; Zhang, et al., 2010) vasorelaxation(PE) glib-sensitive (perfused heart + PE) PGI2 rabCorA vasodilation glib-sensitive responses to PGI2 and iloprost (Jackson, et al., 1993) channel activation(w-c); rTA glib-sensitive response to iloprost (Schubert, et al., 1997) vasodilation(m.t.)

pCorA hyperpolarization glib and IBTX-sensitive response to iloprost (Edwards, et al., 2001) channel activation(c-att); activation of a glib-sensitive current; rAo (Eguchi, et al., 2007) vasorelaxation(PE) glib-sensitive GLP-1 rAo vasorelaxation(PE) glib-sensitive (Green, et al., 2008) (w-c) channel activation ; PACAP suggested to activate a KATP-like current; PACAP rCerebrA (Koide, et al., 2014) vasodilation(m.t.) glib-sensitive response d (c-att.) pCorA, cult channel activation L-arginine stimulated KATP channel in SMCs incubated by endotoxin (Miyoshi, et al., 1994)

d (c-att.) (Kubo, et al., 1994) rAo, cult channel activation activation of a glib-sensitive current by ANP and isosorbide dinitrate (Quayle, et al., 1994; Wellman, et al., rabMA, pCorA channel activation(w-c) SIN-1 or SNP did NOT activate any glib-sensitive current 1998)

Stimulus Model Response studied* Observation / Mechanism Reference NO, NO rabMA hyperpolarization glib-sensitive response to SIN-1 (Murphy & Brayden, 1995) donors, gpCarA hyperpolarization glib-sensitive response to NO donors (Corriu, et al., 1996) ANP rabMA vasorelaxation(NA) responses to NO or nitroglycerin were NOT sensitive to glyburide (Khan, et al., 1993)

rabCorA vasodilation(perfused heart + PE) response to SNP was NOT glib-sensitive (Jackson, et al., 1993)

pRetA vasodilation(in vivo) SNP hyperemic response was NOT glib-sensitive (Gidday, et al., 1996)

pPialA vasodilation(in vivo) glib-sensitive response to SNP (Armstead, 1996)

canMCA vasorelaxation(UTP) response to SNP or SIN-1 was NOT glib-sensitive (Onoue & Katusic, 1997)

gpBA vasorelaxation(PGF2) responses to nerve-released NO or SNP were NOT glib-sensitive (Jiang, et al., 1998)

(NA) NO, NO rabAo vasorelaxation response to NO was NOT glib-sensitive (Ferrer, et al., 1999) donors, pCorA vasodilation(m.t.) response to SNP was NOT glib-sensitive (Hein & Kuo, 1999) ANP pPA vasodilation(in vivo) response to SNP was NOT glib-sensitive (Saqueton, et al., 1999) (conted) canFV vasorelaxation(PGF2) response to NO only slightly inhibited by glib + CBTX (Bracamonte, et al., 1999) pRetA vasodilation(m.t.) dilation to lactate was L-NAME-and glib-sensitive. (Hein, et al., 2006) mMA vasorelaxation(PE) SNP relaxant response was NOT glib-sensitive (Mustafa, et al., 2011) response to ACh was NOT glib-sensitive endoth. rabCorA vasodilation(perfused heart + PE) (Jackson, et al., 1993) NOS-independent response to BK, NOT ACh, was glib sensitive (c-att.; i-o)) channel activation isoflurane activates KATP current via PKA; a role in vasorelaxant isoflurane rAo (Tanaka, et al., 2007) vasorelaxation(PE) response to isoflurane is suggested cilostamide (PDE3 inhibitor) produced a glib-sensitive PDE inhib. rMA hyperpolarization (Kansui, et al., 2009) hyperpolarization

*mode of contraction/constriction (isolated artery); or patch-clamp modes are given in superscript when relevant.

Abbreviations: for species: can: canine; gp: guinea pig; m: mouse; p: porcine; rab: rabbit; r: rat; for vascular beds: Ao: aorta; BA: basilar artery; CarA: carotid artery; CerebrA: cerebral artery; CorA: coronary artery; FV: femoral vein; MA: mesenteric artery; MCA: middle cerebral artery; PialA: pial artery;

PA: pulmonary artery; RetA: retinal artery; SV: saphenous vein; TA: tail artery; endo(-): endothelium disrupted; for patch-clamp modes: c-att.: cell- attached; i-o: inside-out; pp.: perforated patch; w-c: whole-cell; cultd : cultured; for precontraction protocols: m.t.: myogenic tone; ET: endothelin-1; meth.: methoxamine; NA: noradrenaline; PE: phenylephrine; PGF2prostaglandin F2; other abbreviations: 2-k-1clip HT: 2-kidney-1-clip 69 hypertension model; ACh: acetylcholine; BK: bradykinin; CBTX : charybdotoxin; glib: glibenclamide; endoth.: endothelium-dependent vasodilators;

GLP-1: glucagon-like peptide-1; IBTX: iberiotoxin; ISO: isoprenaline; NO: nitric oxide; PACAP: pituitary adenylate cyclase activating polypeptide; PDE inhib.: PDE inhibitor; PGI2: prostacyclin; SMCs: smooth muscle cells; SNP: sodium nitroprusside; R: receptor; UTP: uridine triphosphate.

Table 3: Studies testing the regulation of KV1 and KV7 channels by some cAMP- and cGMP-associated pathways in vasculature.

Channel Model Response studied* Observation / Mechanism Reference (hist.) rabCerebrA vasorelaxation response to PGI2 was blunted by dendrotoxin and IBTX (Dong, et al., 1998) rabAo vasorelaxation(NA) response to NO was attenuated by 4-AP (0.1 mM) (Ferrer, et al., 1999) vasorelaxation(m.t. + U46619) response to ISO displayed sensitivity to 4-AP (3 mM); this was lost following 24 h KV1 rCorA (Li, et al., 2003) channel activation(w-c) exposure to high glucose canCorA vasodilation(m.t.) responses to adenosine and SNP were inhibited by 4-AP (0.3 mM) and correolide (Dick, et al., 2008) (m.t.) rCerebrA vasodilation response to ISO (mainly via 1-AR) was blunted by KV1 blocker Psora4 (Moore, et al., 2015) (meth.) vasorelaxation response to ISO was attenuated by LNP, not by 1 mM 4-AP nor glib (Chadha, et al., 2012b; Stott, et rRenalA (w-c) channel activation ISO elicited a KV7-like current al., 2015) (U46619) vasorelaxation responses to adenosine and A2AR agonist CGS-21680 or ISO were abolished by 10 rCorA (Khanamiri, et al., 2013) µM LNP ; response to SNP was not altered by LNP vasorelaxation(U46619) response to CGRP was inhibited by LNP, siRNA against Kcnq4, but not against rMCA (Chadha, et al., 2014) vasodilation(m.t.) Kcnq5 rCorA(right and left) vasorelaxation(ser.) response to FSK was attenuated by LNP and XE991 (Morales-Cano, et al., 2015) mCorA, vasorelaxation(U46619) response to FSK was slightly attenuated by LNP (10 µM) (Lee, et al., 2015) mCerebrA KV7 LNP partially inhibited responses to ANP (Ao and Ren), CNP (Ao) and SNP (Ao); rAo, rRenalA vasorelaxation(meth.) (Stott, et al., 2015) KV7.1 blocker HMR 1556 had no effect d A7r5 cult , native ISO induced large increase in h KV7.5 current via PKA, but had less effect on h KV channel activation(w-c) (Mani, et al., 2016) or expr. KV7 7.4/7.5, and no effect on KV7.4 rMA, rRenalA vasorelaxation(U46619) relaxation evoked by the EPAC agonist 8-pCPT-AM (5 µM) was blunted by LNP (Stott, et al., 2016) and IBTX; relaxation to ISO was attenuated by LNP channel activation(w-c) NO donors (DEA-NO, SNP) and riociguat activated a KV current, hyperpolarized Em rPA hyperpolarization(w-c) (Mondejar-Parreno, et al., 2019) and produced relaxation in a KV7 blockers (XE991, linopirdine)-sensitive manner vasorelaxation(PE)

*: when isolated artery is studied, mode of contraction/constriction (isolated artery); patch-clamp modes are given in superscript when relevant;

Abbreviations: for species: can: canine; h: human; m: mouse; rab: rabbit; r: rat; for vascular beds: Ao: aorta; CerebrA: cerebral artery; CorA: coronary artery; MA: mesenteric artery; MCA: middle cerebral artery; PA: pulmonary artery; RenalA: renal artery; for patch-clamp modes: w-c: whole-cell; 71 cultd : cultured; for precontraction protocols: hist.: histamine; m.t.: myogenic tone; meth.: methoxamine; NA: noradrenaline; ser.: serotonin; other abbreviations: 4-AP: 4-aminopyridine; A2AR: adenosine A2A receptor; ANP, CNP: A-type and C-type natriuretic peptides; CGRP: calcitonin gene-related protein; DEA-NO: diethylamine NONOate; EPAC: exchange protein activated by cAMP; expr.: heterologously expressing; FSK: forskolin; glib: glibenclamide; IBTX: iberiotoxin; ISO: isoprenaline; LNP: linopirdine; PGI2: prostacyclin; PKA: cAMP-dependent protein kinase; SNP: sodium nitroprusside.

Table 4: Studies testing the regulation of BKCa channels by some cAMP and cGMP –associated pathways in vasculature.

Stimulus Model Response studied* Observation / Mechanism Reference rAo, cultd channel activation(c-att.) ISO induced channel activity (Sadoshima, et al., 1988) Gs protein activated the channel, not via PKA; PKAc activated the pCorA channel activation(mb bil.) (Scornik, et al., 1993) channel. (w-c) gpBA channel activation ISO increased macroscopic IKCa and single channel activity (Song & Simard, 1995) pCorA channel activation(c-att.; i-o) ISO increased channel activity via PKG (White, et al., 2000) gpLV hyperpolarization response to ISO was attenuated by IBTX and CBTX (Von der Weid & Van Helden, 1996) rMA hyperpolarization response to ISO was NOT altered by IBTX nor CBTX (Fujii et al., 1999) -AR hyperpolarization, rMA responses to ISO was NOT altered by IBTX (Garland, et al., 2011) agonist vasorelaxation(PE) (ISO) rCorA vasodilation(m.t. + U46619) response to ISO was attenuated by TEA (1 mM) (Price, et al., 1996) rMA vasorelaxation(PE) response to ISO was attenuated by CBTX (Huang & Kwok, 1997) rAo vasorelaxation(PE) response to ISO was attenuated by IBTX and 4-AP (5 mM) via 2-AR (Satake, et al., 1996b) rMA vasorelaxation(meth.) response to ISO was attenuated by IBTX and was PKA-independent (White, et al., 2001)

response to ISO displayed sensitivity to IBTX; this was reduced rCorA vasorelaxation(m.t. + U46619) (Li, et al., 2003) following 24 h exposure to high glucose rAo vasorelaxation(PE) response to ISO was attenuated by IBTX (Matsushita, et al., 2006)

ISO response was inhibited by IBTX or TRAM-34 + UCL1684 (SKCa and rMA vasorelaxation(U46619) (Matsumoto, et al., 2012) IKCa inhibition), in control but not in a model of HT bAo channel activation(c. att.) increase in channel open probability (Williams, et al., 1988) rCerebrA, rCorA channel activation(pp); increases in STOCs frequency and amplitude (moderate) (Porter, et al., 1998) canCorA vasodilation(m.t. + U46619) response was attenuated by IBTX (Cabell, et al., 1994) adenosine rCerebrA vasodilation(in vivo) response was attenuated by TEA (1 mM) and IBTX (Paterno, et al., 1996) rCorA vasodilation(m.t. + U46619) responses was attenuated by TEA (1 mM) (Price, et al., 1996) pCorA vasodilation(m.t.) response was NOT attenuated by IBTX (Hein & Kuo, 1999)

channel activation(w-c; c-att.; iloprost increased IKCa; (Schubert, et al., 1996) rTA i-o) (m.t.) PGI2 ; vasodilation vasodilatory response to iloprost was slightly inhibited by IBTX (Schubert, et al., 1997) pCorA hyperpolarization response to iloprost were glib- and IBTX-sensitive (Edwards, et al., 2001) d (c-att.; i-o) PGE2 hCorA, cult channel activation PGE2 increased channel activity via PKG (Zhu, et al., 2002)

Stimulus Model Response studied* Observation / Mechanism Reference urocortin rCorAendo(-) vasorelaxation(U46619) response was inhibited by IBTX or TEA (1-3 mM) and PKA inhibitor (Huang, et al., 2003) rCerebrA, rCorA channel activation(pp); FSK increased STOCs frequency, and moderately amplitude; FSK Ca2+ sparks; increased Ca2+ sparks frequency; (Porter, et al., 1998; Wellman, et al., 2001) vasodilation(m.t.) response was attenuated by IBTX and ryanodine FSK pCorA channel activation(c-att.; i-o) FSK increased channel activity via PKG (White, et al., 2000) rabCerebrA vasodilation(in vivo) response was attenuated by IBTX and CBTX (Taguchi, et al., 1995) rCorA vasodilation(m.t. + U46619) response was attenuated by TEA (1 mM) (Price, et al., 1996) rCorAendo(-) vasorelaxation(U46619) response was attenuated by IBTX and TEA (3 mM) (Huang, et al., 2003) pCorA channel activation(c-att.; i-o) dopamine increased channel activity via PKG (White, et al., 2000) dopamine d (c-att.) hCorA, cult channel activation D1R agonists activated BKCa via D5R & PKG, not PKA (Natarajan, et al., 2010) (c-att.; i-o) bAo channel activation SNP increased BKCa channel open probability (Williams, et al., 1988) (w-c) rabPA channel activation SNP stimulated STOCs (Clapp & Gurney, 1991) (c-att.; i-o) pCorA channel activation nitroglycerin and cGMP activated IKCa (Fujino, et al., 1991) rCerebrA channel activation(c-att.; i-o) SIN-1 induced 2-fold increase in channel open probability (Robertson, et al., 1993) (w-c) channel activation NO increased macroscopic, CBTX-sensitive IKCa rPA (Archer, et al., 1994) vasorelaxation(NA) NO-induced response was inhibited by CBTX channel activation(i-o) NO increased NPo in excised patch; vasodilation to NO was CBTX- rabAo (PE) (Bolotina, et al., 1994) vasorelaxation sensitive and was higher in the presence of MB (sGC inhibitor) (c-att.) NO, NO rabCorA channel activation S-nitrosothiol, SNAP and cGMP increased activity, via PKG (George & Shibata, 1995) donors hPA, cultd channel activation(c-att.) NO and SIN-1 increased open probability, via PKG (Peng, et al., 1996) channel activation(c-att.; pp) SNP stimulated channel activity, via PKG; rMA (Carrier, et al., 1997) vasodilation(m.t. + PE) response to SNP was attenuated by TEA (1 mM) and PKG inhibitor channel activation(c-att.) NO donor DETA-NONOate increased channel activity, via sGC; bCorA (Li, et al., 1998; Li, et al., 1997) vasodilation response to DETA-NONOate was attenuated by IBTX rMA channel activation(w-c; i-o) NO and SIN-1 donors increased channel activity, NOT via cGMP (Mistry & Garland, 1998) channel activation(w-c; pp.) SNP stimulated an IBTX-sensitive current and STOCs; hCorA (Bychkov, et al., 1998) vasorelaxation(ser.) responses to SNP was attenuated by IBTX or TEA (1 mM) rCerebrA, rCorA channel activation(pp.); SNP increased STOCs frequency and amplitude (moderate); SNP (Porter, et al., 1998) Ca2+ sparks; increased Ca2+ sparks frequency

Stimulus Model Response studied* Observation / Mechanism Reference channel activation(c-att.) NO donor increased channel activity, this was NOT sensitive to ODQ; rMCA (Sun, et al., 2000) vasodilation(ser.) NO response attenuated by IBTX hRadA channel activation(w-c) SNAP evoked a TEA (1 mM)-sensitive current, NOT sensitive to ODQ (Zhang, et al., 2006) rabMA hyperpolarization response to SIN-1 was NOT altered by IBTX nor CBTX (Murphy & Brayden, 1995) hyperpolarisation (Wellman, et al., 1996) rCorA SNP-evoked responses were attenuated by IBTX or TEA (1mM) vasodilation(m.t.) (Price & Hellermann, 1997) rabMA vasodilation(NA) responses to NO or NO donors were attenuated by IBTX and CBTX (Khan, et al., 1993) canCorA vasodilation(m.t. + U46619) response to SNP was minimally reduced by TEA (1 mM) (Cabell, et al., 1994) gpPA (left or main) response to SNAP in PA was hampered by IBTX, CBTX and TEA (1 vasorelaxation(hist.) (Bialecki & Stinson-Fisher, 1995) gpCarA, gpAo mM), not in Ao or CarA rabCerebrA vasodilation(in vivo) response to SNP was NOT altered by to IBTX (Taguchi, et al., 1995) response to nitroglycerin was attenuated by IBTX but NOT sensitive rAo vasorelaxation(PE) (Satake, et al., 1996a) NO, NO to sGC inhibition with MB donors p vasodilation(in vivo + L-NAME) response to SNAP was absent in the presence of IBTX or CBTX (Zanzinger, et al., 1996) (contd) rCerebrA vasodilation(in vivo) responses to SNP and cGMP were attenuated by IBTX and TEA (Paterno, et al., 1996) rMA vasorelaxation(PE) response to SIN-1 was attenuated by CBTX (Plane, et al., 1996; Plane, et al., 1998) rCarA vasorelaxation(PE) response to NO was attenuated by CBTX, NOT by IBTX canMCA vasorelaxation(UTP) responses to SNP and SIN-1 were attenuated by CBTX (Onoue & Katusic, 1997)

pRetA vasodilation responses to NO donors were attenuated by BKCa blockers, but not in (-) (Hardy, et al., 1998) pChor(endo (+) or (-) vasodilation(m.t. + U46619) endo ; NO stimulated PGI2 production via BKCa channels gpBA vasorelaxation(PGF2) response to nerve-released NO or SNP were blunted by IBTX or CBTX (Jiang, et al., 1998) rabAo vasorelaxation(NA) response to NO was attenuated by CBTX (Ferrer, et al., 1999) canFV vasorelaxation(PGF2) response to NO was only slightly inhibited by CBTX + glib (Bracamonte, et al., 1999) ovPA (perinatal) vasodilation(in vivo) response to NO was reduced by TEA, KT-5823 and ryanodine (Saqueton, et al., 1999) mTibA vasodilation(m.t.) response to DEA-NO (1 µM, NOT 10 µM) was sensitive to IBTX (Sausbier, et al., 2000)

gpAo vasorelaxation(NA) response to NOR3 was partially inhibited by IBTX (Tanaka, et al., 2000) rMA vasorelaxation(PE) response to DEA-NONOate was attenuated by IBTX, if ODQ present (Sampson, et al., 2001) horsePenA vasorelaxation(PE) response to SNAP was attenuated by IBTX and PKG inhibition (Prieto, et al., 2006) ANP; CNP bAo channel activation(c-att.) ANP increased channel open probability (Williams, et al., 1988)

Stimulus Model Response studied* Observation / Mechanism Reference channel activation(w-c) ANP and CNP increased IBTX-sensitive current; pCorAendo(- ) (Liang, et al., 2010) vasorelaxation(PGF2; ET) response to CNP was attenuated by IBTX and glib ANP; CNP canCA vasorelaxation(K(+)) response to ANP was attenuated by CBTX (Taniguchi, et al., 1993) d (cont ) rCorA vasorelaxation(m.t. + U46619) response to ANP was attenuated by TEA (1 mM) (Price & Hellermann, 1997) rMAendo(-) vasorelaxation(PGF2) response to ANP was attenuated by IBTX (Tanaka, et al., 1998) CGRP rPialA vasodilation(in vivo) response was attenuated by CBTX and glib (Hong, et al., 1996) channel activation(w-c); PACAP activated STOCs; PACAP rCerebrA (Koide, et al., 2014) vasodilation(m.t.) response was attenuated by paxilline AM pregnant rMA vasorelaxation(NA) response was attenuated by paxilline and IBTX (Ross & Yallampalli, 2006) VIP rMAendo(-) vasorelaxation(PE) response was attenuated by IBTX (Tanaka, et al., 1999) opioids pPialA vasodilation(in vivo) responses to opioid receptor agonists were attenuated by IBTX (Armstead, 1997a) pCorA, hCorA channel activation(w-c; c-att.) 17-estradiol increased activity, via PKG (White, et al., 1995; White, et al., 2002) estrogen channel activation(w-c; i-o) 17-estradiol slightly decreased macroscopic current; rCorA (Wellman, et al., 1996) vasodilation(m.t.) estrogen response involved an endothelium/NO/PKG pathway

*: when isolated artery is studied, mode of contraction/constriction (isolated artery); patch-clamp modes are given in superscript when relevant;

Abbreviations: for species: b: bovine; can: canine; gp: guinea pig; h: human; ov: ovine; p: porcine; rab: rabbit; r: rat; for vascular beds: Ao: aorta; BA: basilar artery; CarA: carotid artery; CerebrA: cerebral artery; Chor : choroidal vascular bed; CorA: coronary artery; FV: femoral vein; LV: lymphatic vessels; MA: mesenteric artery; MCA: middle cerebral artery; PenA: penial artery; PialA: pial artery; PA: pulmonary artery; RadA: radial artery; RetA: retinal artery; SV: saphenous vein; TA: tail artery; TibA: tibialis artery; for patch-clamp modes: c-att.: cell-attached; i-o: inside-out; mb bil.: channel reconstituted in membrane bilayers; pp.: perforated patch; w-c: whole-cell; cultd : cultured; for precontraction protocols: m.t.: myogenic tone; ET: endothelin-1; hist: histamine; meth.: methoxamine; NA: noradrenaline; PE: phenylephrine; PGF2prostaglandin F2; ser.: serotonin; endo(-): endothelium disrupted; other abbreviations: ACh: acetylcholine; AM: adrenomedullin; glib: glibenclamide; 2-AR: 2-adrenergic receptor; BK: bradykinin; CBTX : charybdotoxin; DEA-NO: diethylamine NONOate; DxR: dopamine receptor (subtype 1 or 2); FSK: forskolin; GLP-1: glucagon-like

76 peptide-1; HT: arterial hypertension; IBTX: iberiotoxin; IKCa: Ca-activated, K+ current; ISO: isoprenaline; MB: methylene blue; PKA: cAMP-dependent protein kinase; PKAc: catalytic subunit of PKA; PKG: cGMP-dependent protein kinase; sGC: soluble guanylyl cyclase; SNP: sodium nitroprusside; STOC: spontaneous transient outward current; TEA: tetraethylammonium salt; UTP: uridine triphosphate.

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Figure legends:

Figure 1: Overview of CN-regulated pathways that regulate vascular tone effectors.

Cellular effector Observation / mechanism (pointed arrows denote activation, blunt-end arrows inhibition) PKG induces phosphorylation of IP3R. (Komalavilas & Lincoln, 1996). (1) Phosphorylation of IRAG by PKG1 reduces agonist-induced Ca2+-release. IP3R, IRAG (Schlossmann, et al., 2000). IRAG ablation reduces ability of cGMP to decrease agonist-evoked contraction or Ca2+ release. (Desch, et al., 2010; Geiselhoringer, et al., 2004). Cyclic GMP-PKG inhibits PLC via RGS2; but inhibition of NOS has no influence on IP3 accumulation in whole

(2) arteries. PLC (Hirata, et al., 1990; Osei-Owusu, et al., 2007; Rapoport, 1986; Tabernero, et al., 1996). SERCA inhibition inhibits relaxation evoked by cGMP- and cAMP-elevating agents. (Andriantsitohaina, et al., 1995; Cohen, et al., 1999; Luo, et al., 1993; Mundina-Weilenmann, et al., 2000). NO reduces SOCE secondarily to an increase in Ca2+ reuptake by SERCA in SMCs.

concentrations

2+ (3) (Cohen, et al., 1999). SERCA, PLB Activation of SERCA by NO involves cGMP-independent mechanisms, including a S-glutathionylation of SERCA. (Adachi, et al., 2004; Van Hove, et al., 2009). Phosphorylation of PLB by PKA and PKG (and also PKC) at ser-16 is associated with vasorelaxation. (Colyer, 1998; Simmerman & Jones, 1998) (Cornwell, et al., 1991; Karczewski, et al., 1998). 8-Br-cGMP increases NCX activity in cultured VSMCs. (Furukawa, et al., 1991). 2+ 2+ + (5) NCX contribution to Ca efflux may be low at physiological [Ca ]i and [Na ]. NCX (Furukawa, et al., 1988; Karaki, et al., 1997). 2+ NCX is not involved in NO-induced decrease in [Ca ]i. (Cohen, et al., 1999). PMCA is activated by cGMP and PKG, but not PKA. The phosphorylation of phosphatidylinositol by PKG is suggested

mechanisms decreasing cytosolic Ca cytosolic decreasing mechanisms 2+ to mediate this stimulation. PMCA is not involved in NO-induced decrease in [Ca ]i. (4) (Cohen, et al., 1999; Furukawa & Nakamura, 1987; Furukawa, et al., 1988; Popescu, et al., 1985; Rashatwar, et al., 1987; Vrolix, et PMCA al., 1988).

PMCA is not a substrate of PKG in SMCs sarcolemma. (Baltensperger, et al., 1988; Yoshida, et al., 1992). (6) See sections 3, 4, 5, Tables 1-4 and Figure 2. Ion channels Phosphorylation of MLCK by PKA (ser-1760) reduces MLCK activity and decreases affinity of calmodulin for MLCK, (7) provided it is not bound to calmodulin. MLCK (Adelstein, et al., 1978; Conti & Adelstein, 1981; de Lanerolle, et al., 1984; Raina, et al., 2009).

2+ PKGI and PKA phosphorylate MYPT1. Phosphorylation by PKG at ser-695 hampers the phosphorylation of thr-696 by Rho kinase. (Nakamura, et al., 2007; Surks, et al., 1999; Wooldridge, et al., 2004). (8) MLCP inhibition abolishes cGMP-mediated relaxation. MLCP (Nakamura, et al., 2007). Activation of MLCP is promoted by an interaction between MYPT1 and PKGI. (Surks, et al., 1999). Inhibitory phosphorylation of RhoA by PKG at ser-188.

mechanisms decreasing mechanisms

sensitivity to cytosolic Ca cytosolic to sensitivity (9) (Sauzeau, et al., 2000). RhoA EPAC signalling inhibits RhoA. (Zieba, et al., 2011). Abbreviations: EPAC: exchange protein activated by cAMP; IP3R: IP3 receptor; IRAG: IP3R-associated cGMP kinase substrate; MLCK: myosin

light chain kinase; MLC20: 20-kDa subunit of myosin light chain; MLCP: myosin light chain phosphatase; NCX: Na+, Ca2+ exchanger; NO:

nitric oxide; NOS: nitric oxide synthase; NPR: natriuretic peptide receptor; PDE: phosphodiesterase; PKA: cAMP-activated protein kinase;

PKG: cGMP-activated protein kinase; PKC: protein kinase C; PLB: phospholamban; PLC: phospholipase C; PMCA: plasma membrane Ca2+-

ATPase; RGS2: regulator of G protein signaling-2; SERCA: sarcoplasmic reticulum Ca2+-ATPase; SMC: smooth muscle cell; SOCE: store-

operated Ca2+ entry.

Figure 2: Ion channel regulation by cGMP-PKG and cAMP-PKA/EPAC signalling in vascular cells.

Legend Ion channel and regulatory mechanism (pointed arrows denote activation, blunt-end arrows inhibition) SOCE (resulting from activities of Orai1, TRP and other channels activity) in SMCs are inhibited by both cAMP-PKA and (1) cGMP-PKG pathways. See Table 1 & sections 3.3, 3.4. 2+ Coupling between TRPC1/TRPV4, Ca sparks and BKCa channel is inhibited by the NO-cGMP-PKG axis. (2) See section 3.4, (Zhang, et al., 2014b). In endothelial cells, NO-cGMP-PKG pathway inhibits SOCE. See section 3.3, (Dedkova & Blatter, 2002; Kwan, et al., 2000). (3) In endothelial cells, TRPV4 is activated by PKA. See section 3.4, (Cao, et al., 2018; Zheng, et al., 2013). Cyclic GMP activates a Ca2+-activated, Cl- channel, possibly made up of bestrophin-3 proteins. (4) See section 4. In vascular SMCs CaV1.2 channel is generally activated by cAMP-PKA, while it is inhibited by cGMP-PKG pathways. (5) See section 3.1.

In vascular SMCs CaV3 channels (TTCC) are inhibited by cAMP-PKA and cGMP-PKG pathways. (6) See section 3.2, (Harraz, et al., 2013; Harraz & Welsh, 2013; Howitt, et al., 2013). In vascular SMCs K+ channels are generally activated by both cAMP-PKA and cGMP-PKG pathways. See section 5 and Tables 2-4. (7) EPAC inhibits KATP channel while it stimulates KV7 activity. See section 5, (Purves, et al., 2009; Stott, et al., 2016). In endothelium, adenosine and CGRP receptors stimulation activate KATP channels. (8) See section 5.2.2, (Aziz, et al., 2017; Norton & Segal, 2018). 2+ In vascular SMCs, cAMP-PKA and NO donors (via PKG?) activates Ca sparks and coupled BKCa channel activity, possibly by stimulating SERCA. (9) See section 5.4.2, (Porter, et al., 1998; Wellman, et al., 2001). 2+ In vascular SMCs, EPAC stimulation activates Ca sparks and associated BKCa channel activity. See section 5.4.2, (Humphries, et al., 2017; Roberts, et al., 2013). KCNK3 channel is stimulated by cAMP-PKA and possibly by cGMP-PKG pathways. (10) See section 5.5. 2+ + Abbreviations: Best3: bestrophin-3; BKCa: large conductance, Ca -activated, K channel; CGRP: calcitonin gene-related peptide; EPAC:

+ + + exchange protein activated by cAMP; KATP: ATP-sensitive, K channel; Kir: inward rectifier, K channel; KV: voltage-dependent K channel; NO

nitric oxide; PKA: cAMP-activated protein kinase; PKG: cGMP-activated protein kinase; RyR: ryanodine receptor; SERCA: sarcoplasmic

reticulum Ca2+-ATPase; SOC: store-operated channel; SOCE: store-operated Ca2+ entry; TRP: transient receptor potential; TTCC: T-type Ca2+

channel.

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