Arteriolar VSMCs express BK_Ca channels that provide negative-feedback regulation of myogenic tone (Figure 1). Both membrane depolarization and increases in intracellular Ca²⁺ activate BK_Ca (Tykocki et al., 2017), and because of their large conductance (~200 pS), they powerfully dampen the excitation of VSMCs, preventing vasospasm. BK_Ca channels consist of a tetramer of K_Ca1.1 α-pore-forming subunits (gene=KCNMA1) which have seven transmembrane spanning domains (Meera et al., 1997; Figure 2A). Voltage is sensed by positively charged amino acids in membrane spanning domains S2, S3, and S4 (Ma et al., 2006; Figure 2A), while Ca²⁺ is sensed by two regulator of conductance of K⁺ (RCK) domains (RCK1 and RCK2) in the long, cytosolic C-terminus of the α-subunit (see (Hoshi et al., 2013a) for references; Figure 2A).

Vascular smooth muscle cells express both β and γ subunits that modulate the function of the BK_Ca channel α-pore-forming subunits (Figure 2A). The primary β subunits in VSMCs are β1 (KCNMB-1, K_Caβ1; Tykocki et al., 2017; Figure 2A). These subunits modulate channel gating kinetics and increase the Ca²⁺ sensitivity of the α-subunit (McCobb et al., 1995; McManus et al., 1995; Meera et al., 1996; Tseng-Crank et al., 1996). They also are dynamically trafficked to the cell membrane from Rab11A-positive recycling endosomes, providing the ability of VSMCs to tune BK_Ca channel function (see (Leo et al., 2014, 2017) for details). The expression of β1-subunits may be downregulated during disease states like hypertension (Amberg et al., 2003; Tajada et al., 2013) and diabetes (McGahon et al., 2007), decreasing the ability to activate VSMC BK_Ca channels, increasing myogenic tone. The BK_Ca channel agonists dehydrosoyasaponin I (McManus et al., 1995) and 17β-estradiol require expression of β1-subunits (Valverde et al., 1999). Thus, β1-subunits control the Ca²⁺ sensitivity and the pharmacology of BK_Ca channels in VSMCs.

Arteriolar VSMC BK_Ca channels have a high Ca²⁺ setpoint requiring >3μM cytosolic Ca²⁺ ([Ca²⁺]_in) to open at negative, physiological membrane potentials (−30 to −40mV; Jackson and Blair, 1998). For reference, global [Ca²⁺]_in measured with Fura-2 in arterioles with myogenic tone is on the order of 300–400nM (Brekke et al., 2006). Patch clamp studies have shown that arteriolar BK_Ca channels are silent when VSMCs are dialyzed with solutions containing 300nM [Ca²⁺]_in (Jackson, 1998), consistent with a high [Ca²⁺]_in threshold for their activation. The high Ca²⁺ setpoint (threshold) in arteriolar VSMCs may be due to lower expression of the β₁-subunits (Yang et al., 2009, 2013) and possible differences in expression of spliced variants of the α-pore-forming subunit (Nourian et al., 2014) compared to VSMCs in larger arteries.

There also are γ-subunits associated with BK_Ca channels that are leucine-rich-repeat-containing proteins (LRRCs; Yan and Aldrich, 2010; Almassy and Begenisich, 2012; Evanson et al., 2014; Gonzalez-Perez et al., 2014; Figure 2A). LRRCs allow activation of BK_Ca channels at negative membrane potentials, even in the absence of Ca²⁺, by shifting their voltage vs. activity relationships to the left (increasing their voltage-sensitivity), facilitating their negative feedback function (Yan and Aldrich, 2010; Gonzalez-Perez et al., 2014). The BK_Ca channel sensitivity to activation by docosahexaenoic acid (DHA) also is increased by LRRCs (Hoshi et al., 2013b). The role played by LRRCs in arteriolar VSMCs has not been studied.

BK_Ca channels provide strong negative feedback regulation of both pressure-induced and agonist-induced tone in resistance arteries and arterioles [see (Tykocki et al., 2017) for numerous references]. However, there is regional heterogeneity in the source of Ca²⁺ that activates BK_Ca channels in resistance arteries versus arterioles. In most resistance arteries, BK_Ca channels are controlled by Ca²⁺ sparks which represent the simultaneous release of Ca²⁺ from the ER through small, clustered groups of RyRs (Nelson et al., 1995). Vascular smooth muscle cells that utilize this mechanism of BK_Ca channel activation display the so-called spontaneous-transient-outward currents (STOCs): bursts of activity of small groups of BK_Ca channels coinciding with the RyR-based Ca²⁺ sparks [(Nelson et al., 1995), see (Tykocki et al., 2017) for additional references]. In VSMCs where this mechanism is active, pharmacological block of RyRs produces the same effect as block of BK_Ca channels.

In contrast to many larger resistance arteries, Ca²⁺ influx through VGCCs directly activates BK_Ca channels in skeletal muscle arteriolar VSMCs; RyRs are silent, at least under the conditions studied (Westcott and Jackson, 2011; Westcott et al., 2012). In resistance arteries immediately upstream from skeletal muscle arterioles, both RyR-dependent and VGCC-dependent control of BK_Ca channels is apparent (Westcott and Jackson, 2011; Westcott et al., 2012). These data suggest that there may be a spectrum of control mechanisms that are involved in Ca²⁺-dependent control of BK_Ca channels in the resistance vasculature. In cerebral penetrating arterioles, both RyRs and BK_Ca channels are silent at rest, but both can be activated by low pH (Dabertrand et al., 2012). The molecular mechanisms underlying pH-sensitive recruitment of RyR-control of BK_Ca channels has not been established. The mechanisms responsible for the differences in Ca²⁺ sources that control BK_Ca channels are not known, but likely relate to the number and location of BK_Ca channels expressed relative to RyRs, VGCCs and other ion channels.

Ryanodine receptors are composed of four, >500kDa subunits that form ryanodine-sensitive Ca²⁺ channels in ER membranes (Figure 2B; Van Petegem, 2015; Yan et al., 2015; Zalk et al., 2015). Increases in [Ca²⁺]_in from resting levels [~300nM in VSMCs with tone (Brekke et al., 2006).] up to ~10μM activate release of Ca²⁺ through RyRs, although high levels of [Ca⁺]_in (>10μM) are inhibitory (Tykocki et al., 2017). Ryanodine receptors also serve as scaffolds for a plethora of signaling proteins [see (Tykocki et al., 2017) for numerous references]. There are three isoforms of RyRs, RyR1, RyR2 and RyR3 [genes=RYR1, RYR2 and RYR3, respectively (Lanner et al., 2010)]: RyR1 is predominantly expressed in skeletal muscle, RyR2 is expressed in cardiac muscle and RyR3 is expressed in the brain and other tissues (Ledbetter et al., 1994; Giannini et al., 1995; Reggiani and te Kronnie, 2006). Vascular smooth muscle expresses multiple isoforms of RYRs with considerable vessel-to-vessel heterogeneity (Vallot et al., 2000; Yang et al., 2005; Salomone et al., 2009; Vaithianathan et al., 2010; Westcott and Jackson, 2011; Westcott et al., 2012). In VSMCs of skeletal muscle arterioles, RyR2 is predominate, and RyR1 is absent (Westcott et al., 2012).

Ryanodine receptors are highly regulated proteins that are modulated by phosphorylation, cellular redox status and interactions with many binding partners in addition to [Ca²⁺]_in (see Tykocki et al., 2017). The overall function of RyRs depends on exactly where they are located in cells and with which ion channels and other proteins they interact.

The elemental Ca²⁺ signal generated by RyRs is the Ca²⁺ spark which represents the simultaneous release of Ca²⁺ from small clusters of RyRs as noted in Section VSMC BK_Ca Channels and the Regulation of Arteriolar Tone. Calcium influx through VGCCs has been shown to indirectly regulate Ca²⁺ spark frequency and amplitude by effects on global [Ca²⁺]_in and ER Ca²⁺ store loading (Essin et al., 2007). Subsequent studies have shown that the magnitude of Ca²⁺ influx through the persistent activity of membrane clusters of VGCCs, that can be recorded as VGCC Ca²⁺ sparklets (Navedo et al., 2005; Amberg et al., 2007), controls the amplitude of Ca²⁺ sparks (Tajada et al., 2013). These data suggest that local influx of Ca²⁺ is a major determinant of RyR activity in VSMCs.

In skeletal and cardiac muscle, RyRs act in a positive-feedback manner through Ca²⁺-induced-Ca²⁺-release (CICR) to cause explosive release of Ca²⁺ from the ER and subsequent muscle contraction. In both skeletal muscle and cardiac muscle, Ca²⁺ sparks form the basis of this positive feedback process. A similar positive feedback role for Ca²⁺ sparks has been proposed for some arteriolar VSMCs (Curtis et al., 2004, 2008; Fellner and Arendshorst, 2005, 2007; Balasubramanian et al., 2007; Tumelty et al., 2007; Kur et al., 2013). In addition to Ca²⁺ sparks, RyRs can cooperate with IP₃Rs and contribute to Ca²⁺ waves and the positive regulation of myogenic tone in some resistance arteries (Jaggar, 2001; Mufti et al., 2010, 2015; Westcott and Jackson, 2011; Westcott et al., 2012). In other VSMCs, RyR-dependent Ca²⁺ sparks may also act in an excitatory fashion by activating plasma membrane CaCCs producing the so-called spontaneous transient inward currents (STICs) that cause membrane depolarization, VGCC activation and an increase in tone (ZhuGe et al., 1998; Cheng and Lederer, 2008).

As outlined in Section VSMC BK_Ca Channels and the Regulation of Arteriolar Tone, in many resistance arteries upstream from the microcirculation, RyRs function as part of a negative-feedback process limiting VSMC excitability. In these vessels, RyR-dependent Ca²⁺ sparks are functionally coupled to BK_Ca channels producing membrane hyperpolarization, VGCC deactivation and a decrease in tone (Nelson et al., 1995; Jaggar et al., 1998; Cheng and Lederer, 2008).

However, in skeletal muscle (Westcott and Jackson, 2011; Westcott et al., 2012), cerebral (Dabertrand et al., 2012), and ureteral (Borisova et al., 2009) arterioles downstream from resistance arteries, RyRs are not active and do regulate myogenic tone. Low pH has been shown to recruit RyR-dependent Ca²⁺ sparks in cerebral arterioles, thereby activating BK_Ca channels and mediating dilation (Dabertrand et al., 2012). Whether RyRs can be recruited by pH or other conditions in skeletal muscle or ureteral VSMCs has not been studied.

The mechanisms responsible for the heterogeneity in RyR function are not known but most likely result from the specific pattern and magnitude of RyR isoform expression, their cellular localization, and the expression and localization of other ion channels (for example, CaCC vs. BK_Ca channels) in the plasma membrane over RyRs. This area of research should be explored in more detail in the future.

Inositol 1,4,5 trisphosphate receptors are homotetramers that, like RyRs, form large (~310kDa) Ca²⁺ release channels in ER membranes (Foskett et al., 2007; Figure 2C). There is one binding site for IP₃ on each IP₃R monomer (Foskett et al., 2007; Seo et al., 2012, 2015; Taylor et al., 2014).

Three isoforms of IP₃Rs (IP₃R1, IP₃R2, and IP₃R3) arise from three genes (ITPR1, ITPR2 and ITPR3 respectively; Foskett et al., 2007). There is regional heterogeneity in VSMC IP₃R expression and multiple isoforms are usually expressed in a given VSMC (see (Narayanan et al., 2012) for review). In VSMCs from skeletal muscle resistance arteries and downstream arterioles, we have found expression of IP₃R1>IP₃R2 >>IP₃R3 (Westcott et al., 2012).

Like RyRs, IP₃Rs can be triggered to open by increases in [Ca²⁺]_in, with IP₃ affecting the sensitivity of the channels to CICR [see (Tykocki et al., 2017) for review]. In the presence of IP₃, IP₃Rs display a bell shaped [Ca²⁺]_in-response relationship with high [Ca²⁺]_in (>1μM) inhibiting Ca²⁺ release through these channels (Tu et al., 2005). IP₃Rs serve as amplifiers of Ca²⁺ signals generated by other ion channels. They have a number of protein binding partners that modulate their function including FKBP12 (MacMillan et al., 2005), RACK1 (Patterson et al., 2004; Foskett et al., 2007), ankyrin (Hayashi and Su, 2001), Homer (Tu et al., 1998; Foskett et al., 2007), Bcl family members (Bcl-x_L, Mcl and Bcl-2; Li et al., 2007; Eckenrode et al., 2010) and, importantly, a number of TRPC channels including TRPC1 (Boulay et al., 1999), TRPC3 (Boulay et al., 1999; Kiselyov et al., 1999), TRPC4 (Mery et al., 2001), TRPC6 (Boulay et al., 1999) and TRPC7 (Vazquez et al., 2006), either directly (Boulay et al., 1999) or as a component of larger protein complexes (Yuan et al., 2003).

Vascular smooth muscle IP₃Rs are essential for the initiation and maintenance of myogenic tone in resistance arteries (Osol et al., 1993; Gonzales et al., 2010, 2014; Garcia and Earley, 2011) and some, but not all arterioles (Jackson and Boerman, 2017). Three mechanisms have been proposed to account for pressure-dependent activation of IP₃Rs in resistance arteries including angiotensin receptor-mediated (Gonzales et al., 2014), or integrin-mediated (Mufti et al., 2015) activation of PLCγ₁, angiotensin receptor-mediated activation of PLCβ (Mederos y Schnitzler et al., 2008; Schleifenbaum et al., 2014), or mechanisms involving membrane depolarization-induced activation of G_q-coupled receptors (Ganitkevich and Isenberg, 1993; del Valle-Rodriguez et al., 2003; Urena et al., 2007; Mahaut-Smith et al., 2008; Liu et al., 2009; Fernandez-Tenorio et al., 2010; Yamamura et al., 2012).

In contrast, myogenic tone in hamster cheek pouch arterioles (Jackson and Boerman, 2017) and in murine 4^th-order mesenteric arteries (Mauban et al., 2015) does not depend on IP₃ and activation of IP₃Rs. Phospholipase-mediated hydrolysis of phosphatidylcholine and subsequent production of diacylglycerol was proposed to participate in the generation and maintenance of myogenic tone in murine 4^th-order mesenteric arteries (Mauban et al., 2015).

Myogenic tone in rat cerebral resistance arteries is accompanied by an increase in the frequency of Ca²⁺ waves (Jaggar, 2001; Mufti et al., 2010, 2015) that involve both IP₃Rs (Mufti et al., 2015) and RyRs (Jaggar, 2001; Mufti et al., 2010, 2015). Similarly, Ca²⁺ waves in skeletal muscle resistance arteries depend on both RyRs and IP₃Rs (Westcott and Jackson, 2011; Westcott et al., 2012). In contrast, Ca²⁺ waves in downstream skeletal muscle arterioles depend only on Ca²⁺ release from IP₃Rs (Westcott and Jackson, 2011; Westcott et al., 2012) that may amplify Ca²⁺ influx through VGCCs (Jackson and Boerman, 2018). However, in rat (Miriel et al., 1999) and mouse (Zacharia et al., 2007) mesenteric resistance arteries, Ca²⁺ waves were inhibited as myogenic tone developed. Thus, there appears to be regional heterogeneity in the role played by IP₃R in the development and maintenance of myogenic tone. The mechanisms responsible for the heterogeneity in function of IP₃Rs among blood vessels has not been established but likely stems from differences in the IP₃R isoforms that are expressed; their localization and interactions with other proteins; and their proximity to other ion channels.

VSMCs also express CaCCs that may contribute to myogenic tone. The protein anoctamin-1 (gene=ANO1), also known as transmembrane member 16A (TMEM16A), appears to be the molecular basis of CaCCs in VSMCs (Ji et al., 2019). This protein exists as a homodimer with each monomer having 10 membrane spanning domains (S1-S10), with the pore being formed by S3-S7 helices which also contains a Ca²⁺ binding domain (Ji et al., 2019; Figure 2E). TMEM16A demonstrates a synergistic dependence on voltage and Ca²⁺ to control its activity, with depolarization and increases in [Ca²⁺]_in leading to opening of these channels (Ji et al., 2019). In vascular smooth muscle, [Cl⁻]_in is elevated due to intracellular Cl⁻ accumulation from the activities of the Cl⁻/HCO₃⁻ exchanger and the Na⁺/K⁺/Cl⁻ co-transporter (Matchkov et al., 2013). The elevated [Cl⁻]_in sets the equilibrium potential for Cl⁻ [−40 to −25mV, (Matchkov et al., 2013)] to be positive to the resting membrane potential [−45 to −30mV, (Tykocki et al., 2017)] of VSMCs that develop myogenic tone. Therefore, opening of a Cl⁻ channel results in an outward Cl⁻ current (an inward current in electrophysiological terms), membrane depolarization, activation of VGCCs and an increase in tone (Matchkov et al., 2013).

Calcium-activated chloride channels contribute to agonist-induced tone in a variety of arteries (Bulley and Jaggar, 2014). In addition, STICs carried by Cl⁻ and coupled to RyR-mediated Ca²⁺ sparks or IP₃-based Ca²⁺ waves have been reported (Bulley and Jaggar, 2014). Cerebral resistance artery VSMCs express TMEM16A that are functionally coupled to transient receptor potential C-family member 6 (TRPC6) channels. Calcium influx through TRPC6 activates TMEM16A contributing to the membrane depolarization, VGCC activation and pressure-induced myogenic tone in these vessels (Bulley et al., 2012; Wang et al., 2016). In hamster cheek pouch arterioles, CaCCs appear to contribute to myogenic tone when VGCCs are active (Jackson, 2020), suggesting that CaCCs may be functionally coupled to VGCCs in those VSMCs. The molecular identity of CaCCs in hamster cheek pouch arteriolar VSMCs has not been established. Additional research on expression and function of CaCCs in resistance arteries and arterioles appears warranted.

VSMCs express many members of the transient receptor potential (TRP) family of ion channels that contribute to myogenic tone [see (Earley and Brayden, 2015; Tykocki et al., 2017) for more information; Figures 1, 3]. Of these, TRPM4 channels are Ca²⁺-activated and are essential for pressure-induced myogenic tone in cerebral resistance arteries (Gonzales et al., 2014). Like all TRP channels, the pore-forming subunit of TRPM4 channels has six transmembrane domains (S1–S6) which assemble as a tetramer to form a functional ion channel with residues in the intramembrane loop between S5 and S6 forming the channel’s pore (Earley and Brayden, 2015; Figure 2D). A conserved TRP domain located distal to S6 and a TRPM homology region in the NH2 terminus (Earley and Brayden, 2015) distinguish all members of the TRPM family (Earley and Brayden, 2015; Figure 2D). TRPM4 channels selectively conduct monovalent cations such that opening of these channels produces membrane depolarization due primarily to the influx of Na⁺ (Earley and Brayden, 2015). Calmodulin binding sites in the C-terminus of TRPM4 are essential for Ca²⁺-dependent activation and the Ca²⁺-sensitivity of these channels is increased by protein kinase C-dependent phosphorylation in their amino terminus (Earley, 2013). Rho kinase also has been reported to increase the Ca²⁺-sensitivity of TRPM4 channels in cerebral parenchymal arterioles (Li and Brayden, 2017).

In cerebral resistance arteries and arterioles, TRPM4 channels are part of the signal transduction pathway for pressure-dependent myogenic tone (Gonzales et al., 2014; Li et al., 2014; Li and Brayden, 2017; see Figure 3 and Section Integration of Ca²⁺-Dependent Ion Channels Into the Mechanisms Underlying Pressure-Induced Myogenic Tone for more details). In this scheme, TRPM4 channels are activated by release of Ca²⁺ through IP₃Rs into the subplasmalemmal space (Gonzales et al., 2010), with the IP₃Rs being activated by IP₃, formed by mechanosensitive G-protein coupled receptor-mediated stimulation of phospholipase C (PLC)γ₁, and Ca²⁺ entry through TRPC6 channels, likely activated by both pressure and PLCγ₁-production of diacylglycerol (DAG; Gonzales et al., 2014; Figure 3). As noted above, in cerebral parenchymal arterioles, rho-kinase, which also is activated and contributes to myogenic tone, appears to modulate the Ca²⁺ sensitivity of TRPM4 channels (Li and Brayden, 2017; Figure 3). The Na⁺ entry through TRPM4 channels, along with the entry of Ca²⁺ and Na⁺ through TRPC6 channels produces membrane depolarization and activation of Ca²⁺ entry through VGCCs, hallmark elements of pressure-dependent myogenic tone (see (Tykocki et al., 2017) for numerous references; Figure 3). The role of TRPM4 in myogenic tone of vessels in other vascular beds has been questioned because global knockout of TRPM4 has no effect on pressure-induced tone in hind limbs of mice (Mathar et al., 2010). However, the details of the mechanisms responsible for pressure-induced tone in the TRPM4 knockout animals was not determined, such that compensation for the global knockout of TRPM4 channels may have occurred. Additional research on TRPM4 and myogenic tone appears warranted.

Another potentially Ca²⁺-activated ion channel that is involved in regulation of myogenic tone are TRPP1 (PKD2) channels. Similar to TRPM4 channels already described, TRPP1 channels are tetramers of 6 membrane spanning domains encoded by the PKD2 gene that have coiled-coil domains in their C-termini and a Ca²⁺-binding EF-hand motif that may be involved in Ca²⁺-dependent activation of these channels (Giamarchi and Delmas, 2007). The channels formed from TRPP1 are non-selective cation channels that conduct both Ca²⁺ and Na⁺ (Giamarchi and Delmas, 2007). The function of TRPP1 in regulation of myogenic tone is unclear. In murine mesenteric arteries, VSMC TRPP1 channels appear to inhibit myogenic tone (Sharif-Naeini et al., 2009), whereas in rat cerebral arteries VSMC TRPP1 channels significantly contribute to myogenic tone (Narayanan et al., 2013). Conditional knockout of TRPP1 from VSMCs decreases blood pressure and substantially reduces myogenic tone in murine skeletal muscle resistance arteries (Bulley et al., 2018). The plasma membrane expression of TRPP1 in VSMCs is controlled by recycling of sumoylated channels and SUMO1 modification of TRPP1 channels is required for pressure-induced myogenic tone (Hasan et al., 2019). How TRPP1 channels “fit” with other channels that have been shown to be involved in initiation and maintenance of myogenic tone (TRPC6 and TRPM4, for example) remains to be established. Nor has it been established that VSMC TRPP1 channels are activated by Ca²⁺ or that Ca²⁺-dependent activation is part of their role in pressure-dependent myogenic tone. It is known that TRPP1 channels can heterodimerize with other members of the TRP family (Giamarchi and Delmas, 2007) such that it is feasible that TRPP1 channels may be part of a multi-channel complex. Additional research will be required to determine how TRPP1 channels and all of the other VSMC ion channels implicated in the generation and maintenance of myogenic tone fit together.

Voltage-gated Ca²⁺ channels composed of CaV1.2 α-subunits (gene=CACNA1C) play a central role myogenic tone as these channels provide the main source of intracellular Ca²⁺, the primary trigger of VSMC contraction (Tykocki et al., 2017). Calcium-dependent inhibition of VGCCs is mediated by calmodulin that binds to the C-terminus of CaV1.2 channels that make up VSMC VGCCs (Shah et al., 2006). Thus, VGCCs themselves may contribute to the negative-feedback regulation of myogenic tone through this process (Figure 3).

Vascular smooth muscle cells express a diverse array of K_V channels that participate in the negative-feedback regulation of myogenic tone (Tykocki et al., 2017). Early studies showed Ca²⁺-dependent inhibition of K_V channel currents in VSMCs from large arteries (Gelband et al., 1993; Ishikawa et al., 1993; Gelband and Hume, 1995; Post et al., 1995; Cox and Petrou, 1999). However, the molecular identity of the K_V channel isoform that was inhibited was not identified: it was only suspected to be a channel inhibited by 4-amino pyridine (4-AP). Block of K_V channels by 4-AP appears to be Ca²⁺-dependent, making interpretation of 4-AP sensitivity difficult (Baeyens et al., 2014). It is well established that increased [Ca²⁺]_in inhibits K_V7.2-7.5 channels via binding to calmodulin associated with these channels (Alaimo and Villarroel, 2018). K_V7 channels contribute substantially to the regulation of myogenic tone in resistance arteries (Mackie et al., 2008; Greenwood and Ohya, 2009; Jepps et al., 2013; Cox and Fromme, 2016). Therefore, it is likely that at least some of the inhibitory effect of elevated [Ca²⁺]_in on whole-cell K_V currents is through inhibition of K_V7 channels. Regardless, Ca²⁺-dependent inhibition of active K_V channels will cause membrane depolarization, activation of VGCCs and a further increase in [Ca²⁺]_in contributing to the positive-feedback regulation of myogenic tone (Figure 3). It should be noted that the density of K_V channels is such that Ca²⁺-dependent inhibition of these channels serves only to blunt the main, negative-feedback role that K_V channels play in the regulation of myogenic tone (Tykocki et al., 2017; Jackson, 2018).

Elevated [Ca²⁺]_in also inhibits ATP-sensitive K⁺ (K_ATP) channels through Ca²⁺-dependent activation of the protein phosphatase, calcineurin (Wilson et al., 2000). These channels are active at rest in the microcirculation of a number of vascular beds (Tykocki et al., 2017). Closure of K_ATP channels by increased Ca²⁺ would contribute to membrane depolarization, activation of VGCCs, and a further increase in [Ca²⁺]_in, a positive-feedback process that would increase myogenic tone (Figure 3).

Endothelial cells express IP₃Rs that contribute to the negative-feedback regulation of arteriolar myogenic tone. Early EC studies demonstrated that the initial increase in [Ca²⁺]_in in response to agonists of EC Gα_q-coupled receptors resulted from Ca²⁺ release from ER stores (Hallam and Pearson, 1986; Colden-Stanfield et al., 1987; Busse et al., 1988; Schilling et al., 1992; Sharma and Davis, 1994, 1995). Subsequent studies pinpointed IP₃Rs as the primary Ca²⁺ release channel involved in this response (Sharma and Davis, 1995; Cohen and Jackson, 2005).

Endothelial cells from arteries (Mountian et al., 1999, 2001; Grayson et al., 2004; Ledoux et al., 2008) and arterioles (Jackson, 2016) appear to express all three isoforms of IP₃R. However, the dominant isoform may display regional- or species-dependent heterogeneity. For example, IP₃R2 is the dominant IP₃R expressed in mouse mesenteric artery ECs (Ledoux et al., 2008), whereas IP₃R3 is the dominant IP₃R in mouse cremaster muscle arteriolar ECs (Jackson, 2016). There is little information about the specific localization of IP₃R in native arteriolar ECs. In both EC-VSMC co-cultures and in intact mouse cremaster arterioles, IP₃R1 localizes at sites of MEGJs (Isakson, 2008). Similarly, in mouse mesenteric resistance arteries, EC IP₃Rs cluster near holes in the internal elastic lamina (Ledoux et al., 2008), that are sites of myoendothelial projections (MEPs) and MEGJs (Sandow and Hill, 2000; Figure 1). Although the IP₃R isoform(s) expressed in these IP₃R clusters has not been identified, they were demonstrated to be the sites of EC Ca²⁺ pulsars, localized IP₃-dependent Ca²⁺ events arising from clusters of IP₃Rs in the ER that extend into MEPs (Kansui et al., 2008; Ledoux et al., 2008; Figure 1).

Myoendothelial projections and MEGJs are important signaling microdomains in resistance arteries and arterioles and contain a growing list of signaling proteins including IP₃Rs (Kansui et al., 2008; Ledoux et al., 2008), IK_Ca channels (Sandow et al., 2006), TRPA1 channels (Earley et al., 2009a), TRPV4 channels (Sonkusare et al., 2012, 2014), anchoring proteins [e.g., AKAP150 (Sonkusare et al., 2014)], protein kinases [e.g., PKC (Sonkusare et al., 2014)], NO synthase (Straub et al., 2011; Wolpe et al., 2021), Na⁺/K⁺ ATPase (Dora et al., 2008) and other proteins (Straub et al., 2014; Wolpe et al., 2021; Figure 1). Calcium influx through TRPA1 and TRPV4, which produce small, localized Ca²⁺ events called Ca²⁺ sparklets, likely serves as the source of Ca²⁺ that actually triggers release of Ca²⁺ through IP₃Rs to form both localized Ca²⁺ pulsars (Kansui et al., 2008; Ledoux et al., 2008), Ca²⁺ wavelets (Tran et al., 2012) and larger Ca²⁺ waves (Duza and Sarelius, 2004; Kansui et al., 2008) found in ECs of resistance arteries and arterioles. These Ca²⁺ events are then translated into several signals that are vasodilatory and tend to reduce or temper myogenic tone. Activation of EC sK_Ca and IK_Ca channels (Section EC sK_Ca and IK_Ca Channels and Arteriolar Tone, below) leads to EC hyperpolarization, which can be conducted through MEGJs to overlying VSMCs, deactivating VGCCs, reducing VSMC Ca²⁺ influx and decreasing myogenic tone (Figure 3). Endothelial cell IP₃R Ca²⁺ signals also activate EC NO synthase and production of other EC autacoids (PGI₂, EETs, H₂O₂, etc.) that diffuse to overlying VSMCS and reduce myogenic tone (Figure 3).

Global increases in [Ca²⁺]_in reported for ECs in intact resistance arteries or arterioles exposed to endothelium-dependent vasodilators (Dora et al., 1997; Marrelli, 2000; Cohen and Jackson, 2005; Socha et al., 2011) are a complicated blend of IP₃R-mediated Ca²⁺ pulsars, Ca²⁺ wavelets and Ca²⁺ waves. Both the number and frequency of Ca²⁺ pulsars (Ledoux et al., 2008) and both synchronous (Duza and Sarelius, 2004; Socha et al., 2012) and asynchronous (Ledoux et al., 2008; Socha et al., 2012) Ca²⁺ waves are increased by endothelium-dependent vasodilators, such as acetylcholine (Ledoux et al., 2008; Socha et al., 2012) or adenosine (Duza and Sarelius, 2004). Additional research will be required to discover the precise IP₃R isoform expression, location and function related to endothelium-dependent vasomotor activity and modulation of myogenic tone.

Early studies of ECs from large arteries provided evidence for expression of functional RyRs (Lesh et al., 1993; Graier et al., 1994, 1998; Ziegelstein et al., 1994; Rusko et al., 1995; Kohler et al., 2001b). In contrast, there is a lack of evidence for expression of RyRs in resistance artery and arteriolar ECs. Mouse mesenteric resistance artery ECs do not express mRNA for the three RyR isoforms, whereas transcripts for IP₃Rs are readily detected (Ledoux et al., 2008). In addition, resting Ca²⁺ levels or acetylcholine-evoked Ca²⁺ events in mouse (Ledoux et al., 2008) or rat (Kansui et al., 2008) mesenteric resistance artery ECs are unaffected by concentrations of ryanodine that block RyRs. Similarly, mouse cremaster arteriolar ECs do not express message for RyRs (Jackson, 2016), and the RyR agonist, caffeine (10mM), has no effect on [Ca²⁺]_in in these ECs (Cohen and Jackson, 2005). These data do not support a role for RyRs in resistance artery or arteriolar EC Ca²⁺ signals.

Resistance artery and arteriolar ECs express both sK_Ca (K_Ca2.3; gene=KCNN3) and IK_Ca (K_Ca3.1; gene=KCNN4) channels (Kohler et al., 2001a; Eichler et al., 2003; Taylor et al., 2003; Sandow et al., 2006; Si et al., 2006; Grgic et al., 2009). These channels are a tetramer of six transmembrane domain subunits with cytosolic N- and C-termini (Adelman et al., 2012; Figure 2F). The ion conducting pore is formed from a pore loop between membrane spanning domains 5 and 6, as in voltage-gated K⁺ channels (Adelman et al., 2012). Calmodulin interacts with the intracellular C-terminus to gate opening of both channels (Xia et al., 1998; Fanger et al., 1999; Adelman et al., 2012; Sforna et al., 2018). The Ca²⁺ sensitivity of sK_Ca and IK_Ca channels is an order of magnitude higher than for BK_Ca channels. The threshold for activation by Ca²⁺ binding to calmodulin occurs at 100nM, 50% of maximal activation at 300nM and maximal activation at 1μM for both sK_Ca channels (Xia et al., 1998) and IK_Ca channels (Ishii et al., 1997). The distinct pharmacology of sK_Ca and IK_Ca channels has helped to define their function in intact vessels (Jackson, 2016).

Endothelial cell sK_Ca and IK_Ca channels are not distributed uniformly in the plasma membrane of ECs: IK_Ca channels cluster at MEPs (Sandow et al., 2006; Ledoux et al., 2008; Earley et al., 2009a), the site of MEGJs (Sandow and Hill, 2000), whereas sK_Ca channels are more distributed around the cell periphery (Sandow et al., 2006). Both channels appear to reside in macromolecular signaling complexes. At MEPs and near MEGJ’s, IK_Ca channels localize with IP₃Rs (Ledoux et al., 2008), TRPA1 channels (Earley et al., 2009a), TRPV4 channels (Sonkusare et al., 2012, 2014), anchoring proteins [e.g., AKAP150 (Sonkusare et al., 2014)], protein kinases [e.g., PKC (Sonkusare et al., 2014)], nitric oxide synthase (Straub et al., 2011; Wolpe et al., 2021), Na⁺/K⁺ ATPase (Dora et al., 2008), likely G-protein coupled receptors (Sonkusare et al., 2014) and other proteins (Straub et al., 2014; Wolpe et al., 2021; Figure 1). Local Ca²⁺ signals through TRPA1 channels (Earley et al., 2009a), TRPV4 channels (Sonkusare et al., 2012, 2014), and/or IP₃Rs (Ledoux et al., 2008) activate IK_Ca (and sK_Ca) channels, leading to EC hyperpolarization and conduction of this signal to overlying VSMCs. Hyperpolarization then deactivates VSMC VGCCs reducing myogenic tone (Figure 3). EC hyperpolarization also may amplify Ca²⁺ influx through TRPA1 and TRPV4 channels by increasing the electrochemical gradient for Ca²⁺ influx (Qian et al., 2014).

Endothelial cell sK_Ca channels also exist in macromolecular signaling microdomains around the EC periphery. They are found in cholesterol-rich areas (caveolae or lipid rafts) and colocalize with caveolin-1 (Absi et al., 2007). Ca²⁺ influx through TRPC3 channels selectively activates sK_Ca channels in rat cerebral arteries (Kochukov et al., 2014), suggesting that TRPC3 and sK_Ca channels exist in the same microdomain. In mouse carotid arteries, sK_Ca channels are in caveolae adjacent to EC-EC gap junction plaques (Brahler et al., 2009). Conditional knockout of sK_Ca channels attenuates shear-stress-induced vasodilation in these arteries, suggesting that sK_Ca channel localization has functional consequences (Brahler et al., 2009). The respective EC localization of sK_Ca and IK_Ca channels and their signaling microdomains explain how these two channels mediate different facets of EC hyperpolarization and the regulation of myogenic tone (Crane et al., 2003; Si et al., 2006).

Because ECs are electrically coupled to VSMCs via MEGJs, resting membrane potential of ECs can impact myogenic tone. Resting EC membrane potential is determined, in part, by the activity of sK_Ca and IK_Ca channels. Overexpression of sK_Ca channels (which hyperpolarizes ECs) reduces myogenic tone of mesenteric resistance arteries (Taylor et al., 2003). In contrast, conditional knockout of sK_Ca channels has the opposite effect (EC depolarization and an increase in myogenic tone; Taylor et al., 2003). Consistent with these data, pharmacological inhibition of sK_Ca and IK_Ca channels, or both channels augment(s) myogenic tone in rat cerebral parenchymal arterioles (Cipolla et al., 2009; Hannah et al., 2011). Endothelial cell sK_Ca and IK_Ca channels seem to play a smaller role in modulating myogenic tone of larger cerebral resistance arteries, although they remain important in endothelium-dependent agonist-induced vasodilation (Cipolla et al., 2009). Nonetheless, sK_Ca and IK_Ca channels significantly contribute to EC-dependent negative-feedback regulation of myogenic tone.

Endothelium-dependent vasodilators that act through G_q-coupled receptors also activate sK_Ca and IK_Ca channels. In some vessels, such as guinea-pig carotid artery (Corriu et al., 1996), rat mesenteric arteries preconstricted with phenylephrine (Crane et al., 2003) and porcine coronary arteries (Bychkov et al., 2002) both channels appear to be involved because block of both sK_Ca and IK_Ca channels is necessary to inhibit agonist-induced EC hyperpolarization. In contrast, IK_Ca channels mediate endothelium-dependent hyperpolarization and vasodilation in rat cerebral arteries (Marrelli et al., 2003) and in murine arteries and arterioles (Brahler et al., 2009). The reason for this heterogeneity in the roles played by sK_Ca and IK_Ca channels between vascular beds is not apparent and will require further research.

The expression and function of BK_Ca channels in ECs remains debatable (Sandow and Grayson, 2009). As described for VSMCs, BK_Ca channels are activated by both voltage and Ca²⁺, have a much larger conductance (~250 pS) than sK_Ca and IK_Ca channels, do not require association with calmodulin, and display pharmacology distinct from sK_Ca and IK_Ca channels (Hoshi et al., 2013a; Tykocki et al., 2017). Cultured large artery ECs have been reported to express BK_Ca channels (see (Sandow and Grayson, 2009) for references). Native ECs isolated from hypoxic rats (Hughes et al., 2010; Riddle et al., 2011) or cholesterol depleted ECs (Riddle et al., 2011) express functional BK_Ca channels. In cultured ECs, BK_Ca channels are located in caveolae and caveolin inhibits their function (Wang et al., 2005). These studies open the possibility that EC BK_Ca channels are normally inhibited. Conversely, chronic hypoxia, and potentially other stresses or pathologies, that alter membrane lipid domains may upregulate EC BK_Ca channel function (Sandow and Grayson, 2009).

Electrophysiological studies of freshly isolated bovine coronary artery (Gauthier et al., 2002), mouse carotid artery (Brahler et al., 2009), and rat cerebral parenchymal arteriolar (Hannah et al., 2011) ECs found only sK_Ca channel and IK_Ca channel currents; no BK_Ca channel currents were detected. While it has been reported that ECs in freshly isolated rat cremaster arterioles express protein for BK_Ca channels (Ungvari et al., 2002), neither mRNA nor protein for this channel were detected in bovine coronary artery ECs (Gauthier et al., 2002). Murine skeletal muscle resistance artery and arteriolar ECs lack BK_Ca channel mRNA (Jackson, 2016). Thus, there may be regional or species heterogeneity in EC expression of BK_Ca channels. Additional research appears to be warranted to define if and where EC BK_Ca are expressed, how they are regulated and their function in the regulation of myogenic tone.

Electrophysiological studies of bovine pulmonary artery and human umbilical vein ECs demonstrate the functional expression of CaCCs (Nilius et al., 1997; Zhong et al., 2000). Unlike VSMCs (see Section VSMC Ca²⁺-Activated Cl⁻ Channels and Arteriolar Tone), initial studies did not report expression of TMEM16A in ECs in lung sections (Huang et al., 2009; Ferrera et al., 2011). However, more recent studies have identified TMEM16A expression and function in human pulmonary artery ECs and have shown that over expression of these channels leads to EC dysfunction (Skofic Maurer et al., 2020). In hypertension, EC TMEM16A also contributes to endothelial dysfunction (Ma et al., 2017). TMEM16A is expressed in murine cerebral capillary ECs where it regulates membrane potential, Ca²⁺ signaling, proliferation, migration, and blood brain barrier permeability (Suzuki et al., 2020). Block of TMEM16A preserves blood brain barrier function after ischemic stroke (Liu et al., 2019). Hypoxia stimulates proliferation of brain capillary ECs via increased expression of TMEM16A (Suzuki et al., 2021). Hypoxia also increases expression of TMEM16A in mouse cardiac ECs (Wu et al., 2014).

The function of TMEM16A in arteriolar ECs related to regulation of myogenic tone is not clear. In murine capillary ECs, block of TMEM16A results in membrane hyperpolarization suggesting that in ECs, like in VSMCs (see Section VSMC Ca²⁺-Activated Cl⁻ Channels and Arteriolar Tone), activation of these CaCCs leads to membrane depolarization, counter to the effects of activation of EC sK_Ca and IK_Ca channels which produce robust EC hyperpolarization. Thus, it may be that CaCCs in ECs are part of a negative feedback mechanism to dampen membrane hyperpolarization induced by EC sK_Ca and IK_Ca channels when intracellular Ca²⁺ is elevated.

Transient receptor potential vanilloid-family member 4 channels are another prominent Ca²⁺-modulated ion channel expressed in ECs (Sonkusare et al., 2012, 2014; Hong et al., 2018; Chen and Sonkusare, 2020). These channels are formed from a tetramer of six membrane spanning domain subunits, with the pore of the channel formed by a pore-loop between domains 5 and 6 like many other ion channels (Figure 2G). They conduct primarily Ca²⁺ and are activated by a diverse array of chemicals including EETs (Nilius et al., 2004). In ECs, TRPV4 channels exist in signaling complexes near MEGJ’s along with IK_Ca channels, IP₃Rs and other proteins (Sonkusare et al., 2012, 2014; Hong et al., 2018; Chen and Sonkusare, 2020; Figures 1, 3). Intracellular Ca²⁺ potentiates the activation of TRPV4 channels through calmodulin that binds to the C-terminal region of this channel (Strotmann et al., 2003).

Endothelial TRPV4 channels mediate agonist-induced, endothelium-dependent vasodilation, particularly in arterioles where activation of these receptors leads to activation of IK_Ca channels, EC hyperpolarization and conduction of this hyperpolarization to overlying VSMCs to induce vasodilation (Marrelli et al., 2007; Earley et al., 2009b; Sonkusare et al., 2012, 2014; Zhang et al., 2013; Zheng et al., 2013; Du et al., 2016; Diaz-Otero et al., 2018; Figure 3). In addition, TRPV4 channels play a central role in myoendothelial negative-feedback that tempers vascular tone in the absence of an endothelial agonist. Agonist-induced activation of VSMC Gq-coupled receptors leads to a global increase in EC intracellular Ca²⁺(Dora et al., 1997; Schuster et al., 2001; Tuttle and Falcone, 2001; Jackson et al., 2008; Kansui et al., 2008) that contributes to the negative-feedback regulation of vascular tone (Lemmey et al., 2020). Studies in murine mesenteric resistance arteries have shown that endothelial TRPV4 channels are activated during this process through a mechanism involving Ca²⁺ release through IP₃Rs, resulting in activation of IK_Ca channels blunting agonist-induced vasoconstriction (Hong et al., 2018; Figure 3). Similarly, studies in rat cremaster arterioles have shown that endothelial TRPV4 channels are activated at low intravascular pressure, leading to TRPV4 Ca²⁺ sparklets (localized [Ca²⁺]_in signals through small groups of TRPV4 channels), activation of IK_Ca channels and dampening of myogenic tone (Bagher et al., 2012). The precise signal that is communicated from VSMCs to ECs to initiate myoendothelial feedback remains in question, with data supporting Ca²⁺ as the signal (Garland et al., 2017) and other findings supporting IP₃ as the signal (Tran et al., 2012; Hong et al., 2018). Additional research will be required to determine whether Ca²⁺ or IP₃ mediates myoendothelial negative-feedback and whether there is heterogeneity among vessels in which signal (Ca²⁺ or IP₃) is used.

Endothelial cells also express TRPP1 channels where they function in shear-stress dependent vasodilation (MacKay et al., 2020). Shear-stress-induced increases in EC [Ca²⁺]_in that activate sK_Ca channels, IK_Ca channels and EC nitric oxide synthase were shown to be substantially impaired by conditional knockout of EC TRPP1 with no change in Ca²⁺ signals activated by muscarinic receptor activation (MacKay et al., 2020). Calcium-dependent activation of TRPP1 channels was not established in these studies, so [Ca²⁺]_in modulation of these channels in ECs and their role in regulating myogenic tone other than when activated by shear-stress remains to be established.

Multiple mechano-sensors of wall stress (or strain) initiate the myogenic response culminating in steady-state myogenic tone (Figure 3). Putative sensors (in green font in Figure 3) include: several G-protein coupled receptors (Brayden et al., 2013; Narayanan et al., 2013; Schleifenbaum et al., 2014; Storch et al., 2015; Kauffenstein et al., 2016; Mederos et al., 2016; Hong et al., 2017; Pires et al., 2017; Chennupati et al., 2019), various cation channels (Welsh et al., 2002; Jernigan and Drummond, 2005; Gannon et al., 2008; VanLandingham et al., 2009; Narayanan et al., 2013; Nemeth et al., 2020), integrins (Davis et al., 2001; Martinez-Lemus et al., 2005; Colinas et al., 2015), matrix metalloproteinases and epidermal growth factor receptors (EGFR; Lucchesi et al., 2004; Amin et al., 2011); and membrane-bound tumor necrosis factor α (mTNF α), TNF α receptor (TNFR) and downstream sphingosine-1-phosphate (S1P) signaling (Kroetsch et al., 2017; Figure 3). Pressure-dependent stimulation of these putative mechano-sensors activates phospholipase C (PLC) catalyzing hydrolysis of membrane phosphatidyl inositol 4,5 bisphosphate (PIP₂) to form IP₃ and DAG (Figure 3).

Pressure- and likely DAG-induced activation of plasma membrane TRPC6 channels results in Ca²⁺ influx through these channels (Slish et al., 2002; Welsh et al., 2002). The resultant local [Ca²⁺]_in increase, along with IP₃, activates IP₃Rs to release Ca²⁺ from the ER, amplifying the local [Ca²⁺]_in increase. This subplasmalemmal increase in [Ca²⁺]_in then activates overlying plasma membrane TRPM4 channels. Calcium influx through TRPC6 channels also activates plasma membrane Ca²⁺-activated Cl⁻ channels (CaCCs; Bulley et al., 2012; Wang et al., 2016). The cation influx through TRPC6 and TRPM4 channels, and Cl⁻ efflux through CaCCs causes membrane depolarization (Figure 3). As noted in Section Pressure-Dependent Activation of Mechanosensors Leads to Formation of IP₃ and DAG and shown in Figure 3, additional cation channels including TRPP1 channels may contribute to the pressure-induced depolarization.

Membrane depolarization induced by ionic currents through TRPC6 channels, TRPM4 channels, CaCCs and other ion channels activates plasma membrane VGCCs resulting in Ca²⁺ influx. VGCC-mediated Ca²⁺ influx across the plasma membrane, along with IP₃R-mediated Ca²⁺ release from ER Ca²⁺ stores, increases cytoplasmic (global) [Ca²⁺]_in levels, leading to calmodulin-mediated myosin light-chain kinase (MLCK) activation, phosphorylation of the myosin light-chains (MLC), actin-myosin cross-bridge formation, cross bridge cycling and an increase in myogenic tone (vasoconstriction; Cole and Welsh, 2011; Figure 3).

Membrane depolarization-induced activation of VGCCs is inherently a positive-feedback process because the Ca²⁺ influx through these channels will itself lead to depolarization and further activation of VGCCs. This process is limited in VSMCs by activation of at least three negative-feedback processes. Membrane depolarization activates K_V channels, and membrane depolarization along with increased [Ca²⁺]_in activates BK_Ca channels. The K⁺ efflux through these two K⁺ channels (which by themselves would cause membrane hyperpolarization) blunts and limits depolarization-induced activation of VGCC (Figure 3; Jackson, 2017, 2020). Additional negative feedback arises from Ca²⁺-dependent inactivation of VGCCs (Shah et al., 2006; Figure 3).

In addition to activating TRPC6 channels, the DAG formed from the activity of PLC along with elevated [Ca²⁺]_in activates protein kinase C (PKC) supporting the increase in tone by increasing the activity of TRPM4 channels (supporting depolarization) and VGCCs (promoting Ca²⁺ influx) while blunting the activity of several K⁺ channels (also supporting membrane depolarization; Jackson, 2020, 2021; Figure 3). The negative feedback involving K_V channels is blunted by Ca²⁺-dependent inhibition of these channels (Gelband et al., 1993; Ishikawa et al., 1993; Gelband and Hume, 1995; Post et al., 1995; Cox and Petrou, 1999; Figure 3). Ca²⁺-dependent activation of the protein phosphatase, calcineurin, inhibits ATP-sensitive K⁺ (K_ATP) channels, limiting their activity and promoting depolarization (Wilson et al., 2000; Figure 3).

Stimulation of the mechano-sensors in vascular smooth muscle also activates the small G-protein rhoA, which, in turn, activates rho-kinase (Chennupati et al., 2019; Figure 3). Rho kinase phosphorylates a number of substrates that also support myogenic tone including inhibition of myosin light chain phosphatase (MLCPPT; Cole and Welsh, 2011), stimulation of actin cytoskeleton remodeling that accompanies activation of the contractile machinery (Loirand et al., 2006; Moreno-Dominguez et al., 2013), inhibition of K_V channels as a consequence of actin remodeling (Luykenaar et al., 2009) and increasing the Ca²⁺ sensitivity of TRPM4 channels (Li and Brayden, 2017; Figure 3). Activated PKC also may inhibit MLCPPT through phosphorylation of the inhibitory protein, CPI₁₇ (Cole and Welsh, 2011; Figure 3).

Endothelial cells lining resistance arteries and arterioles play a negative-feedback role, dampening myogenic tone both through the Ca²⁺-dependent production of vasodilator autacoids (PGI₂, NO, EETS, etc.) and by conduction of Ca²⁺-dependent membrane hyperpolarization from the endothelium to overlying VSMCs via MEGJs (Figures 1, 3). Endothelial cells chemically and electrically converse with VSMCs through MEGJs that may form at myoendothelial projections that penetrate holes in the internal elastic lamina and contact the overlying VSMCs. Heterocellular gap junctions (MEGJs) between ECs and VSMCs form and allow small molecules (like IP₃) and ionic currents (including Ca²⁺) to move between the cells. Pressure-induced increases in VSMC [Ca²⁺]_in or IP₃ can pass to endothelial cells leading to EC IP₃R-induced Ca²⁺ signals (Ca²⁺ pulsars and wavelets) that can increase the production of Ca²⁺-dependent EC vasodilator autacoids that feedback to the VSMCs reducing myogenic tone (Figure 3). In addition, increased EC [Ca²⁺]_in will activate EC sK_Ca and IK_Ca channels causing EC membrane hyperpolarization. Myoendothelial gap junctions allow this hyperpolarization to be passed from ECs to VSMCs, producing VSMC hyperpolarization, deactivation of VSMC VGCCs and reduced myogenic tone (Figure 3). Thus, the production of EC autacoids and EC membrane potential are both strongly dependent on the activity of Ca²⁺-dependent ion channels in the endothelium including IP₃Rs, TRPV4 channels, sK_Ca channels and IK_Ca channels (Lemmey et al., 2020).