Ion channels and arteriolar tone: Ion channels and the regulation of myogenic tone in peripheral arterioles

Abstract

Myogenic tone is a hall-mark feature of arterioles in the microcirculation. This pressure-induced, contractile activation of vascular smooth muscle cells (VSMCs) in the wall of these microvessels importantly contributes to the regulation and maintenance of blood pressure; blood flow to and within organs and tissues; and capillary pressure and fluid balance. Ion channels play a central role in the genesis and maintenance of myogenic tone. Mechanosensitive ion channels such as TRPC6 may serve as one of the sensors of pressure-induced membrane stress/strain, and TRPC6 along with TRPM4 channels are responsible for pressure-induced VSMC depolarization that may be bolstered by the activity of Ca²⁺-activated Cl⁻ channels and inhibition of voltage-gated K⁺ (K_V) channels, inwardly-rectifying K⁺ (K_IR) channels and ATP-sensitive K⁺ (K_ATP) channels. Membrane potential depolarization activates voltage-gated Ca²⁺ channels (VGCCs), with CaV1.2 channels playing a central role. Calcium entry through CaV1.2 channels, which is amplified by Ca²⁺ release through IP₃ receptors in the form of Ca²⁺ waves in some arterioles, provides the major source of activator calcium responsible for arteriolar myogenic tone. Stabilizing negative-feedback comes from depolarization- and Ca²⁺-induced activation of large-conductance Ca²⁺-activated K⁺ channels and depolarization-induced activation of K_V channels. Myogenic tone also is dampened by tonic activity of K_IR and K_ATP channels. While much has been learned about ion channel expression and function in myogenic tone, additional studies are required to fill in our knowledge gaps due to significant regional differences in ion channel expression and function and a lack of data specifically from VSMCs in arterioles.

Introduction

Arterioles in the peripheral microcirculation substantially contribute to total peripheral resistance and blood pressure (Pries & Secomb, 2011; Renkin, 1984; Zweifach & Lipowsky, 1984); control blood flow to and within organs and tissues; and modulate the pressure in capillaries and venules contributing to tissue fluid filtration/reabsorption. These arteriolar functions are effected by vascular smooth muscle cells (VSMCs) (Davis, Hill, & Kuo, 2011; Renkin, 1984) that wrap in a single circumferential layer around the endothelial cell “tube” that forms the basic structure of arterioles (Rhodin, 2014; Simionescu & Simionescu, 1984). For the purpose of this chapter, arterioles are defined as arterial vessels with a single layer of VSMCs and that are embedded within the parenchyma which they perfuse. Feed arteries (or resistance arteries), the last branch of the arterial system before arterioles, are external to the parenchyma and generally have more than one layer of VSMCs overlying the endothelium. Changes in the contractile activity of the VSMCs (contraction or relaxation) produce coordinated constriction or dilation of the arteriolar lumen resulting in changes in the hydraulic resistance to fluid flow and changes in the pressure drop along the length of these vessels (Zweifach & Lipowsky, 1984). A hall-mark feature of arterioles is pressure-induced myogenic tone, the steady-state contractile activity of vascular smooth muscle cells in the arteriolar wall that sets the baseline degree of contraction of the VSMCs at a given blood pressure (Davis & Hill, 1999; Davis et al., 2011; Hill, Zou, Potocnik, Meininger, & Davis, 2001; Johnson, 1980). Arterioles can either constrict or dilate in response to physiological or pathophysiological stimuli from this baseline level of tone (Davis et al., 2011; Renkin, 1984).

Vascular smooth muscle cells express a diverse array of ion channels that contribute to myogenic tone and its regulation (Tykocki, Boerman, & Jackson, 2017). This includes more than 5 classes of K⁺ channels (Tykocki et al., 2017), 1 or more classes of voltage-gated Ca²⁺ channels (Tykocki et al., 2017), several types of Cl⁻ channels (Bannister et al., 2012; Dam, Boedtkjer, Aalkjaer, & Matchkov, 2014; Dam, Boedtkjer, Nyvad, Aalkjaer, & Matchkov, 2014; Heinze et al., 2014; Hubner, Schroeder, & Ehmke, 2015; Leblanc et al., 2015; Matchkov, Boedtkjer, & Aalkjaer, 2015) and a variety of transient-receptor-potential (TRP) channels (Tykocki et al., 2017) expressed in the plasma membrane and 2 or more Ca²⁺ permeable channels in the endoplasmic reticulum (Tykocki et al., 2017) that can be involved in the generation and modulation of myogenic tone. The purpose of this chapter is to review the functional expression of a number of these channels in VSMCs that appear to importantly contribute to the regulation of myogenic tone in arterioles in the peripheral microcirculation. The reader is directed to a recent extensive review of ion channel expression and function in arterioles and resistance arteries for a more general treatment of this topic (Tykocki et al., 2017). Ion channel expression and function in endothelial cells will not be discussed and the reader is directed to recent reviews for information and references on that topic (Jackson, 2016b; Thakore & Earley, 2019).

Overview of myogenic tone in arterioles and resistance arteries

The precise mechanism by which blood pressure activates arteriolar VSMCs to generate myogenic tone remains in question, largely because there appear to be multiple mechanisms involved that depend on the species studied, tissue location and the experimental conditions under which this process is examined. When pressure is increased in a resistance artery or arteriole, this radial force will be transmitted to the wall of the arteriole increasing tangential wall stress (T) given by the law of Laplace: T = Pr/Δ, where P = pressure in the lumen of the vessel, r is the lumen radius and Δ is the thickness of the wall. The increase in T will cause an initial passive dilation of the vessel resulting in “stretch” of the VSMCs, until the VSMCs can generate sufficient active tension to overcome the new pressure-related T. The increased T or the strain (“stretch”) that ensues then activates some sensing mechanism in the membrane of the VSMCs to activate the VSMCs to actively contract to overcome the pressure-induced T and develop myogenic tone. Studies in cerebral resistance arteries suggest the increased T (or the strain that ensues) is sensed, perhaps, by a mechanosensitive-G_q-coupled receptor (msGPCR) such as the Angiotensin II Receptor 1B (ATR1B) (Mederos, Storch, & Gudermann, 2016; Pires et al., 2017; Schleifenbaum et al., 2014), Cysteinyl Leukotriene 1 Receptors (CysLT1) (Mederos et al., 2016; Storch, Blodow, Gudermann, & Mederos, 2015), or P2Y6 Purinergic Receptors (Brayden, Li, & Tavares, 2013) as well as mechanosensitive ion channels such as TRPC6 (Welsh, Morielli, Nelson, & Brayden, 2002) (Figure 1). Signaling mechanisms involving integrins and myogenic tone also have been proposed (Davis et al., 2001; Martinez-Lemus, Crow, Davis, & Meininger, 2005). The precise mechanism by which these receptors and ion channels are activated by the increased stress or strain is not yet known. However studies in other systems suggest that increased membrane stress/strain may be transmitted to membrane proteins to activate channels/receptors by: 1.) transmission of force via connections with the extracellular matrix and the cytoskeleton, 2.) changes in membrane curvature, 3.) tension caused by membrane thinning, and/or membrane tension-induced conformational changes (Leiphart, Chen, Peredo, Loneker, & Janmey, 2019). Regardless of the specifics, it is proposed that activation of the msGPCR leads to activation of Phospholipase C_γ1 (PLCγ1), hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP₂) and formation of inositol 1,4,5 trisphosphate (IP₃) and diacylglycerol (DAG) (Gonzales et al., 2014). The DAG produced by this process may contribute to the activation of TRPC6 channels (Inoue et al., 2009; Mederos y Schnitzler et al., 2008) although evidence against this idea has been presented (Anfinogenova, Brett, Walsh, Harraz, & Welsh, 2011) (Figure 1). TRPC3 also can be activated by DAG produced during agonist-induced activation of G_q-protein coupled receptors (Reading, Earley, Waldron, Welsh, & Brayden, 2005), but these channels have not been implicated in the signaling pathway of myogenic tone. Phosphatidyl choline-specific PLC also has been proposed to participate in myogenic tone of murine mesenteric resistance arteries (Mauban, Zacharia, Fairfax, & Wier, 2015).

Figure 1: Ion channels and signaling pathways in myogenic tone.

Schematic diagram of some of the major ion channels and signaling pathways involved in myogenic tone in resistance arteries and arterioles. Black arrows show stimulation, increases or activation of signaling molecules, ion channels or enzymes that participate in myogenic tone. Red capped lines indicate inhibition, decreases or deactivation of signaling molecules, ion channels or enzymes involved in myogenic tone. Also shown are pharmacological agents that we have used to interrogate the ion channels and signaling pathways in arteriolar myogenic tone. Abbreviations: CaCC = Ca²⁺-activated Cl⁻ channel; VGCC = voltage-gated Ca²⁺ channel; BK_Ca = large-conductance Ca²⁺-activated K⁺ channel; K_V = voltage-gated K⁺ channel; K_IR = inwardly-rectifying K⁺ channel; K_ATP = ATP-sensitive K⁺ channel; msGPCR = mechanosensitive G-protein-coupled receptor; DAG = diacylglycerol; PKC = protein kinase C; NFA = niflumic acid; DTZM = diltiazem; NIF = nifedipine; TEA = tetraethylammonium; IBTX = iberiotoxin; 4-AP = 4-aminopyridine; GLIB = glibenclamide; BIM I = Bisindolylmaleimide I; PLC = phospholipase C; PIP₂ = phosphatidylinositol bisphosphate; IP₃ = inositol, 1,4,5 trisphosphate; IP₃R1 = IP₃ receptor 1; RyR = ryanodine receptor; CICR = Ca²⁺-induced-Ca²⁺ release; LARG = Guanine Nucleotide Exchange Factor LARG; RhoA = small G-protein Rho; 2-APB = 2-Aminoethoxydiphenyl borate; RhoK = Rho kinase; LIMK = LIM kinase; CPI₁₇ = C-kinase potentiated Protein phosphatase-1 Inhibitor; MLCPPT = myosin light-chain phosphatase; MLC = myosin light-chain; MLCK = myosin light-chain kinase; See text for more details and references.

The IP₃ formed from hydrolysis of PIP₂ then binds to IP₃ receptors (IP₃R) in the peripheral endoplasmic reticulum (ER). Coupled with the Ca²⁺ entry through TRPC6 channels, this results in release of Ca²⁺ from the ER through the IP₃R via Ca²⁺-induced Ca²⁺ release. The Ca²⁺ released through the IP₃R then activates TRPM4 channels (Gonzales, Amberg, & Earley, 2010; Gonzales et al., 2014), a process that may involve protein kinase C which is activated by PLC-dependent production of DAG and Ca²⁺ (Earley, Straub, & Brayden, 2007). Along with the inward cation current through TRPC6 channels, the cation influx through TRPM4 channels results in membrane depolarization, activation of voltage-gated Ca²⁺ channels and Ca²⁺ influx (Gonzales, Garcia, Amberg, & Earley, 2010; Gonzales et al., 2014) (Figure 1). Increased activity of PKC tethered to CaV1.2 by A-kinase Anchoring Proteins (AKAPs) may facilitate this process (Navedo, Amberg, Votaw, & Santana, 2005).

The local increase in Ca²⁺ from TRPC6, IP₃R and CaV1.2 may also activate Ca²⁺-activated Cl⁻ channels (CaCC) contributing to pressure-induced membrane depolarization in some vessels (Bulley et al., 2012; Heinze et al., 2014), particularly small arterioles (Heinze et al., 2014) (Figure 1). Membrane depolarization is also supported by inhibition of several K⁺ channels (Figure 1). The PLC-dependent production of DAG and the increase in local Ca²⁺ concentration activate PKCs. Increased PKC activity inhibits basal K_V channel (Aiello, Clement-Chomienne, Sontag, Walsh, & Cole, 1996; Clement-Chomienne, Walsh, & Cole, 1996; Hayabuchi, Standen, & Davies, 2001; Kidd, Bulley, & Jaggar, 2017; Ko et al., 2010) and K_ATP channel (Hayabuchi, Davies, & Standen, 2001; Hu, Huang, Jan, & Jan, 2003; Jiao, Garg, Yang, Elton, & Hu, 2008; Sampson, Davies, Barrett-Jolley, Standen, & Dart, 2007) activity, promoting membrane depolarization. The increase in intracellular Ca²⁺ associated with stretch-induced activation of VSMCs also inhibits K_V channel activity promoting membrane depolarization and increased myogenic tone (Cox & Petrou, 1999; Gelband, Ishikawa, Post, Keef, & Hume, 1993; Ishikawa, Hume, & Keef, 1993). Increased intracellular Ca²⁺ acting through the Ca²⁺-activated phosphatase, calcineurin, also inhibits K_ATP activity (Wilson, Jabr, & Clapp, 2000). Vascular smooth muscle stretch also has been shown to inhibit inwardly rectifying K⁺ (K_IR) channels in vascular smooth by a mechanism involving PKC (Wu et al., 2007). Membrane depolarization induced by activation of TRPC6, TRPM4, VGCCs and CaCCs also will decrease the activity of open K_IR due to the voltage-dependent block of these channels by polyamines and Mg²⁺ (Tykocki et al., 2017). Thus, closure of open K⁺ channels reduces outward current and enhances the pressure-induced membrane depolarization and myogenic tone.

The global increase in intracellular Ca²⁺ promotes Ca²⁺ binding to the calcium binding protein, calmodulin (CaM), and the Ca²⁺-CaM complex activates myosin light-chain kinase (MLCK), resulting in phosphorylation of the 20 KD myosin light-chains (Cole & Welsh, 2011; Zou, Ratz, & Hill, 1995). Phosphorylated myosin light chains then allow the formation of actin-myosin cross bridges, the cross-bridge cycle and smooth muscle contraction (increased myogenic tone) (Cole & Welsh, 2011; Zou et al., 1995) (Figure 1). The myosin light chains are dephosphorylated by myosin light-chain phosphatase (MLCPPT); the ratio of the activity of MLCK/MLCPPT determines the Ca²⁺ sensitivity of the contractile machinery (Cole & Welsh, 2011). Activation of G-proteins activates the small GTPase, RhoA, via guanine nucleotide exchange factors (GEFs) such as LARG which couples G_12/13 to activation of RhoA (Chennupati et al., 2019) (Figure 1). RhoA activation then activates Rho Kinase that has several effects in VSMCs related to myogenic tone. First, activated Rho kinase phosphorylates and inhibits MLCPPT (Cole & Welsh, 2011). This results in Ca²⁺ sensitization of the contractile machinery as the ratio of the activity of MLCK/MYPPT increases. Second, activated Rho kinase also modulates the actin cytoskeleton through LIM kinase (LIMK)-mediated inhibition of Cofilin (Loirand, Guerin, & Pacaud, 2006). Remodeling of cortical actin induced by this process inhibits K_V channels and promotes membrane depolarization and increased myogenic tone (Luykenaar, El-Rahman, Walsh, & Welsh, 2009). Finally, the RhoA/Rho kinase pathway has also been implicated in the modulation of TRP channel function (Li & Brayden, 2017) and voltage-gated Ca²⁺ channel activity (Guan, Baty, Zhang, Remedies, & Inscho, 2019) (Figure 1).

In addition to modulating ion channel activity, activated PKC also can affect the Ca²⁺ sensitivity of the contractile machinery in VSMCs (Cole & Welsh, 2011). Activated PKC phosphorylates the protein CPI₁₇ and phosphorylated CPI₁₇ inhibits MLCPPT to promote Ca²⁺ sensitization of the contractile machinery supporting increased myogenic tone (Cole & Welsh, 2011).

Mechanisms of myogenic tone in peripheral arterioles: Role of mechanosensitive G-protein-coupled receptors and inositol 1,4,5 trisphosphate receptors

Myogenic tone in 2^nd-order cremaster arterioles, as a “model” peripheral microvessel, appears to follow the general pathway outlined above. Consistent with this mechanism, both hamster and mouse cremaster arterioles display asynchronous, IP₃-dependent Ca²⁺ waves that appear to contribute to global Ca²⁺ levels and maintenance of myogenic tone: Inhibition of PLCs with U73122 inhibits Ca²⁺ waves, decreases global intracellular Ca²⁺ levels and causes vasodilation in both hamster (Westcott & Jackson, 2011) (Figure 2) and mouse (Westcott, Goodwin, Segal, & Jackson, 2012) cremaster arterioles. Identical results were obtained by blockade of IP₃R with either 2-APB or xestospongin D or depletion of internal Ca²⁺ stores with thapsigargin (Westcott et al., 2012; Westcott & Jackson, 2011). These findings support the general mechanisms shown in Figure 1. Despite the asynchronous Ca²⁺ waves that are observed in pressurized hamster and mouse cremaster arterioles studied by pressure myography, vasomotion (oscillations in diameter), associated with the Ca²⁺ waves, is not usually observed (at least on a detectable scale). We think that this may mean that the contractile machinery “filters” these oscillations, responding instead to a time-averaged value of the global intracellular Ca²⁺ concentration. In contrast, when vasoconstriction is induced with a G-protein coupled agonist, such as phenylephrine, synchronous Ca²⁺ waves are observed in the VSMCs that are associated with diameter oscillations in synchrony with the Ca²⁺ waves (Jackson, unpublished observations) as has been reported in mesenteric arteries (Mauban, Lamont, Balke, & Wier, 2001). The increase in Ca²⁺ sensitivity induced by phenylephrine acting at α₁-adrenoreceptors may partially explain why vasomotion is observed in the presence of an agonist rather than pressure alone. Additional research will be required to resolve this issue.

Figure 2: Smooth muscle cells in cremaster arterioles display Ca²⁺ waves sensitive to phospholipase C inhibition.

Panel A: Image of a tangential, confocal slice through a hamster cremaster arteriole loaded with Fluo-4 and studied by pressure myography, along with 9 regions of interest (ROI’s) that were placed at the peak of Ca²⁺ waves as described (Jackson & Boerman, 2017). The scale bar equals 20 μm. Reproduced from (Jackson & Boerman, 2017) with permission. Panel B: Plot of relative fluorescence (F/F_o) for the 9 ROI’s in panel A showing the amplitude and duration of Ca²⁺ events at these locations. Application of SparkAn software to these data with a threshold of 15% above baseline revealed 19 Ca²⁺ waves with mean amplitude = 1.52 ± 0.07 F/F_o (range = 1.2 – 2.6) during the 16 s recording period shown for this vessel. Similar results were obtained in 4 additional cremaster arterioles, confirming prior studies (Westcott & Jackson, 2011). Reproduced from (Jackson & Boerman, 2017) with permission. Panels C–E: Data from Westcott and Jackson (Westcott & Jackson, 2011), used with permission. Panel C: Shows inhibition of the occurrence (Occ – number of cells with waves), the amplitude (Amp) and the frequency (Hz) of Ca²⁺ waves by the PLC inhibitor, U-73122 (n = 8). Panel D: In addition to inhibition of Ca²⁺ waves, U-73122 also caused a decrease in global Ca²⁺ levels measured as the average Fluo-4 fluorescence in projections of 500 images before and after treatment with U-73122 relative to Control fluorescence (F/F_o) (Westcott & Jackson, 2011) (n = 5). Panel E: Associated with the U-73122-induced fall in global Ca²⁺ was a significant dilation (Westcott & Jackson, 2011) (n = 4). Similar results were obtained with the IP₃ receptor antagonists 2-APB and xestospongin or depletion of Ca²⁺ stores using thapsigargin in both hamster (Westcott & Jackson, 2011) and mouse (Westcott et al., 2012) second order cremaster arterioles. These data support the hypothesis that PLC, IP₃, IP₃R and the resultant Ca²⁺ waves contribute to global Ca²⁺ levels and myogenic tone in these arterioles. * = p < 0.05 by paired t-tests.

Where hamster cremaster arterioles deviate from the model shown in Figure 1 is in the reliance on ATR1: the ATR1 antagonist, Losartan, which inhibits myogenic tone in the renal circulation (Mederos y Schnitzler et al., 2008) and effectively blocks Ang II-induced vasoconstriction in hamster cremaster arterioles (Jackson & Boerman, 2017), has no effect on basal myogenic tone in 2^nd-order hamster cremaster arterioles (Jackson & Boerman, 2017). In support of our findings with Losartan, Hong and colleagues (Hong et al., 2016) have shown that the ATR1 inverse agonist, Candesartan (1 μM), has no effect on myogenic tone in 1^st-order rat cremaster arterioles. However, they went on to demonstrate that a higher concentration (10 μM) of Candesartan does inhibit myogenic tone in these arterioles (Hong et al., 2016). Interpretation of these data are difficult because of the high concentration of Candesartan used, but they suggest that there may be regional or species differences in the details of the mechanisms underlying myogenic tone. Consistent with the notion of regional heterogeneity in mechanisms of myogenic tone, Brayden and colleagues have shown that 1 μM Candesartan inhibits myogenic tone of rat pial arteries, but has no significant effect on myogenic tone of downstream parenchymal arterioles, microvessels that appear to use P2Y6 receptors as the sensors responsible for myogenic tone (Brayden et al., 2013). The msGPCR responsible for activation of PLC in 2^nd-order cremaster arterioles remains to be established.

Hamster cheek pouch arterioles develop myogenic tone similar in magnitude to that in cremaster arterioles, however, PLC, IP₃ and IP₃R do not appear to be involved in this process (Jackson & Boerman, 2017). Cheek pouch arterioles do not display Ca²⁺ waves when studied under conditions identical to those in cremaster arterioles and effective inhibition of PLC with U73122 (Figure 3) or of IP₃R with 2-APB has no effect on myogenic tone in these vessels (Jackson & Boerman, 2017). These data demonstrate that there are substantial regional differences in the mechanisms responsible for myogenic tone in peripheral arterioles, and in particular that msGPCR-PLC-IP₃-IP₃R-dependent mechanisms are not an essential component of the process in all vessels. They also open the possibility that the mechanisms may not be fixed, but able to vary as the conditions demand. The membrane signaling mechanism responsible for myogenic tone in hamster cheek pouch arterioles remains to be established, but mechanosensitive ion channels, such as TRPC6 (Welsh et al., 2002) or mechanisms involving integrins (Davis et al., 2001; Martinez-Lemus et al., 2005) may be speculated.

Figure 3: Smooth muscle cells in cheek pouch arterioles do not display Ca²⁺ waves and myogenic tone is insensitive to phospholipase C inhibition.

Panel A: Image of a tangential, confocal slice through a hamster cheek pouch arteriole loaded with Fluo-4 and studied by pressure myography, along with 9 regions of interest (ROI’s) that were placed on the cells as described (Jackson & Boerman, 2017). The scale bar equals 20 μm. Reproduced from (Jackson & Boerman, 2017) with permission. Panel B: In the 9 ROI’s shown, no events with amplitude greater than 15% above baseline were detected during the 16 s recording period shown (Jackson & Boerman, 2017) with permission. Panel C: U-73122 had no effect on global Ca²⁺ levels in hamster cheek pouch measured as the average Fluo-4 fluorescence from projections of 500 images as described (Jackson & Boerman, 2017) (n = 3). Panel D: U-73122 also had no significant effect on the diameter of cheek pouch arterioles (n = 3). Panels C and D were reproduced from (Jackson & Boerman, 2017) with permission. Data identical to Panels C and D were obtained for the IP3R antagonist, 2-APB indicating that PLC and IP₃R do not contribute to global Ca²⁺ levels or resting myogenic tone in cheek pouch arterioles (Jackson & Boerman, 2017). Panels E and F show, as a positive control, that removal of extracellular Ca²⁺ decreases global Ca²⁺ levels and dilates cheek pouch arterioles (n = 3). * = p < 0.05 by paired t-tests.

In VSMCs of resistance arteries upstream from the microcirculation, Ca²⁺ waves that contribute to global Ca²⁺ levels and myogenic tone depend on both IP₃R and RyR (Figure 4); inhibition of RyR reduces the occurrence and frequency of Ca²⁺ waves (Westcott et al., 2012; Westcott & Jackson, 2011). However, in contrast to the effects of inhibition of IP₃R, which also inhibits Ca²⁺ waves, inhibition of RyR leads to an increase in global intracellular Ca²⁺ and an increase in myogenic tone (Figure 4B) (Westcott et al., 2012; Westcott & Jackson, 2011). The increase in Ca²⁺ and tone with inhibition of RyR results from inhibition of RyR-mediated Ca²⁺ sparks and the loss of Ca²⁺ spark-mediated activation of BK_Ca channels (Westcott et al., 2012; Westcott & Jackson, 2011), as will be discussed below. In downstream cremaster arterioles, in contrast to effects of inhibition of IP₃R signaling (Figure 4C) (Westcott et al., 2012; Westcott & Jackson, 2011), effective inhibition of RyR has no effect on Ca²⁺ waves or myogenic tone (Figure 4D) (Westcott et al., 2012; Westcott & Jackson, 2011) in the arterioles. RyR appear to be silent in cremaster arterioles and do not contribute to Ca²⁺ waves or the regulation of arteriolar myogenic tone (Westcott et al., 2012; Westcott & Jackson, 2011). It should be noted that RyR are not silent in all arterioles. In retinal arterioles, RyR-dependent Ca²⁺ sparks contribute to contraction of VSMCs in this microcirculation (See Chapter 7 in this volume). In cerebral parenchymal arterioles, RyR contribute to Ca²⁺ waves under resting conditions, but can generate Ca²⁺ sparks when extracellular pH is reduced leading to activation of BK_Ca channels in VSMCs from these arterioles (Dabertrand, Nelson, & Brayden, 2012). Thus, there appear to be significant regional differences in RyR function.

Figure 4: Ryanodine inhibits Ca²⁺ waves in hamster cremaster feed arteries, but not in downstream arterioles.

Shown are data from (Westcott & Jackson, 2011) used with permission. Data are means ± 95% confidence intervals for occurrence (Occ) of Ca²⁺ waves (Panels A–D). All other data are means ± SE for amplitude (Amp, F/F_o), and frequency (Freq, Hz) of Ca²⁺ waves (Panels A–D); Global Fluo-4 intensity, an index of global intracellular Ca²⁺ (Panels A–D); and diameter (panels A–D) in the absence (Control) or presence of the IP₃ receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB, 100 μM; Panels A and C, n = 6); or the ryanodine receptor antagonist, ryanodine (RYR, 10 μM; Panels B, and D). * = significantly different from Control value, p < 0.05, n = 6 by paired t-tests. Dashed lines in Panels A–D represent the maximum diameters of the vessels in 0 Ca²⁺ PSS. Panels A and C: 2-APB inhibits Ca²⁺ waves in both feed arteries and downstream arterioles. Similar results were obtained with another IP₃R antagonist, xestospongin (Westcott & Jackson, 2011) or depletion of Ca²⁺ stores with thapsigargin (Westcott & Jackson, 2011). These findings demonstrate that Ca²⁺ waves are dependent on IP₃R in both cremaster feed arteries and arterioles. In feed arteries, Ca²⁺ waves are also inhibited by ryanodine supporting a role for RyR in these resistance arteries. However, in downstream arterioles, even 50 μM ryanodine has no effect on Ca²⁺ waves. See (Westcott & Jackson, 2011) and text for more information.

VGCCs and myogenic tone in peripheral arterioles

There is considerable evidence, mostly from ex vivo studies of resistance arteries and arterioles studied by pressure myography, that CaV1.2, which form L-type VGCCs, provide a significant source of activator Ca²⁺ responsible for myogenic tone: in hamster cremaster and hamster cheek pouch arterioles maximal inhibition of CaV1.2 VGCC with diltiazem produces 86% dilation in cremaster arterioles vs. 54% dilation in cheek pouch arterioles both studied by pressure myography under identical conditions (Jackson & Boerman, 2017). These data support a major role for CaV1.2 VGCC in myogenic tone, but also suggest that other sources of Ca²⁺ significantly contribute to the process, particularly in cheek pouch arterioles. Likely sources include TRP channels expressed in these VSMCs such as TRPC1, TRPC3, or TRPC6 (although this needs to be studied), or Ca²⁺ entry through transporters such as the Na⁺/Ca²⁺ exchanger (Zhang et al., 2010). Measurement of membrane potential in pressurized cremaster and cheek pouch arterioles indicates that the latter VSMCs are slightly hyperpolarized (−33 mV vs. −28 mV in cremaster arterioles) (Figure 5A) that might account for lower activity of CaV1.2 in the cheek pouch. In addition, Ba²⁺ current density through nifedipine-sensitive CaV1.2 channels are smaller in VSMCs from cheek pouch than cremaster arterioles (Figure 5B). Despite these differences, intracellular Ca²⁺ measured with Fura 2 (Figure 5C) and myogenic tone (Figure 5D) are similar in cremaster and cheek pouch arterioles.

Figure 5: Cheek pouch arteriolar VSMCs are slightly hyperpolarized and have smaller VGCC currents than cremaster VSMCs.

Panel A: Sharp electrode membrane potential recorded from VSMCs in cremaster and cheek pouch arterioles studied by pressure myography at 80 cm H₂O. See (Burns et al., 2004) for detailed methods. Data for cremaster replotted from (Burns et al., 2004). Under similar recording conditions, cheek pouch arterioles were more hyperpolarized −33 ± 1 mV (n = 63 cells in 8 arterioles) than cremaster arterioles (−27 ± 1 mV (n = 34 arterioles; p < 0.05 by t-test). Panel B: Whole cell Ba²⁺ current density (pA/pF) recorded in isolated VSMCs as described in (Cohen & Jackson, 2003) (n = 5 cells for each group; * = p < 0.05 by two-way ANOVA followed by Sidak test for comparison of means). Panel C: Intracellular Ca²⁺ concentration measured with FURA 2 as described (Burns et al., 2004; Jackson, Boerman, Lange, Lundback, & Cohen, 2008; Westcott & Jackson, 2011). Data are replotted from (Burns et al., 2004) for cremaster (n = 7) and (Brekke, Jackson, & Segal, 2006) for cheek pouch arterioles (n = 7) (p > 0.05 by t-test). Panel D: Data are replotted from (Jackson & Boerman, 2017) for n = 38 arterioles for both cremaster and cheek pouch (p > 0.05 by t-test). Despite the apparent difference in membrane potential (Panel A) and the activity of VGCCs (Panel B) intracellular Ca²⁺ and resting myogenic tone are similar in these two classes of arterioles.

In cremaster arterioles, we think that IP₃R serve to amplify Ca²⁺ signals through VGCC (Jackson & Boerman, 2018). We have shown that activation of VGCC by the CaV1.2 agonist Bay K8644 or by membrane depolarization increases the frequency and occurrence of Ca²⁺ waves and increases global myoplasmic Ca²⁺, whereas removal of extracellular Ca²⁺ or deactivation of VGCC by reduced pressure or membrane hyperpolarization reduce the frequency and occurrence of Ca²⁺ waves and decrease global myoplasmic Ca²⁺ (Jackson & Boerman, 2018). These data suggest that IP₃R serve to amplify Ca²⁺ signals through CaV1.2 VGCC in cremaster arterioles. This process appears to be lacking in cheek pouch arterioles studied under identical conditions and may also help explain why CaV1.2 VGCC contribute less to myogenic tone in these microvessels (Jackson & Boerman, 2017).

In vivo, the role played by CaV1.2 in arteriolar myogenic tone appears more variable. We (Jackson, 2012; Welsh, Jackson, & Segal, 1998) and others (Hill & Meininger, 1994) have shown that in pentobarbital-anesthetized rodents, inhibition of CaV1.2 channels with either diltiazem or nifedipine has no steady-state effect on arteriolar myogenic tone in hamster cheek pouch (Welsh et al., 1998), hamster cremaster (Jackson, 2012) (Figure 6A) and rat cremaster arterioles (Hill & Meininger, 1994). This lack of effect of CaV1.2 VGCC blockers does not appear to be due to a lack of efficacy because the blockers inhibited oxygen-induced arteriolar constriction (Jackson, 2012; Welsh et al., 1998) (Figure 6B) and vasomotion (Hill & Meininger, 1994; Jackson, 2012) (Figure 6C) processes dependent on CaV1.2 VGCCs. The source of activator Ca²⁺ after blockade of CaV1.2 VGCC in vivo remains to be established. It is likely that some other VGCC such as CaV3.X T-type VGCC (Abd El-Rahman et al., 2013; Dam, Boedtkjer, Aalkjaer, et al., 2014; Gustafsson, Andreasen, Salomonsson, Jensen, & Holstein-Rathlou, 2001; Hansen, Jensen, Andreasen, & Skott, 2001; Morita, Cousins, Onoue, Ito, & Inoue, 1999) or other classes of VGCC can take over this important role and maintain arteriolar tone within a homeostatic range. In vivo, arterioles are exquisitely sensitive to the ambient PO₂: increased PO₂ results in profound arteriolar constriction in peripheral arterioles (Jackson, 2016a) (Figure 6B). At rest, arterioles are exposed to much lower PO₂ values than the systemic value of 100 mm Hg. In 2^nd-order cheek pouch and cremaster arterioles, we estimate that the resting PO₂ is about 30 mm Hg, and not 100 mm Hg as seen in large, systemic arteries, due to substantial pre-capillary diffusion of O₂ out of blood into the surrounding tissue (Jackson, 2016a). Experimentally, by superfusion of tissues with physiological salt solution equilibrated with elevated PO₂, we and others have shown that arteriolar tone can be substantially increased and that this is the result of membrane depolarization and activation of CaV1.2 VGCC due to O₂-dependent production of vasoconstrictors like leukotrienes and 20-HETE (Jackson, 2016a). It is worthy to note that most pressure myography studies of resistance arteries and arterioles are performed at PO₂’s in excess of 100 mm Hg (usually air saturated with a PO₂ about 150 mm Hg). This theoretically could explain why ex vivo studies of resistance arteries and arterioles consistently show a major role for CaV1.2 VGCC, whereas in vivo studies, particularly where the PO₂ is controlled to much lower physiological levels, the contribution of CaV1.2 VGCC to resting tone appears less. However, despite the high PO₂, the degree of tone in pressure myograph systems with high PO₂ is quite similar to the level of tone observed in arterioles studied in vivo at much lower PO₂ values. If the ambient PO₂ were the sole explanation of the higher activity of VGCC ex vivo compared to in vivo, one would expect that myogenic tone would be substantially higher in the pressure myograph than is typically observed. In addition, we have previously shown that changes in PO₂ between 15 and 150 mm Hg do not appear to have effects on VSMC membrane potential (Jackson, 2000a), outward K⁺ currents (Jackson, 2000a), currents through VGCCs (Cohen & Jackson, 2003) or IP₃-mediated Ca²⁺ transients (Cohen & Jackson, 2003) in hamster cremaster VSMCs, in vitro. Therefore, why CaV1.2 VGCCs appear to be silent, in vivo, in cheek pouch and cremaster arterioles, but active in isolated vessels studied in the pressure myograph remains unknown.

Figure 6: Lack of effect of diltiazem on resting myogenic tone in hamster cremaster arterioles, in vivo.

Data are replotted from (Jackson, 2012). Panel A: Data are relative diameters ± SE (n = 4) of arterioles in superfused hamster cremaster muscles studied by intravital microscopy as described (Jackson, 1986, 1993a, 1993b) at rest (Control – open bar), after topical application of the endothelium-dependent dilator, methacholine (10 μM) (Maximum – black bar), or in the presence of 10 μM diltiazem (grey bar), as indicated. Note that the vessels developed myogenic tone (Control diameters less than Maximum), yet diltiazem no effect on vessel diameter. * = significantly different from Control, p < 0.05. Panel B: Data are mean diameters ± SE (n = 4) of hamster cremaster muscle arterioles studied by intravital microscopy. Shown are arteriolar diameters at rest and in the presence of elevated superfusate O₂ tension, before (Control) and during superfusion with diltiazem, as indicated. As described previously (Jackson, 1986, 1993b, 1998), under control conditions, elevated O₂ tension caused significant arteriolar constriction. However, this response was abolished by diltiazem. These data demonstrate the efficacy of diltiazem in this preparation. * = significantly different from Rest, p < 0.05. C. Shown is a digitized trace of arteriolar internal diameter in a hamster cremaster studied by intravital microscopy before and during superfusion with diltiazem as indicated. As shown, the arteriole displayed vasomotion, the oscillatory diameter shown at the beginning of the trace. This behavior was abolished when the muscle was superfused with diltiazem. These data also demonstrate the efficacy of diltiazem in this preparation.

Non-selective cation channels and myogenic tone in peripheral arterioles

As outlined in Figure 1, cation influx through TRPC6 and TRPM4 channels appear to be an integral part of myogenic tone in cerebral resistance arteries (Gonzales et al., 2014). Cation channels inhibitable by the lanthanide Gd³⁺ (10 nM−1 μM) and the Chilean rose tarantula venom toxin GsMTx-4 (5 μM) also appear essential for the development of myogenic tone in both 2^nd-order hamster cremaster and cheek pouch arterioles (Jackson & Boerman, 2017). For example, Gd³⁺ inhibited 67 ± 4% of Ca²⁺-dependent myogenic tone with a logIC₅₀ = −7.3 ± 0.1 (~55 nM) in both cheek pouch and cremaster arterioles (Jackson & Boerman, 2017). While the precise identity of the cation channels that contribute to membrane depolarization and activation of VGCC in these vessels remain to be established, the strong inhibition of myogenic tone by Gd³⁺ and GsMTx-4 suggests that TRPC6 or other mechanosensitive ion channels inhibited by these substances also importantly contribute to myogenic tone in these hamster arterioles. A role for TRPM4 in myogenic tone of cremaster and cheek pouch arterioles has not been directly established. It is also not known if the apparent activity of TRP channels contributes to myogenic tone only through effects on membrane potential (increased activity will depolarize VSMCs and activate CaV1.2 VGCCs) or if Ca²⁺ influx through these channels contributes to activator Ca²⁺ in the VSMCs. Additional research will be required to answer these questions.

Ca²⁺ activated Cl⁻ channels and myogenic tone in peripheral arterioles

Pressure-induced membrane depolarization of arteriolar VSMCs may also be supported by CaCCs (Bulley et al., 2012; Heinze et al., 2014). Consistent with this hypothesis, a CaCC blocker, niflumic acid, dilates isolated cheek pouch arterioles studied by pressure myography (Figure 7), where CaV1.2 VGCCs contribute to resting myogenic tone (Jackson & Boerman, 2017). We suspect that it is Ca²⁺ entry through CaV1.2 VGCC that activates the CaCCs in these vessels, because we have also found that niflumic acid dilates O₂-constricted cheek pouch arterioles, in vivo, in a concentration-dependent manner (Figures 8A and B), and attenuates the O₂-induced VSMC depolarization that drives this constriction (Figures 8C and D). However, niflumic acid had no significant effect on resting diameter, in vivo, a condition where CaV1.2 channels do not contribute to resting myogenic tone: arterioles were 28 ± 3 μm at rest and were 32 ± 5 μm in the presence of 100 μM niflumic acid (maximum diameter = 40 ± 3 μm, n = 4, p > 0.05 by paired t-test). A role for CaCC in 2^nd-order cremaster arterioles has not been directly studied.

Figure 7: Block of Ca²⁺-activated Cl⁻ channels dilates hamster cheek pouch arterioles studied by pressure myography.

Data are mean dilations relative to maximal diameter ± SE (n = 3) in the presence of 30–100 μM niflumic acid in isolated cheek pouch arterioles studied by pressure myography as in (Jackson & Boerman, 2017). Resting diameters were 66 ± 3.2 μm and maximum diameters in 0 mM Ca²⁺ were 91± 8.9 μm. As shown, niflumic acid produced concentration-dependent dilation of cheek pouch arterioles studied by pressure myography. These data suggest that Ca²⁺-activated Cl⁻ channels (CaCCs) contribute to pressure-induced myogenic tone in cheek pouch arterioles, in vitro.

Figure 8: Ca²⁺-activated Cl⁻ channels (CaCC) contribute to O₂-induced constriction of hamster cheek pouch arterioles, in vivo.

Panel A: digitized diameter trace of a hamster cheek pouch arteriole studied by intravital microscopy as described (Jackson, 1993b). As indicated, superfusion of the preparation with solution equilibrated with 21% O₂ caused sustained vasoconstriction. Addition of niflumic acid (NFA) from 30–100 μM produced concentration-dependent arteriolar dilation. Arteriolar diameter recovered after washout of niflumic acid and removal of O₂ from the superfusate. Sodium nitroprusside (SNP) applied as a 200 nmol bolus dilated the arteriole demonstrating significant resting tone. Panel B: Summary data for experiments as shown in Panel A. Data are mean diameters ± SE (n = 3) of O₂-constricted arterioles in the absence (0 μM) or presence (30 – 100 μM) of niflumic acid in the superfusate. Niflumic acid produced concentration-dependent dilation of O₂ constricted arterioles supporting a role for CaCC in O₂-induced constriction of hamster cheek pouch arterioles. Panel C: Digitized trace of arteriolar VSMC membrane potential recorded with sharp electrodes as described (Welsh et al., 1998). From a resting membrane potential of about −33 mV, increasing superfusate O₂ content to 21% depolarized the cell to about −20 mV as we have previously described (Welsh et al., 1998). Addition of 100 μM niflumic acid repolarized the cell to about −30 mV. Panel D: Summary data for experiments as in Panel C. Data are means ± SE (n = 8) under control conditions with 0% O₂ (PO₂ = 15 mm Hg), with 21% O₂ in the superfusate and with 21% O₂ and 100 μM niflumic acid in the superfusate. Elevated O₂ tension depolarized the VSMCs in cheek pouch arterioles as reported (Welsh et al., 1998), and niflumic acid (inhibition of CaCC) lead to significant repolarization (* = p < 0.05 vs. 0% O₂; ** = p < 0.05 vs. 21% O₂ but not different from 0% O₂ by Tukey’s test after ANOVA).

Lack of a role for PKC in arteriolar myogenic tone in peripheral arterioles

There is considerable evidence in resistance arteries for a role of PKC in myogenic tone, with studies in cerebral arteries (Earley et al., 2007; Osol, Laher, & Cipolla, 1991; Slish, Welsh, & Brayden, 2002; Yeon et al., 2002), coronary arteries (Miller, Dellsperger, & Gutterman, 1997), subcutaneous resistance arteries (Coats, Johnston, MacDonald, McMurray, & Hillier, 2001), small arteries from rat gracilis muscle (Massett, Ungvari, Csiszar, Kaley, & Koller, 2002), rabbit facial vein (Laher & Bevan, 1989), murine mesenteric resistance arteries (Mauban et al., 2015) and ferret aorta (Pawlowski & Morgan, 1992) showing that inhibition of PKC inhibits myogenic tone. In contrast, studies in 2^nd-order hamster cremaster and cheek pouch arterioles using the PKC inhibitor, bisindolylmaleimide I (BIM I), which effectively blocks phorbol ester-induced arteriolar constriction in these preparations, has no significant effect on myogenic tone (Jackson & Boerman, 2017). Similarly, in 1^st-order rat cremaster arterioles (Hill, Falcone, & Meininger, 1990) and rat ophthalmic arteries (Ito, Jarajapu, Grant, & Knot, 2007) effective inhibition of PKC had no effect on myogenic tone. The cause of these regional differences in the role played by PKC has not been established. Nonetheless, these data support the hypothesis that there are significant regional- and perhaps species-dependent differences in the mechanisms of myogenic tone and specifically the role played by PKC.

Rho kinase and myogenic tone in peripheral arterioles

Rho kinase has been proposed to be involved in the regulation of Ca²⁺ entry into rat aortic and mesenteric VSMCs (Ghisdal, Vandenberg, & Morel, 2003). Rho kinase has also been proposed to affect VSMC membrane potential by affecting the activity of K_V channels (Luykenaar et al., 2009) and TRPM4 channels (Li & Brayden, 2017) in cerebral arteries, which in turn, affect VSMC Ca²⁺ signaling through voltage-dependent activation of VGCC. In second order hamster cremaster and cheek pouch arterioles, we found that the Rho kinase inhibitors Y27632 (Jackson & Boerman, 2017) and H1152 (Figure 9) inhibited >80% of Ca²⁺-dependent myogenic tone (Jackson & Boerman, 2017). However, the loss of tone was associated with inhibition of IP₃R-dependent Ca²⁺ waves in cremaster arterioles and a substantial decrease in global myoplasmic Ca²⁺ in both cremaster (Figure 9) and cheek pouch arterioles (Jackson & Boerman, 2017). These data support a significant role for Rho kinase in arteriolar VSMC Ca²⁺ signaling, in addition to possible effects on Ca²⁺ sensitization. Studies in renal arterioles have shown that Y27632 and another Rho kinase inhibitor, RKI 1447, both inhibit myogenic tone and Ca²⁺ signaling through voltage-gated Ca²⁺ channels (Guan et al., 2019). In cerebral VSMCs, Y27632 produces a small (~15%) inhibition of currents through CaV1.2 VGCC by (Wu et al., 2009), although it is not known if this was a direct effect of the drug on the channel, or because Rho kinase modulates the function of these ion channels. Our findings that Y27632 and H1152 dilated cheek pouch arterioles (Jackson & Boerman, 2017), which do not appear to rely on a msGPCR-G-protein-PLC-dependent pathway to develop myogenic tone, suggest that either these drugs act off target to inhibit Ca²⁺ influx through VGCCs, or that there is, perhaps, a Ca²⁺-dependent pathway for activation of RhoA and Rho kinase that does not require activation of a GPCR.

Figure 9: Rho-kinase antagonist inhibits Ca²⁺ signaling and myogenic tone in hamster cremaster arterioles.

Data are from (Jackson & Boerman, 2017) with permission. Panel A: Shown are maximum intensity projections of 500 images of a hamster cremaster arteriole loaded with Fluo-4 before (Control) and in the presence of the Rho kinase inhibitor, H1152, showing the decrease in global Ca²⁺ produced by this inhibitor. The scale bar in the Control image is 20 μm and applies to the H1152 image as well. Panel B: Summary data showing that H1152 inhibited Ca²⁺ waves, reduced global Ca²⁺ and dilated hamster cremaster arterioles studied by pressure myography (Jackson & Boerman, 2017). Data are the occurrence of Ca²⁺ waves (Occ; number of cells showing waves/total number of cells studied) ± 95% confidence interval (n = 4), or means ± SE (n = 4) for the amplitude (Amp; F/F_o) or frequency (Freq; Hz) of Ca²⁺ waves; global Fluo-4 levels (an index of global Ca²⁺) and arteriolar internal diameter. Identical results were obtained using the Rho kinase inhibitor,10 μM Y-27632 (Jackson & Boerman, 2017). * = significantly different from Control value, p < 0.05 by paired t-tests.

Ion channels opposing myogenic tone in peripheral arterioles

Potassium channel activity, in general, opposes myogenic tone (Jackson, 2000b, 2005, 2017, 2018a; Jackson & Blair, 1998; Tykocki et al., 2017). Negative feedback regulation of myogenic tone is achieved by the activity of large-conductance, Ca²⁺ activated K⁺ (BK_Ca) channels and K_V channels, essentially preventing vasospasm. The model of myogenic tone presented in Figure 1 is inherently a positive feedback process. Pressure-induced “stretch” of VSMCs leading to activation of TRPC6 and TRPM4 channels, membrane depolarization and activation of CaV1.2 VGCC, if left unchecked, would strongly depolarize the plasma membrane of the VSMCs theoretically approaching the equilibrium potential for Ca²⁺ (~+120 mV) and leading to maximal activation of the contractile machinery in these cells. This does not happen because there is strong expression of both BK_Ca and K_V channels in the sarcolemma of the VSMCs that provides a negative-feedback signal to dampen and stabilize myogenic tone to maintain homeostasis (Jackson, 2000b, 2005, 2017, 2018a; Jackson & Blair, 1998; Tykocki et al., 2017).

The increase in intracellular Ca²⁺ (more on this topic below) and membrane depolarization associated with the myogenic response activate BK_Ca channels (Jackson, 2000b, 2005, 2017, 2018a; Jackson & Blair, 1998; Tykocki et al., 2017). The opening of only a few of these high conductance channels (~250 pS in cremaster arteriolar VSMCs; (Jackson & Blair, 1998)) leads to K⁺ efflux from the VSMCs down the K⁺ electrochemical gradient, limiting the pressure-induced depolarization of the VSMCs and preventing pressure-induced vasospasm (Jackson, 2000b, 2005, 2017, 2018a; Jackson & Blair, 1998; Tykocki et al., 2017). However, the source of Ca²⁺ that contributes to the activation of BK_Ca channels in peripheral arterioles appears to be different from what has been reported in upstream resistance arteries (Westcott et al., 2012; Westcott & Jackson, 2011). In cerebral (Nelson et al., 1995) and other resistance arteries (Westcott et al., 2012; Westcott & Jackson, 2011), Ca²⁺ released from groups of ryanodine receptors (RyR) into the subsarcolemmal space in the form of Ca²⁺ sparks appear to provide a major source of Ca²⁺ controlling the activity of BK_Ca channels. In these upstream resistance arteries, inhibition of ryanodine receptors with ryanodine or tetracaine, for example, eliminates Ca²⁺ sparks and increases myogenic tone to the same extent as blocking BK_Ca channels directly (Nelson et al., 1995; Westcott et al., 2012; Westcott & Jackson, 2011). In contrast, some arteriolar VSMCs do not display Ca²⁺ sparks at resting myogenic tone (Dabertrand et al., 2012; Westcott et al., 2012; Westcott & Jackson, 2011). In addition, inhibition of RyR with ryanodine or tetracaine which should inhibit RyR-dependent activation of BK_Ca channels, has no significant effect on myogenic tone implying that RyR are silent in these microvessels, as noted earlier in the chapter. For example, blockade of RyR in cremaster (Westcott et al., 2012; Westcott & Jackson, 2011) or cheek pouch arterioles (Figure 10A and C) studied by pressure myography, has no significant effect on myogenic tone, whereas, direct block of BK_Ca channels produces the expected constriction (increase in myogenic tone) (Westcott et al., 2012; Westcott & Jackson, 2011) (Figure 10B and C). Thus, BK_Ca channels do not appear to be controlled by RyR-related Ca²⁺ sparks in cremaster or cheek pouch arterioles. Instead, we think that Ca²⁺ entry through CaV1.2 VGCCs provides the major source of Ca²⁺ controlling the activity of BK_Ca channels. As has been reported in VSMCs from rabbit coronary arteries (Guia, Wan, Courtemanche, & Leblanc, 1999) and mouse mesenteric arteries (Suzuki, Yamamura, Ohya, & Imaizumi, 2013), BK_Ca channels are co-localized with VGCC in hamster cremaster arteriolar VSMCs (Jackson, 2018b). In vivo studies also support the hypothesis that Ca²⁺ entry through CaV1.2 VGCCs activate BK_Ca channels in cremaster arterioles (Jackson & Blair, 1998) and in cheek pouch arterioles (Figure 11). The lack of role for RyR in control of BK_Ca channels may be due to the level of expression of RyR isoforms and apparent lack of clustering of these channels in endoplasmic reticulum compared to RyR in upstream resistance arteries (Westcott et al., 2012).

Figure 10: Ryanodine receptors are silent, whereas BK_Ca channels are active and contribute to the negative-feedback regulation of myogenic tone in hamster cheek pouch arterioles, in vitro.

Panels A and B: Digitized diameter records demonstrating the lack of effect of ryanodine (Panel A) but significant constriction produced by the BK_Ca channel blocker TEA (1 mM) in isolated cheek pouch arterioles studied by pressure myography as described (Jackson & Boerman, 2017). Panel C: Summary data for experiments as in Panels A and B showing that TEA but not ryanodine constricts hamster cheek pouch arterioles. Data are means ± SE (n = 15 arterioles). * = p < 0.05 by Sidak’s multiple comparison method after two-way ANOVA.

Figure 11: Block of BK_Ca channels has no effect on resting diameter but potentiates O₂-induced arteriolar constriction, in vivo.

Panel A: Digitized diameter record of a hamster cheek pouch arteriole studied by intravital microscopy as in (Jackson, 1993b). At rest, 1 mM TEA had no effect on resting diameter but augmented constriction when the arterioles are exposed to elevated O₂ levels as shown. After washout of O₂ and TEA, elevated of O₂ levels constricted the arterioles, but to a larger diameter than in the presence of 1 mM TEA. Subsequent addition of TEA further constricted the arteriole as in the first trial of 21% O₂ + 1 mM TEA shown in the left side of Panel A. Panel B: Summary data for experiments as in Panel A. Data are mean diameters ± SE (n = 5). Under control conditions, TEA had no effect on arteriolar diameter, but in O₂ constricted arterioles, TEA caused further constriction confirming prior studies in hamster cremaster muscle that BK_Ca channels are active when VGCC are active (Jackson & Blair, 1998).

The BK_Ca channels expressed by cremaster arteriolar VSMCs also have a substantially higher Ca²⁺ set point (the minimum Ca²⁺ concentration required for activity at physiological membrane potentials) (Jackson & Blair, 1998). This appears to be due to a lower expression of modulatory β-subunits (Yang et al., 2009) but could also be due to differences in spliced variant expression of the α-subunits that also can affect the response of these channels Ca²⁺ (Nourian et al., 2014) or differences in expression of other modulatory subunits (Evanson, Bannister, Leo, & Jaggar, 2014). The consequence of this high Ca²⁺ set point is that very high local levels of Ca²⁺ appear to be required to activate these channels in peripheral arteriolar VSMCs (>3 μM). We suspect that Ca²⁺ that enters through VGCC adjacent to a BK_Ca channel, perhaps in the form of Ca²⁺ sparklets (Amberg, Navedo, Nieves-Cintron, Molkentin, & Santana, 2007), is sufficient to activate these channels, tuning the activity of the BK_Ca channels to match the activity of the VGCCs as previously proposed in coronary VSMCs (Guia et al., 1999) and mesenteric VSMCs (Suzuki et al., 2013). Calcium released, for example, through IP₃R in the form of Ca²⁺ waves in mouse and hamster feed arteries and downstream cremaster arteriolar VSMCs do not appear to activate BK_Ca channels because block of IP₃R reduces myogenic tone rather than increases tone as expected if BK_Ca channels were activated by the IP₃R-dependent Ca²⁺ waves (Westcott et al., 2012; Westcott & Jackson, 2011). In contrast, studies of cerebral arteries suggest that IP₃R are coupled to BK_Ca channels and contribute to the negative-feedback regulation of myogenic tone (Zhao et al., 2010). Why Ca²⁺ waves appear not to be coupled to BK_Ca channels in feed arteries and cremaster arterioles remains to be established. Most likely the Ca²⁺ waves do not raise subsarcolemmal Ca²⁺ to a level sufficient to activate arteriolar BK_Ca channels.

Studies of cerebral arteries and mesenteric arteries also have suggested a role for Ca²⁺ influx through CaV3.2 channels in controlling BK_Ca channel activity (Abd El-Rahman et al., 2013; Harraz et al., 2014; Harraz et al., 2015). However, studies in a murine skeletal muscle feed artery have failed to replicate their findings (Mullan, Pettis, & Jackson, 2017) suggesting that there may be regional differences in coupling between CaV3.2 and BK_Ca channels.

Voltage-gated K⁺ channels also contribute to the negative-feedback regulation of myogenic tone in peripheral arterioles in addition to BK_Ca channels (see (Jackson, 2018a; Tykocki et al., 2017) for reviews). Studies have shown that VSMCs from resistance arteries and arterioles display a diverse array of K_V channels that include members of the K_V 1, 2, 3, 4, 7 and 11 families (Tykocki et al., 2017). These channels are activated by membrane depolarization and we showed that block of members of this large family of K⁺ channels with 4-aminopyridine (4-AP) substantially reduces whole-cell outward K⁺ currents at physiological membrane potentials (−90 – 0 mV) in cremaster arteriolar VSMCs and depolarizes isolated VSMCs (Jackson, Huebner, & Rusch, 1997) (Figure 12A–D). In pressure myography experiments of hamster cremaster arterioles, 3 mM 4-AP constricts arterioles by about 20% (Jackson, 2018a) (Figure 12E). Similar effects of 4-AP are seen in pressurized hamster cheek pouch arterioles (Figure 12F). Thus, as was shown in cerebral resistance arteries (Knot & Nelson, 1995) and elsewhere (Tykocki et al., 2017), these data provide compelling evidence that in addition to BK_Ca channels, K_V channels also importantly contribute to the negative feedback regulation of myogenic tone in peripheral arterioles. Although Ca²⁺ and PKC-dependent inhibition of K_V may dampen this negative feedback, there appears to be sufficient K_V channel activity so that these channels still importantly contribute to the negative-feedback of VSMC membrane potential and myogenic tone.

Figure 12: K_V channels contribute to the negative feedback regulation of arteriolar myogenic tone.

Panels A–D are replotted from (Jackson et al., 1997) with permission. Panel A: Digitized Whole-cell currents recorded from an enzymatically isolated rat cremaster VSMC using the perforated patch technique (Jackson et al., 1997). Shown are outward currents elicited by 400 mV voltage pulses between −90 and 0 mV in 10 mV increments recorded under Control conditions. Panel B: Whole-cell currents as in Panel A, but in the presence of 3 mM 4-AP to block K_V channels. Note that the majority of outward current was inhibited by 4-AP. Panel C: Summary data for experiments as in Panels A and B. Data are mean current densities (pA/pF) ± SE (n = 8 cells) showing substantial inhibition of currents by 4-AP. Panel D: Expanded scale of mean current densities for data shown in Panel C showing that 4-AP shifted the whole-cell reversal potential (membrane potential where current density = 0), a measure of membrane potential, from −44 ± 2.7 mV to −23 ± 4.4 mV (n = 8, p < 0.05 by paired t-test) (Jackson et al., 1997). Panels E and F: In addition to inhibition of K_V currents and membrane depolarization (Panels A–E), 4-AP (3 mM) constricted hamster cremaster (Panel E) and cheek pouch (Panel F) arterioles studied by pressure myography as in (Jackson & Boerman, 2017). Data are mean diameters ± SE (n = 8 for cremaster and n = 4 for cheek pouch arterioles), * = p < 0.05 by paired t-tests.

Additional K⁺ channels also contribute to the regulation of membrane potential of arteriolar VSMCs and hence myogenic tone. In mouse skeletal muscle resistance arteries and downstream arterioles, 100 μM Ba²⁺, a selective blocker of inwardly-rectifying K⁺ (K_IR) channels (Quayle, McCarron, Brayden, & Nelson, 1993), constricts these vessels by about 20% (Figure 13A and B). These data suggest that K_IR channels are open under resting conditions and contribute to the regulation of resting membrane potential and myogenic tone. In 1^st order hamster cremaster arterioles, 100 μM Ba²⁺ was reported to produce VSMC depolarization (4.4 ± 1.5 mV; p = 0.035, n = 6 by paired t-test) and a small (10.4 ± 6.5%; p = 0.17, n = 6 by paired t-test), but not statistically significant constriction (Burns, Cohen, & Jackson, 2004). However, reexamination of these data indicated that one arteriole lost tone during the experiment. Elimination of this vessel from the analysis revealed that 100 μM Ba²⁺ constricted the remaining arterioles by 15.3 ± 4.6% (n = 5, p = 0.03 by paired t-test) (Figure 13C and D), consistent with data from mouse cremaster arterioles (Figure 13A and B). Barium also constricts murine skeletal muscle resistance arteries in pressure myography experiments by a similar amount (Hayoz, Pettis, Bradley, Segal, & Jackson, 2017). As with K_V channels, despite the potential for inhibition of the activity of K_IR channels by PKC and membrane depolarization, there are sufficient K_IR channels open at rest that they contribute to membrane potential and dampen pressure-induced membrane depolarization and myogenic tone.

Figure 13: K_IR channels are open under resting conditions and contribute to regulation of myogenic tone in cremaster arterioles.

Panel A: digitized diameter recording from a C57BL6 mouse cremaster arteriole studied by pressure myography as in (Westcott et al., 2012). Addition of the K_IR blocker, Ba²⁺ (100 μM) constricted the arteriole about 20%. Similar results were obtained in mouse skeletal muscle feed arteries (Hayoz et al., 2017). Panel B: Summary data for experiments as in Panel A. Data are mean diameters ± SE (n = 5), * = p < 0.05 by paired t-test. Panel C: Data are replotted from data in (Burns et al., 2004) with one observation from a vessel that lost tone during the experiment removed as noted in the text. Data are mean membrane potentials or diameters ± SE (n = 5), * = p < 0.05 by paired t-test.

In vivo studies in hamster cheek pouch and cremaster muscle suggest that ATP-sensitive K⁺ (K_ATP) channels also are active at resting myogenic tone and blunt the pressure-induced VSMC activity (Jackson, 1993a): the K_ATP antagonist, glibenclamide, significantly constricts arterioles in these preparations supporting the hypothesis that these channels can be open at rest and contribute to the determination of membrane potential and myogenic tone (Jackson, 1993a) (Figure 14). In addition, patch clamp studies of enzymatically isolated hamster cremaster arteriolar VSMCs suggest that K_ATP channels contribute to resting membrane potential within the physiological range (Jackson et al., 1997) (Figure 15). However, ex vivo, in pressure myography experiments, glibenclamide did not produce significant membrane depolarization or constriction of hamster cremaster arterioles (Burns et al., 2004). The mechanism by which K_ATP channels are open in vivo and when cells are isolated from these arterioles but are silent when the arterioles are studied by pressure myography, remains to be established. Nonetheless, these channels may contribute to the regulation of resting membrane potential, particularly in vivo, and also dampen pressure-induced membrane depolarization and myogenic tone.

Figure 14: Glibenclamide constricts hamster cremaster arterioles, in vivo.

Data shown are from (Jackson, 1998) with permission. Panel A: Digitized diameter record of a small third-order arteriole in a superfused hamster cremaster preparation. Starting at the top left of the diameter recording, topical application of 2.3 nmol of the K_ATP agonist, pinacidil, dilated the arteriole indicating that there are closed, recruitable K_ATP channels in this preparation. Elevation of the oxygen in the superfusate to 7% (PO₂ = 49 mm Hg) and 21% (PO₂ = 150 mm Hg) produced arteriolar constriction to demonstrate the reactivity of this arteriole. After return to the control condition (0% O₂, PO₂ = 15 mm Hg) and diameter recovery, 2 μM glibenclamide was added to the superfusate, as indicated. This produced substantial constriction of the arteriole that was maintained as long as glibenclamide was present. The arteriole was then challenged again with pinacidil and no dilation was observed demonstrating the efficacy of glibenclamide for block of K_ATP channels. Panel B: Summary data from 5 similar preparations from (Jackson, 1993a) showing that glibenclamide significantly constricts hamster cremaster arterioles, in vivo. The data are means ± SE. * = p < 0.05 by paired t-test.

Figure 15: K_ATP channels are open at rest in isolated cremaster VSMCs.

Data are replotted from (Jackson, 1998; Jackson et al., 1997) with permission. Panel A shows a family of whole cell currents in a cremaster VSMC studied by the perforated-patch technique as described (Jackson et al., 1997) in response to 400 ms voltage pulses from −90 to −30 mV in 10 mV increments from a holding potential of −60 mV. The arrow next to the current axis and the white dotted line indicate the holding current at −60 mV. Panel B: As in Panel A in the presence of the K_ATP channel antagonist, glibenclamide (1 μM). The arrow next to current axis is the holding current at −60 mV; the dotted white line is the Control holding current. Glibenclamide made the holding current more negative and inhibited currents between −60 and −30 mV. Panel C: Summary data showing that glibenclamide significantly inhibited currents between −60 and −30 mV and reduced the slope of the I–V relationship in this range, resulting in a shift of the whole-cell reversal potential from about −50 mV to about −35 mV, consistent with membrane depolarization. Data are mean current densities ± SE (n = 6 cells), * = p < 0.05 by Tukey’s test after 2-Way ANOVA. Panel D: Summary data as mean membrane potentials ± SE (n = 6 cells) recorded in current clamp as described (Jackson et al., 1997); * = p < 0.05 by paired t-test. Consistent with the changes in the I–V relationship shown in Panel C, glibenclamide significantly depolarized cremaster VSMCs. These data support the hypothesis that there are open K_ATP channels at rest in these VSMCs that contribute to resting membrane potential and the regulation of myogenic tone.

Summary and Perspectives

Ion channels contribute substantially to myogenic tone of peripheral arterioles as outlined in the chapter. They provide the major source of activator Ca²⁺ and also play a dominant role in setting and modulating membrane potential that, in turn, controls the open-state probability of VGCCs, BK_Ca channels, K_V channels and K_IR channels. While much has been learned about ion channel expression and function in VSMCs (Tykocki et al., 2017), few of these studies have extended to VSMCs from arterioles in the microcirculation. The established heterogeneity in the mechanisms regulating myogenic tone between upstream arteries and downstream arterioles (Brayden et al., 2013; Cipolla et al., 2014; Westcott et al., 2012; Westcott & Jackson, 2011) and between arterioles in different regions of the body (Jackson & Boerman, 2017; Kur, Bankhead, Scholfield, Curtis, & McGeown, 2013) imply that extrapolations cannot be made. Direct measurement of ion channel expression and function in arteriolar VSMCs from the specific organ/tissue of onterest must be made. With the explosion in methods for single cell Omics (Chen et al., 2019), this should be possible, but will require modification of existing methods or development of new approaches to isolate specific segments of arterioles in the microvascular bed to be studied.

Three areas warrant further research. First, why are CaV1.2 relatively silent at rest in arterioles, in vivo? Second, what is the genesis of the regional differences in ion channel expression and mechanisms of myogenic tone and its regulation? Third, what is the physiological significance of the redundancy in function of ion channels in VSMCs? All of these questions will require a better understanding of the pattern of expression of all ion channels, their binding partners and the signaling pathways that control these channels along with details of their physiological functions in the regulation of membrane potential and Ca²⁺ signaling in the context of the cells and vessels in which they are expressed. This will require application of isolated VSMC approaches (single cell Omics; patch clamp electrophysiology; Ca²⁺ imaging, etc.), the use of isolated arterioles studied by pressure myography (arteriolar tone, VSMC membrane potential, local and global Ca²⁺ imaging, etc.), and also intravital imaging (arteriolar tone, membrane potential and Ca²⁺ imaging) of the same arterioles as studied ex vivo and from which the VSMCs were isolated to close the loop. Knock-out models while useful, may not be helpful here except perhaps as controls for single cell electrophysiology and expression studies, because deletion of a channel, for example, may disrupt signaling complexes associated with those channels and have significant off-target effects; knock-in experiments using ion channels with non-functional pores might be a better approach to exclude off-target effects of disrupting signaling complexes and other ion channel functions. Selective pharmacological blockade of specific ion channels is also a viable approach because of the paired experimental design that can be applied provided that such agents exist or can be developed through high-throughput screening approaches that are now available.