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The SMCs of skeletal muscle arterioles are intricately sensitive to changes in membrane potential. Upon increasing luminal pressure, the SMCs depolarize, thereby opening VDCCs, which leads to contraction. Mechanisms that oppose this myogenic tone can involve voltage-dependent and independent dilator pathways, and can be endothelium-dependent or independent. Of particular interest are the pathways leading to hyperpolarization of SMCs, as these can potentially evoke both local and conducted dilation. This review focuses on three agonists that cause local and conducted dilation in skeletal muscle: ACh, ATP, and KCl. The mechanisms for the release of these agonists during motor nerve stimulation and/or hypoxia, and their actions to open either Ca 2+ -activated K + channels (K C a ) or inwardly rectifying K + channels (K IR ) are described. By causing local and conducted dilation, each agonist has the ability to improve skeletal muscle blood flow during exercise and ischemia., (© 2016 John Wiley & Sons Ltd.)

Our understanding of the relationship between EC membrane potential and Ca(2+) entry has been shaped historically by data from cells in culture. Membrane hyperpolarization was associated with raised cytoplasmic [Ca(2+) ] ascribed to the increase in the inward electrochemical gradient for Ca(2+) , as ECs are generally thought to lack VGCC. Ca(2+) influx was assumed to reflect the presence of an undefined Ca(2+) "leak" channel, although the original research articles with isolated ECs did not elucidate which Ca(2+) influx channel was involved or indeed if a transporter might contribute. Overall, these early studies left many unanswered questions, not least whether a similar mechanism operates in native ECs that are coupled to each other and, in many smaller arteries and arterioles, to the adjacent vascular SMCs via gap junctions. This review discusses whether Ca(2+) leak through constitutively active EC Ca(2+) channels or a more defined, gated pathway might underlie the reported link between enhanced Ca(2+) entry and hyperpolarization. Electrophysiological evidence from ECs in isolation is compared with those in intact arteries and arterioles and the possible physiological relevance of EC Ca(2+) entry driven by hyperpolarization discussed., (© 2013 John Wiley & Sons Ltd.)

Objective:   To assess the influence of blocking smooth muscle large conductance Ca(2+) -activated K+ channels and voltage-gated K+ channels on the conducted dilation to ACh and isoproterenol., Materials and Methods:   Rat mesenteric arteries were isolated with a bifurcation, triple-cannulated, pressurized and imaged using confocal microscopy. Phenylephrine was added to the superfusate to generate tone, and agonists perfused into a sidebranch to evoke local dilation and subsequent conducted dilation into the feed artery., Results:   Both ACh- and isoproterenol-stimulated local and conducted dilation with similar magnitudes of decay with distance along the feed artery (2000μm: ∼15% maximum dilation). The gap junction uncoupler carbenoxolone prevented both conducted dilation and intercellular spread of dye through gap junctions. IbTx, TEA or 4-AP, blockers of large conductance Ca(2+) -activated K+ channels and voltage-gated K+ channels, did not affect conducted dilation to either agonist. A combination of either IbTx or TEA with 4-AP markedly improved the extent of conducted dilation to both agonists (2000μm: >50% maximum dilation). The enhanced conducted dilation was reflected in the hyperpolarization to ACh (2000μm: Control, 4±1 mV, n = 3; TEA with 4-AP, 14±3mV, n=4), and was dependent on the endothelium., Conclusions:   These data show that activated BK(Ca) and K(V) -channels serve to reduce the effectiveness of conducted dilation., (© 2011 John Wiley & Sons Ltd.)

Objective: Changes in smooth muscle cell (SMC) membrane potential (Em) are critical to vasomotor responses. As a fluorescent indicator approach would lessen limitations of glass electrodes in contracting preparations, we aimed to develop a Forster (or fluorescence) resonance energy transfer (FRET)-based measurement for Em., Methods: The FRET pair used in this study (donor CC2-DMPE [excitation 405 nm] and acceptor DisBAC(4) (3)) provide rapid measurements at a sensitivity not achievable with many ratiometric indicators. The method also combined measurement of changes in Ca(2+) (i) using fluo-4 and excitation at 490 nm., Results: After establishing loading conditions, a linear relationship was demonstrated between Em and fluorescence signal in FRET dye-loaded HEK cells held under voltage clamp. Over the voltage range from -70 to +30 mV, slope (of FRET signal vs. voltage, m) = 0.49 ± 0.07, r(2)  = 0.96 ± 0.025. Similar data were obtained in cerebral artery SMCs, slope (m) = 0.30 ± 0.02, r(2)  = 0.98 ± 0.02. Change in FRET emission ratio over the holding potential of -70 to +30 mV was 41.7 ± 4.9% for HEK cells and 30.0 ± 2.3% for arterial SMCs. The FRET signal was also shown to be modulated by KCl-induced depolarization in a concentration-dependent manner. Further, in isolated arterial SMCs, KCl-induced depolarization (60 mM) measurements occurred with increased fluo-4 fluorescence emission (62 ± 9%) and contraction (-27 ± 4.2%)., Conclusions: The data support the FRET-based approach for measuring changes in Em in arterial SMCs. Further, image-based measurements of Em can be combined with analysis of temporal changes in Ca(2+) (i) and contraction., (© 2010 John Wiley & Sons Ltd.)

As smooth muscle cell (SMC) membrane potential (E(m)) is critical for vascular responsiveness, and arteriolar SMCs are depolarized at physiological intraluminal pressures, we hypothesized that myogenic tone impacts on dilation mediated by endothelium-derived hyperpolarization (EDH). Studies were performed on cannulated mouse cremaster arterioles [diameter, 33+/-2 microm (n=23) at 60 mmHg; SMC Em -34.6+/-1.2 mV (n=7)]. Myogenic activity was assessed as tone developed in response to intraluminal pressure. Functional observations were related to mRNA, protein expression, and anatomy. Acetylcholine concentration-response curves showed a modest shift following indomethacin (10 microM) and L-NAME (100 microM), although maximal vasodilation was achieved. Residual dilation was removed by apamin (1 microM) in combination with TRAM-34 (1 microM) or charybotoxin (0.1 microM), indicating the requirement of small (S) and intermediate (I) calcium-activated potassium channels (K(Ca)). Charybdotoxin, but not TRAM-34, caused vasoconstriction, presumably through the inhibition of SMC BK(Ca). Expression of SK3 and IK1 was confirmed by immunohistochemistry and polymerase chain reaction, while myoendothelial junctions were common, suggesting a high degree of cell coupling. Also consistent with a role for endothelial K(Ca) channels, acetylcholine increased endothelium [Ca(2 +)](i). Apamin and TRAM-34 similarly blocked EDH-mediated dilation at intraluminal pressures of 30 and 90 mmHg, suggesting that in mouse arterioles, SK(Ca -) and IK(Ca -) mediated mechanisms predominate and operate independently of physiological levels of myogenic activation.

Objective: The authors probed endothelium-dependent dilation and endothelial cell Ca2+ handling in myogenically active resistance arteries., Methods: First-order arteries were removed from rat cremaster muscles, cannulated, and pressurized (75 mmHg). Vessel diameter and endothelial cell Ca2+ were monitored using confocal microscopy, and arterial ultrastructure was determined using electron microscopy., Results: Acetylcholine (ACh) stimulated elevations and oscillations in endothelial cell Ca2+, and concentration-dependently dilated arteries with myogenic tone. NO-independent dilation was blocked by 35 mM K+. Combined IK(Ca) (1 microM TRAM-34) and SK(Ca) (100 nM apamin) blockade partially inhibited NO-independent relaxations, with residual relaxations sensitive to BK(Ca) or cytochrome P-450 inhibition (100 nM iberiotoxin, and 20 microM 17-ODYA or 10 microM MS-PPOH). 11,12-EET stimulated iberiotoxin-sensitive dilation, but did not affect endothelial cell Ca2+. 15 mM K+ evoked dilation sensitive to inhibition of K(IR) (30 microM Ba2+) and Na+/K+-ATPase (10 microM ouabain), whereas these blockers did not affect ACh-mediated dilations. Homo- and heterocellular gap junctions were identified in radial sections through arteries., Conclusion: These data suggest that rises in endothelial cell Ca2+ stimulate SK(Ca) and IK(Ca) channels, leading to hyperpolarization and dilation, likely due to electrical coupling. In addition, a component was unmasked following SK(Ca) and IK(Ca) blockade, attributable to activation of BK(Ca) channels by cytochrome P-450 metabolites.