Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation

Key points Endothelial function in resistance vessels entails Ca2+ and electrical signalling to promote vasodilatation and increase tissue blood flow. Whether membrane potential (Vm) governs intracellular calcium concentration ([Ca2+]i) of the endothelium remains controversial. [Ca2+]i and Vm were evaluated simultaneously during intracellular current injection using intact endothelial tubes freshly isolated from mouse skeletal muscle resistance arteries. [Ca2+]i did not change during hyperpolarization or depolarization under resting conditions. However in the presence of 100 nM ACh (∼EC50), [Ca2+]i increased during hyperpolarization and decreased during depolarization. These responses required extracellular Ca2+ and were attenuated by half with genetic ablation of TRPV4 channels. In native microvascular endothelium, half‐maximal stimulation of muscarinic receptors enables Vm to govern [Ca2+]i by activating Ca2+‐permeable channels in the plasma membrane. This effect of Vm is absent at rest and can be masked during maximal receptor stimulation. Abstract In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca2+]i) to endothelial cell hyperpolarization underlies smooth muscle cell relaxation and vasodilatation, thereby increasing tissue blood flow and oxygen delivery. A controversy persists as to whether changes in membrane potential (Vm) alter endothelial cell [Ca2+]i. We tested the hypothesis that Vm governs [Ca2+]i in endothelium of resistance arteries by performing Fura‐2 photometry while recording and controlling Vm of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca2+]i did not change when Vm shifted from baseline (∼−40 mV) via exposure to 10 μM NS309 (hyperpolarization to ∼−80 mV), via equilibration with 145 mm [K+]o (depolarization to ∼−5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while Vm changed linearly between ∼−80 mV and +10 mV. In contrast, during the plateau (i.e. Ca2+ influx) phase of the [Ca2+]i response to approximately half‐maximal stimulation with 100 nm ACh (∼EC50), [Ca2+]i increased as Vm hyperpolarized below −40 mV and decreased as Vm depolarized above −40 mV. The magnitude of [Ca2+]i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca2+]i response to ACh. The effect of hyperpolarization on [Ca2+]i was abolished following removal of extracellular Ca2+, was enhanced subtly by raising extracellular [Ca2+] from 2 mm to 10 mm and was reduced by half in endothelium of TRPV4−/− mice. Thus, during submaximal activation of muscarinic receptors, Vm can modulate Ca2+ entry through the plasma membrane in accord with the electrochemical driving force.