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

Key points Endothelial function in resistance vessels entails Ca 2+ and electrical signalling to promote vasodilatation and increase tissue blood flow. Whether membrane potential ( V m ) governs intracellular calcium concentration ([Ca 2+ ] i ) of the endothelium remains controversial. [Ca 2+ ] i and V m were evaluated simultaneously during intracellular current injection using intact endothelial tubes freshly isolated from mouse skeletal muscle resistance arteries. [Ca 2+ ] i did not change during hyperpolarization or depolarization under resting conditions. However in the presence of 100 nM ACh (∼EC 50 ), [Ca 2+ ] i increased during hyperpolarization and decreased during depolarization. These responses required extracellular Ca 2+ and were attenuated by half with genetic ablation of TRPV4 channels. In native microvascular endothelium, half‐maximal stimulation of muscarinic receptors enables V m to govern [Ca 2+ ] i by activating Ca 2+ ‐permeable channels in the plasma membrane. This effect of V m is absent at rest and can be masked during maximal receptor stimulation.Abstract In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca 2+ ] 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 ( V m ) alter endothelial cell [Ca 2+ ] i . We tested the hypothesis that V m governs [Ca 2+ ] i in endothelium of resistance arteries by performing Fura‐2 photometry while recording and controlling V m of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca 2+ ] i did not change when V m shifted from baseline (∼−40 mV) via exposure to 10 μM NS309 (hyperpolarization to ∼−80 mV), via equilibration with 145 m m [K + ] o (depolarization to ∼−5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while V m changed linearly between ∼−80 mV and +10 mV. In contrast, during the plateau (i.e. Ca 2+ influx) phase of the [Ca 2+ ] i response to approximately half‐maximal stimulation with 100 n m ACh (∼EC 50 ), [Ca 2+ ] i increased as V m hyperpolarized below −40 mV and decreased as V m depolarized above −40 mV. The magnitude of [Ca 2+ ] i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca 2+ ] i response to ACh. The effect of hyperpolarization on [Ca 2+ ] i was abolished following removal of extracellular Ca 2+ , was enhanced subtly by raising extracellular [Ca 2+ ] from 2 m m to 10 m m and was reduced by half in endothelium of TRPV4 −/− mice. Thus, during submaximal activation of muscarinic receptors, V m can modulate Ca 2+ entry through the plasma membrane in accord with the electrochemical driving force.