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

Endothelial function in resistance vessels entails Ca²⁺ and electrical signalling to promote vasodilatation and increase tissue blood flow. Whether membrane potential (V_m) governs intracellular calcium concentration ([Ca²⁺]_i) of the endothelium remains controversial. [Ca²⁺]_i and V_m 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 V_m to govern [Ca²⁺]_i by activating Ca²⁺-permeable channels in the plasma membrane. This effect of V_m is absent at rest and can be masked during maximal receptor stimulation. 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 (V_m) alter endothelial cell [Ca²⁺]_i. We tested the hypothesis that V_m governs [Ca²⁺]_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, [Ca²⁺]_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 mm [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. Ca²⁺ influx) phase of the [Ca²⁺]_i response to approximately half-maximal stimulation with 100 nm ACh (∼EC₅₀), [Ca²⁺]_i increased as V_m hyperpolarized below -40 mV and decreased as V_m 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, V_m can modulate Ca²⁺ entry through the plasma membrane in accord with the electrochemical driving force.