Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation
Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation
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DOI：
10.1113/jp271102
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发表时间：
2015-10-15
影响因子：
5.5
通讯作者：
Segal, Steven S.
中科院分区：
文献类型：
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作者：
Behringer, Erik J.;Segal, Steven S.
要点 阻力血管中的内皮功能需要 Ca2+ 和电信号传导来促进血管舒张并增加组织血流量。膜电位 (V-m) 是否控制内皮细胞内钙浓度 ([Ca2+](i)) 仍存在争议。使用从小鼠骨骼肌阻力动脉新鲜分离的完整内皮管，在细胞内电流注射期间同时评估[Ca2+](i)和V-m。在静息条件下，[Ca2+](i) 在超极化或去极化过程中没有变化。然而，在 100nM ACh（大约 EC50）存在的情况下，[Ca2+](i) 在超极化期间增加，在去极化期间减少。这些反应需要细胞外 Ca2+，并且随着 TRPV4 通道的基因消融而减弱一半。在天然微血管内皮中，毒蕈碱受体的半最大刺激使 V-m 通过激活质膜中的 Ca2+ 通透性通道来控制 [Ca2+](i)。 V-m 的这种作用在休息时不存在，并且在最大受体刺激期间可能被掩盖。 摘要在阻力动脉中，细胞内钙浓度 ([Ca2+](i)) 的升高与内皮细胞超极化相结合，导致平滑肌细胞松弛和血管舒张，从而增加组织血流量和氧输送。关于膜电位 (V-m) 的变化是否会改变内皮细胞 [Ca2+](i) 仍存在争议。我们通过进行 Fura-2 光度测定，同时记录和控制从 C57BL/6 小鼠上腹壁动脉新鲜分离的完整内皮管的 V-m，测试了 V-m 控制阻力动脉内皮细胞中 [Ca2+](i) 的假设。在静息条件下，当 V-m 通过暴露于 10M NS309（超极化至约 -80mV）、通过用 145mm [K+](o) 平衡（去极化至约 -5mV）或在细胞内电流注入期间（+/- 0.5 至 5nA，20 秒脉冲）从基线偏移（约 -40mV）时，[Ca2+](i) 没有变化而V-m 在大约-80mV 和+10mV 之间线性变化。相反，在 [Ca2+](i) 对 100nm ACh 的大约半最大刺激（近似于 EC50）响应的平台期（即 Ca2+ 流入）期间，[Ca2+](i) 随着 V-m 超极化低于 -40mV 而增加，随着 V-m 去极化高于 -40mV 而减少。去极化电流注入期间 [Ca2+](i) 减少的幅度与对 ACh 的平台 [Ca2+](i) 响应幅度相关。去除细胞外 Ca2+ 后，超极化对 [Ca2+](i) 的影响被消除，通过将细胞外 [Ca2+] 从 2mm 提高到 10mm，超极化的影响被微妙地增强，而在 TRPV4(-/-) 小鼠的内皮细胞中，超极化的影响减少了一半。因此，在毒蕈碱受体的次最大激活过程中，V-m 可以根据电化学驱动力调节 Ca2+ 穿过质膜的进入。
Key points 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 100nM ACh (approximate to 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.AbstractIn 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 (approximate to-40mV) via exposure to 10M NS309 (hyperpolarization to approximate to-80mV), via equilibration with 145mm [K+](o) (depolarization to approximate to-5mV), or during intracellular current injection (+/- 0.5 to 5nA, 20s pulses) while V-m changed linearly between approximate to-80mV and +10mV. In contrast, during the plateau (i.e. Ca2+ influx) phase of the [Ca2+](i) response to approximately half-maximal stimulation with 100nm ACh (approximate to EC50), [Ca2+](i) increased as V-m hyperpolarized below -40mV and decreased as V-m depolarized above -40mV. 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 2mm to 10mm 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.