Kremer S G, Breuer W V, Skorecki K L
Department of Medicine, University of Toronto, Canada.
J Cell Physiol. 1989 Jan;138(1):97-105. doi: 10.1002/jcp.1041380114.
Mesangial cells are smooth muscle-like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential-sensitive fluorescent dye bis-oxonol and the intracellular fluorescent calcium-sensitive probe Indo-1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]i was 227 +/- 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4-6-fold rise. Resting membrane potential was 45.8 +/- 0.9 mV and vasoconstrictor hormones caused a depolarization of 14-18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: 1) depolarization persisted when sodium in the medium was substituted with N-methylglucamine; 2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor-stimulated depolarization; 3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and 4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]i by prior exposure to ionomycin in calcium-free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium-dependent and -independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to modulate the calcium signal.
系膜细胞是肾小球的平滑肌样细胞，对血管加压素和血管紧张素产生收缩反应并分泌前列腺素。这些反应有助于调节肾小球毛细血管滤过表面积。我们使用膜电位敏感荧光染料双苯磺酰草酰二苯胺和细胞内荧光钙敏感探针Indo-1来研究培养的大鼠系膜细胞在血管收缩激素作用下膜电位（Em）和细胞内游离钙浓度（[Ca2+]i）的变化。基础[Ca2+]i为227±4 nM，血管加压素或血管紧张素的最大浓度刺激导致[Ca2+]i瞬时升高4 - 6倍。静息膜电位为45.8±0.9 mV，血管收缩激素引起14 - 18 mV的去极化。以下细胞外离子替代表明氯离子外流是导致去极化的主要离子流：1）用N-甲基葡糖胺替代培养基中的钠时，去极化持续存在；2）用葡萄糖酸钠替代培养基中的氯化钠，增强了氯离子外流梯度，增强了血管收缩激素刺激的去极化；3）将细胞悬浮在氯化钾培养基中导致去极化，随后，血管加压素或血管紧张素刺激导致超极化；4）当使用葡萄糖酸钾培养基使细胞去极化时，这种超极化不发生。钙离子载体离子霉素也导致膜去极化。然而，通过预先在无钙培养基中暴露于离子霉素或用细胞内钙缓冲剂BAPTA加载系膜细胞来防止[Ca2+]i升高，并没有消除对血管收缩激素的去极化反应。这表明细胞内钙升高对于去极化不是必需的。相反，使用不同浓度的氯化钾在外部培养基中预先使细胞去极化，这消除了氯离子外流的电化学梯度，导致对血管加压素和血管紧张素的瞬时钙反应相应延长。这些发现表明，血管紧张素和血管加压素通过激活氯离子通道使系膜细胞去极化，并且这种激活可以通过钙依赖和非钙依赖机制发生。此外，氯离子通道的激活导致去极化可能有助于调节钙信号。
