Gardner J P, Tokudome G, Tomonari H, Maher E, Hollander D, Aviv A
Department of Pediatrics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103.
Am J Physiol. 1992 Jan;262(1 Pt 1):C148-55. doi: 10.1152/ajpcell.1992.262.1.C148.
The effects of endothelin-1 (ET) on the cytosolic free Ca (Cai) and cytosolic pH (pHi) were examined in primary cultures of human umbilical artery (HUA) vascular smooth muscle cells (VSMCs), respectively, loaded with fura-2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In 1 mM Ca, ET produced a dose-dependent, biphasic increase in the signal with a maximal effect at 400 nM ET. At this concentration, ET produced a Cai transient (mean +/- SE; a rise from basal Cai of 86 +/- 16 to 216 +/- 33 nM) that lasted for approximately 50-60 s. The Cai transient was followed by a slow but sustained increase in Cai. Both ET-induced Cai transient and posttransient Cai were attenuated in Ca deficient medium or by verapamil and nicardipine. In contrast to ET, thrombin elicited only a monophasic Cai response in HUA VSMCs. This response was also partially sensitive to Ca removal or verapamil. KCl (45 mM) depolarization did not elicit a Cai response. However, the presence of voltage sensitive Ca channels in HUA VSMCs was demonstrated by enhanced Mn uptake in cells depolarized with KCl. Both ET and thrombin treatment did not alter pHi. HUA VSMCs demonstrated a single class of ET receptors (approximately 13,000 sites/cell) with an equilibrium dissociation constant of 0.34 nM. Nicardipine did not alter ET binding. These observations suggest a dual effect of ET on the Cai profile in HUA VSMCs that is mediated by Ca mobilization and Ca entry through Ca channels. The Ca entry could include influx through receptor-operated Ca channels, voltage-sensitive Ca channels, or both, but without a direct interaction between ET and these channels.
在内皮素 -1（ET）对人脐动脉（HUA）血管平滑肌细胞（VSMC）胞质游离钙（Cai）和胞质pH（pHi）的影响的研究中，分别使用负载有fura -2和2',7'-双（羧乙基）-5（6）-羧基荧光素的细胞进行了实验。在1 mM钙浓度下，ET呈剂量依赖性地使信号出现双相增加，在400 nM ET时达到最大效应。在此浓度下，ET引起Cai瞬变（平均值±标准误；Cai从基础值86±16 nM升至216±33 nM），持续约50 - 60秒。Cai瞬变之后是Cai的缓慢但持续的增加。ET诱导的Cai瞬变和瞬变后Cai在缺钙培养基中或通过维拉帕米和尼卡地平处理后均减弱。与ET相反，凝血酶在HUA VSMC中仅引起单相Cai反应。该反应对钙去除或维拉帕米也部分敏感。45 mM KCl去极化未引起Cai反应。然而，通过KCl去极化的细胞中锰摄取增加证明了HUA VSMC中存在电压敏感钙通道。ET和凝血酶处理均未改变pHi。HUA VSMC显示出一类单一的ET受体（约13,000个位点/细胞），平衡解离常数为0.34 nM。尼卡地平不改变ET结合。这些观察结果表明ET对HUA VSMC中Cai分布具有双重作用，这是由钙动员和通过钙通道的钙内流介导的。钙内流可能包括通过受体操纵性钙通道、电压敏感钙通道或两者的内流，但ET与这些通道之间没有直接相互作用。
