Bataller R, Nicolás J M, Gineès P, Görbig M N, Garcia-Ramallo E, Lario S, Tobías E, Pinzani M, Thomas A P, Arroyo V, Rodés J
Department of Medicine, Hospital Clínic i Provincial, University of Barcelona School of Medicine, Catalunya, Spain.
J Hepatol. 1998 Sep;29(3):398-408. doi: 10.1016/s0168-8278(98)80057-3.
BACKGROUND/AIMS: Voltage-operated calcium channels are essential for the regulation of vascular tone and are potential targets for vasodilating agents. They regulate calcium entry and thereby cell contraction in vascular cell types. Hepatic stellate cells in the activated phenotype have contractile properties and could participate in the regulation of sinusoidal blood flow. Thus, this study was aimed at investigating the presence of voltage-operated calcium channels in human hepatic stellate cells activated in culture and the effects of their stimulation on intracellular calcium concentration ([Ca2+]i) and cell contractility.
Binding studies using [3H]-nitrendipine were performed to demonstrate the presence of voltage-operated calcium channels. Voltage-operated calcium channels were stimulated by causing cell membrane depolarization either by electrical field stimulation or extracellular high potassium. [Ca2+]i and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a charge-coupled device-imaging system.
Binding studies demonstrated the existence of voltage-operated calcium channels in human activated hepatic stellate cells (7.1+/-1.4x10(4) sites/cell with a Kd of 2.1+/-0.1 nM). Both electrical field stimulation and potassium chloride-induced cell depolarization resulted in a marked and prolonged increase in [Ca2+]i followed by intense cell contraction. The degree of cell contraction correlated with the intensity of calcium peaks. Removal of extracellular calcium or preincubation of cells with nitrendipine, a specific antagonist of voltage-operated calcium channels, completely blocked the effects on [Ca2+]i and cell contraction, whereas preincubation of cells with BayK-8644, a specific agonist of voltage-operated calcium channels, increased calcium peaks and contraction.
Activated human hepatic stellate cells have a large number of voltage-operated calcium channels, the activation of which is associated with an increase in [Ca2+]i followed by marked cell contraction. Voltage-operated calcium channels probably play an important role in the regulation of activated hepatic stellate cells contractility.
背景/目的：电压门控钙通道对于血管张力的调节至关重要，并且是血管舒张剂的潜在作用靶点。它们调节钙内流，从而调控血管细胞类型中的细胞收缩。具有激活表型的肝星状细胞具有收缩特性，可能参与肝血窦血流的调节。因此，本研究旨在探究培养中激活的人肝星状细胞中电压门控钙通道的存在情况，以及刺激这些通道对细胞内钙浓度（[Ca2+]i）和细胞收缩性的影响。
采用[3H]-尼群地平进行结合研究，以证明电压门控钙通道的存在。通过电场刺激或细胞外高钾使细胞膜去极化来刺激电压门控钙通道。使用配备电荷耦合器件成像系统的落射荧光显微镜，采用形态测量法，对加载了fura-2的单个细胞中的[Ca2+]i和细胞收缩进行测量。
结合研究表明，人激活的肝星状细胞中存在电压门控钙通道（7.1±1.4x10(4)个位点/细胞，解离常数Kd为2.1±0.1 nM）。电场刺激和氯化钾诱导的细胞去极化均导致[Ca2+]i显著且持续增加，随后细胞强烈收缩。细胞收缩程度与钙峰强度相关。去除细胞外钙或用电压门控钙通道的特异性拮抗剂尼群地平对细胞进行预孵育，可完全阻断对[Ca2+]i和细胞收缩的影响，而用电压门控钙通道的特异性激动剂BayK-8644对细胞进行预孵育，则会增加钙峰和收缩。
激活的人肝星状细胞具有大量电压门控钙通道，其激活与[Ca2+]i增加及显著的细胞收缩相关。电压门控钙通道可能在激活的肝星状细胞收缩性调节中起重要作用。
