Roth-Eichhorn S, Eberheim A, Bode H P, Gressner A M
Department of Clinical Chemistry, Philipps University, Marburg, Germany.
J Hepatol. 1999 Apr;30(4):612-20. doi: 10.1016/s0168-8278(99)80191-3.
BACKGROUND/AIMS: The transformation of hepatic stellate cells into myofibroblasts is a key step in the pathogenesis of fibrotic liver diseases. The intracellular signaling associated with hepatic stellate cell transformation becomes a point of interest, especially the role of cytosolic free calcium concentration ([Ca2+]i). The aim of the study was to investigate possible differences between various transformation phenotypes of hepatic stellate cells with regard to the calcium influx mediated by L-type voltage-operated calcium channels (L-type VOC).
Hepatic stellate cells were isolated from rat liver by pronase-collagenase reperfusion and cultured under standard conditions. The transformation of hepatic stellate cells was stimulated by treatment with transforming growth factor-beta (TGF-beta) or inhibited with interferon-gamma (IFN-gamma) and characterized by immunocytochemistry for smooth muscle alpha-actin and determination of hyaluronan in the culture media with a ligand binding assay. [Ca2+]i was measured in individual cells with fluorescence microscopy using fura-2. VOCs were activated by the standard procedure of extracellular potassium elevation, to achieve depolarization, and identified by various controls.
In transformed myofibroblasts the activation of VOCs by potassium elevation from 5.4 mmol/l to 50.4 mmol/l led to a 19% increase in [Ca2+]i in contrast to 0.2% in hepatic stellate cells cultured for 3 days. In 7-day old hepatic stellate cells, after stimulation of cell transformation with TGF-beta-1, an enhanced [Ca2+]i response to potassium elevation was detected, while inhibition of transformation with IFN-gamma for the same time caused a decreased calcium signal compared with untreated control cultures. Short-term treatment with the cytokines (1 day) did not influence depolarization-dependent calcium signals.
The results show the [Ca2+]i increase via L-type VOCs to be dependent on the transformation level of hepatic stellate cells into myofibroblasts which can be influenced by the long-term treatment of hepatic stellate cells with TGF-beta or IFN-gamma. In contrast, there is no evidence for direct regulation of VOC activity by TGF-beta or IFN-gamma after short-term exposure.
背景/目的：肝星状细胞向肌成纤维细胞的转变是肝纤维化疾病发病机制中的关键步骤。与肝星状细胞转变相关的细胞内信号传导成为研究热点，尤其是胞质游离钙浓度（[Ca2+]i）的作用。本研究旨在探讨肝星状细胞不同转变表型在L型电压门控钙通道（L型VOC）介导的钙内流方面可能存在的差异。
通过链霉蛋白酶 - 胶原酶再灌注从大鼠肝脏分离肝星状细胞，并在标准条件下培养。用转化生长因子 - β（TGF - β）刺激肝星状细胞的转变，或用干扰素 - γ（IFN - γ）抑制，通过平滑肌α - 肌动蛋白免疫细胞化学和配体结合测定法测定培养基中的透明质酸来进行表征。使用fura - 2通过荧光显微镜在单个细胞中测量[Ca2+]i。通过细胞外钾升高的标准程序激活VOC以实现去极化，并通过各种对照进行鉴定。
在转化的肌成纤维细胞中，钾浓度从5.4 mmol/l升高到50.4 mmol/l激活VOC导致[Ca2+]i增加19%，而培养3天的肝星状细胞中仅增加0.2%。在7日龄肝星状细胞中，用TGF - β - 1刺激细胞转变后，检测到对钾升高的[Ca2+]i反应增强，而同时用IFN - γ抑制转变导致与未处理的对照培养物相比钙信号降低。细胞因子短期处理（1天）不影响去极化依赖性钙信号。
结果表明，通过L型VOCs使[Ca2+]i增加取决于肝星状细胞向肌成纤维细胞的转变水平，这可受到用TGF - β或IFN - γ对肝星状细胞长期处理的影响。相比之下，短期暴露后没有证据表明TGF - β或IFN - γ直接调节VOC活性。
