Transformation-dependent calcium influx by voltage-operated calcium channels in stellate cells of rat liver

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). Methods: 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-β) or inhibited with interferon-gamma (IFN-γ) and characterized by immunocytochemistry for smooth muscle α-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. Results: 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-β-1, an enhanced [Ca2+]i response to potassium elevation was detected, while inhibition of transformation with IFN-γ 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. Conclusion: 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-β or IFN-γ. In contrast, there is no evidence for direct regulation of VOC activity by TGF-β or IFN-γ after short-term exposure.