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

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).

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-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.

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-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.

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-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.

MeSH terms

  • Actins / metabolism
  • Animals
  • Calcium / metabolism*
  • Calcium Channels / physiology*
  • Calcium Channels, L-Type
  • Cell Differentiation / drug effects
  • Cells, Cultured
  • Cytosol / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / physiology
  • Fura-2
  • Hyaluronic Acid / metabolism
  • Interferon-gamma / pharmacology
  • Kinetics
  • Liver / cytology*
  • Liver / drug effects
  • Liver / physiology*
  • Male
  • Membrane Potentials
  • Microscopy, Fluorescence
  • Muscle, Smooth / cytology
  • Muscle, Smooth / physiology
  • Potassium / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Recombinant Proteins / pharmacology
  • Transforming Growth Factor beta / pharmacology*


  • Actins
  • Calcium Channels
  • Calcium Channels, L-Type
  • Recombinant Proteins
  • Transforming Growth Factor beta
  • Interferon-gamma
  • Hyaluronic Acid
  • Potassium
  • Calcium
  • Fura-2