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


CONCLUSIONS
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.
Roth-Eichhorn
S
S
Department of Clinical Chemistry, Philipps University, Marburg, Germany. roths@mailer.uni-marburg.de
Eberheim
A
A
Bode
H P
HP
Gressner
A M
AM
D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D020746:Calcium Channels, L-Type; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D016257:Fura-2; D006820:Hyaluronic Acid / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000166:cytology / Q000502:physiology; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D000199:Actins; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D007371:Interferon-gamma; D006820:Hyaluronic Acid; D011188:Potassium; D002118:Calcium; D016257:Fura-2