Effects of endothelin-1 on hepatic stellate cell proliferation, collagen synthesis and secretion, intracellular free calcium concentration

Abstract

Aim: To explore the effects of endothelin-1 (ET-1) on hepatic stellate cells (HSCs) DNA uptake, DNA synthesis, collagen synthesis and secretion, inward whole-cell calcium concentration ([Ca2+]i) as well as the blocking effect of verapamil on ET-1-stimulated release of inward calcium (Ca2+) of HSC in vitro. Methods: Rat hepatic stellate cells (HSCs) were isolated and cultivated. 3H-TdR and 3H-proline incorporation used for testing DNA uptake and synthesis, collagen synthesis and secretion of HSCs cultured in vitro; Fluorescent calcium indicator Fura-2/AM was used to measure [Ca2+]i inward HSCs. Results: ET-1 at the concentration of 5×10-8 mol/L, caused significant increase both in HSC DNA synthesis (2 247±344 cpm, P<0.05) and DNA uptake (P<0.05) when compared with the control group. ET-1 could also increase collagen synthesis (P<0.05 vs control group) and collagen secretion (P<0.05 vs control group). Besides, inward HSC [Ca2+] i reached a peak concentration (422±98 mol/L, P< 0.001) at 2 min and then went down slowly to 165±51 mol/L (P< 0.01) at 25 min from resting state (39±4 mol/L) after treated with ET-1. Verapamil (5 mol/L) blocked ET-1-activated [Ca2+]i inward HSCs compared with control group (P<0.05). Fura-2/AM loaded HSC was suspended in no Ca2+ buffer containing 1 mol/L EGTA, 5 min later, 10-8 mol/L of ET-1 was added, [Ca2+]i inward HSCs rose from resting state to peak 399±123 mol/L, then began to come down by the time of 20 min. It could also raise [Ca2+]i inward HSCs even without Ca2+ in extracellular fluid, and had a remarkable dose-effect relationship(P<0.05). Meanwhile, verapamil could restrain the action of ET-1(P<0.05). Conclusion: Actions of ET-1 on collagen metabolism of HSCs may depend on the transportation of inward whole-cell calcium. Copyright © 2004 by The WJG Press.