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

摘要

AIM: To explore the effects of endothelin-l(ET-l) on hepatic stellate cells (HSCs) DNA uptake, DNA synthesis, collagen synthesis and secretion, inward whole-cell calcium concentration ([Ca~(2+)]_i) as well as the blocking effect of verapamil on ET-1-stimulated release of inward calcium (Ca~(2+)) 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 [Ca~(2+)]_i inward HSCs. RESULTS: ET-1 at the concentration of 5x10~(-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 [Ca~(2+)]_ 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 [Ca~(2+)]_i inward HSCs compared with control group (P < 0.05). Fura-2/AM loaded HSC was suspended in no Ca~(2+) buffer containing 1 mol/L EGTA, 5 min later, 10~(-8) mol/L of ET-1 was added, [Ca~(2+)]_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 [Ca~(2+)]_i inward HSCs even without Ca~(2+) 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.