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

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 ([Ca^2+]i) as well as the blocking effect of verapamil on ET-l-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 [Ca^2+]i inwardHSCs.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 vscontrol 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 to165+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+li 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 rain 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 wholecell calcium.

Antiproliferative and proapoptotic effects of somatostatin on activated hepatic stellate cells

AIM: To assess the effects of somatostatin on proliferation and apoptosis of activated rat hepatic stellate cells (HSCs). METHODS: HSCs isolated from the livers of adult SpragueDawley rats (weighing 400-500 g) by in situ perfusion and purified by single-step density gradient centrifugation with Nycodenz, became activated after 10 days' cultivation. Then the apoptotic rate of HSCs treated with different doses of somatostatin for 72 h, was assayed by acridine orange/ethidium bromide fluorescent staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, transmission electron microscopy and flow cytometry, while the proliferation of HSCs was measured by MTT assay. Furthermore, the mechanisms of somatostatin were investigated by cytodynamic analysis. RESULTS: Somatostatin at the concentration of 10^-6-10^-99 mol/L could decrease the proliferative rate, and promote the apoptosis of activated rat HSCs in a dose-dependent way. Its action was most significant when the concentration reached 10^-6 mol/L or 10^-7 mol/L (P<0.05-0.01). An obvious cell-cycle arrest (G0/G1 arrest) was the important way for somatostatin to exert its action. CONCLUSION: Antiproliferative and proapoptotic effects of low-dose somatostatin on activated rat HSCs can be obtained. These findings reveal its potential antifibrotic action.