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


CONCLUSIONS
Actions of ET-1 on collagen metabolism of HSCs may depend on the transportation of inward whole-cell calcium.
Department of Gastroenterology, Shanghai Tenth People Hospital of Tongji University, Shanghai 200072, China. guochuanyong@hotmail.com
Chuan-Yong
CY
Guo
Jian-Ye
JY
Wu
Yun-Bin
YB
Wu
Min-Zhang
MZ
Zhong
Han-Ming
HM
Lu
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D019332:Endothelin-1 / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D011392:Proline / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar; D013936:Thymidine / Q000493:pharmacokinetics; D014316:Tritium; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D019332:Endothelin-1; D014316:Tritium; D003094:Collagen; D004247:DNA; D011392:Proline; D014700:Verapamil; D002118:Calcium; D013936:Thymidine