Pancreatitis-associated ascitic fluid increases intracellular Ca2+ concentration on hepatocytes

Background. We have reported that pancreatitis-associated ascitic fluid (PAAF) contains a cytotoxic factor(s) inducing apoptosis on renal tubular cells and hepatocytes, It has been suggested that elevation of intracellular Ca2+ concentration ([Ca2+](i)) is associated with the development of cell damage and apoptosis, Methods. To clarify the mechanism of hepatocellular injury in acute pancreatitis, the effect of PAAF on hepatocyte [Ca2+](i) was investigated. Primary cultures of rat hepatocytes were loaded with Fura-2/ acetoxymethyl, and the changes of [Ca2+](i) were measured using spectrofluorometer. Results, The baseline of hepatocyte [Ca2+](i) was 172 +/- 17 nM. [Ca2+](i) increased from 1 min after the addition of PAAF in a dose-dependent manner. Fractionation of PAAF revealed only one fraction (molecular weight 5 x 10(4)) possessed both [Ca2+](i) elevation activity and cytotoxic activity. Neither 8-(N,N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) nor thapsigargin inhibited the PAAF-evoked [Ca2+](i) elevation. Chelation of extracellular Ca2+ by ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) prevented the elevation of [Ca2+](i), but verapamil did not prevent it. Platelet-activating factor antagonist (TCV-309) blocked the PAAP-elicited [Ca2+](i) elevation, Pancreatitis-associated serum also increased hepatocyte [Ca2+](i) Moreover, PAAF increased [Ca2+](i) on Madin-Darby canine kidney cells in a dose-dependent manner, Conclusions. These results suggest, that the dramatic elevation of hepatocyte [Ca2+](i) due to PAAF may be closely related to the hepatocellular injury in severe acute pancreatitis and that platelet-activating factor may play a pivotal role in increasing hepatocyte [Ca2+](i), Elevation of [Ca2+](i) in various cells may be involved in the mechanism of multiple organ failure in this disease. (C) 2000 Academic Press.