Pancreatitis-associated ascitic fluid increases intracellular Ca(2+) 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 Ca(2+) concentration (Ca(2+)) 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 Ca(2+) was investigated. Primary cultures of rat hepatocytes were loaded with Fura-2/acetoxymethyl, and the changes of Ca(2+) were measured using spectrofluorometer.

RESULTS

The baseline of hepatocyte Ca(2+) was 172 +/- 17 nM. Ca(2+) 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 Ca(2+) elevation activity and cytotoxic activity. Neither 8-(N,N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) nor thapsigargin inhibited the PAAF-evoked Ca(2+) elevation. Chelation of extracellular Ca(2+) by ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) prevented the elevation of Ca(2+), but verapamil did not prevent it. Platelet-activating factor antagonist (TCV-309) blocked the PAAF-elicited Ca(2+) elevation. Pancreatitis-associated serum also increased hepatocyte Ca(2+). Moreover, PAAF increased Ca(2+) on Madin-Darby canine kidney cells in a dose-dependent manner.

CONCLUSIONS

These results suggest that the dramatic elevation of hepatocyte Ca(2+) 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 Ca(2+). Elevation of Ca(2+) in various cells may be involved in the mechanism of multiple organ failure in this disease.