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+)](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 [Ca(2+)](i) was investigated. Primary cultures of rat hepatocytes were loaded with Fura-2/acetoxymethyl, and the changes of [Ca(2+)](i) were measured using spectrofluorometer.


RESULTS
The baseline of hepatocyte [Ca(2+)](i) was 172 +/- 17 nM. [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](i) 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+)](i), but verapamil did not prevent it. Platelet-activating factor antagonist (TCV-309) blocked the PAAF-elicited [Ca(2+)](i) elevation. Pancreatitis-associated serum also increased hepatocyte [Ca(2+)](i). Moreover, PAAF increased [Ca(2+)](i) on Madin-Darby canine kidney cells in a dose-dependent manner.


CONCLUSIONS
These results suggest that the dramatic elevation of hepatocyte [Ca(2+)](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 [Ca(2+)](i). Elevation of [Ca(2+)](i) in various cells may be involved in the mechanism of multiple organ failure in this disease.
Ueda
T
T
The First Department of Surgery, Kobe University School of Medicine, Kobe, 650-0017, Japan.
Takeyama
Y
Y
Hori
Y
Y
Takase
K
K
Goshima
M
M
Kuroda
Y
Y
D000208:Acute Disease; D000818:Animals; D001202:Ascitic Fluid / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D004533:Egtazic Acid / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008297:Male; D009102:Multiple Organ Failure / Q000209:etiology; D010195:Pancreatitis / Q000150:complications*; D010972:Platelet Activating Factor / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D014700:Verapamil / Q000494:pharmacology
D010972:Platelet Activating Factor; D004533:Egtazic Acid; D014700:Verapamil; D002118:Calcium