INTERACTION OF PLATELET-ACTIVATING-FACTOR WITH RAT HEPATOCYTES - UPTAKE, TRANSLOCATION, METABOLISM, AND EFFECTS ON PAF-ACETYLHYDROLASE SECRETION AND PROTEIN-TYROSINE PHOSPHORYLATION

In the present study the interaction of the phospholipid mediator plat elet-activating factor (PAF) with rat hepatocytes in primary culture w as examined, Following exposure to hepatocytes, exogenous [H-3]alkyl-P AF was metabolized rapidly to [SH]lyso-PAF, the content of which was r aised in the outer leaflet of the plasma membrane within the initial 5 min of incubation, Thereafter [H-3]lyso-PAF was translocated into cel ls with concomitant reacylation to [H-3]alkyl-acyl-glycerophosphocholi ne, A portion of untransformed [H-3]PAF accumulated in the outer leafl et, and only a small amount of the [H-3]PAF was translocated into the inner leaflet of the plasma membrane, Detectable levels of [H-3]lyso-P AF were found in the medium of hepatocyte cultures at all times of inc ubation, These findings suggest that at least a portion of the cellula r PAF-acetylhydrolase (PAF-AH) activity is located in the outer leafle t of the plasma membrane and can be secreted into the medium, Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time d ependent fashion, Incubation of hepatocytes with exogenous PAF increas ed secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analo g methylcarbamyl-PAF significantly reduced secretion, The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activi ty of PAF-AH and also diminished its release by hepatocytes, In contra st, BN 50739 and WEB 2170, thienotriazolodiazepine PAF receptor antago nists, did not affect the PAF-AH activity, but increased its secretion by the cells, A full-length 3,8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot anal ysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was re adily detected in Kupffer cells, Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120-130 and 160-180 kDa and dephosphorylation of 80- to 90-kDa protei ns; these responses were not inhibited by WEB 2170 and BN 50739, The p rotein tyrosine kinase inhibitor genistein abolished the release of fr ee arachidonic acid, suggesting a crucial role for tyrosine phosphoryl ation in PAF-induced phospholipase Az activation in rat hepatocytes. T aken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosin e phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells, This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions. (C) 1996 Academic Press, Inc.