2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) ALTERS PANCREATIC MEMBRANE TYROSINE PHOSPHORYLATION FOLLOWING ACUTE TREATMENT

To understand the basic mechanisms of TCDD's action to cause hypoinsulinemia in several experimental animals, we have studied TCDD-induced changes in various protein kinase activities in membrane preparations of guinea pig pancreas. For this purpose, young male guinea pigs were treated through a single intraperitoneal injection with 1 or 3 mug/kg of TCDD in vivo, and, after given time periods, pancreas samples were obtained and membranes were isolated through homogenization and centrifugation procedures. Several sets of incubation conditions were selected for protein kinase activity assay, each favoring a specific type of protein kinase. It was found that overall protein phosphorylation activities were higher in the preparation from TCDD-treated animals as compared to those found in pair-fed controls and that this trend was more pronounced when he assay medium contained Mn2+ in place of Mg2+ and EGTA. These are the conditions that are known to favor protein tyrosine kinases. Other types of protein kinases from the treated animals did not show any significant differences from the pair-fed control animals, though that of protein kinase C in the treated preparation showed a modest increase. To establish that the type of protein kinases stimulated by TCDD are protein tyrosine kinases, we have carried out phosphoamino acid analyses, KOH digestion, and western blot analyses using an antibody to phosphotyrosine. All the results were consistent in supporting the idea that TCDD causes a rise in protein-tyrosine kinases in pancreas at early stages of poisoning.