Identification of calcium-dependent phospholipase A2 isoforms in human and rat pancreatic islets and insulin secreting beta-cell lines.

Abstract

Phospholipase A2 (PLA2) and its end product, arachidonic acid, are thought to be important signaling components in insulin secretion from pancreatic beta-cells. Because there are multiple Ca2+ -dependent and independent PLA2 biochemical activities in beta-cells, we have used a combination of molecular and immunological techniques to identify the isoforms of Ca2+ -dependent PLA2 present in pancreatic beta-cells. Total RNA extracted from the purified rat and human pancreatic islets and from insulin-secreting beta-TC3 and beta-HC6 cells was used as a template for complementary DNA (cDNA) synthesis. The RT-PCR was performed based on the oligonucleotide primers designed for the 14- kDa type II PLA2 and the 85-kDa cytosolic PLA2. The PCR products for both enzymes yielded single bands (375 bp and 910 bp for type II PLA2 and cytosolic PLA2, respectively). The PCR-generated cDNA fragments were confirmed to be identical to the type II and cytosolic PLA2 isoforms expressed in rat tissues, U937 cells, and A9 cells by DNA sequencing of the PCR products. The presence of these two isoforms of PLA2 was further confirmed by immunoblotting of extracts of pancreatic islets and beta-cells using specific antibodies directed toward each type of PLA2. Demonstration of the presence of type II and cytosolic PLA2 isoforms in islets provides the framework for further investigation of the regulation of PLA2 isoforms and their role in insulin secretion.