IDENTIFICATION AND LOCALIZATION OF A TYPE-IV CYTOSOLIC PHOSPHOLIPASE A(2) IN RAT PANCREATIC BETA-CELLS

The liberation of arachidonic acid (AA), by phospholipase A(2) (PLA(2) ), is the rate-limiting step in a number of cell signalling pathways. In the pancreatic beta-cell, AA itself is thought to participate in th e regulation of insulin secretion. Recently a Ca2+-sensitive, AA-selec tive cytosolic PLA(2) (type IV cPLA(2)) has been isolated from the hum an monocyte U937 cell line. Although the DNA sequence of this enzyme i mplies a molecular weight of 85 kDa, the protein migrates with a molec ular weight of 100-110 kDa on SDS-PAGE. In many cell types, cPLA(2)s w hich are reactive towards antibodies raised against the type IV cPLA(2 ) have been shown to hydrolyse AA from membrane glycerophospholipids. Using a polyclonal antibody raised against a recombinant form of type IV cPLA(2), we have detected an immunoreactive protein with a molecula r weight of 93.5 kDa in rat islets of Langerhans. Furthermore, we have detected similar immunoreactive proteins in insulin-secreting beta-ce ll lines and have shown co-expression of type IV cPLA(2) immunoreactiv ity and insulin immunoreactivity in rat pancreatic beta-cells. Under n on-stimulatory conditions the 93.5 kDa immunoreactive protein detected in rat islets of Langerhans was located predominantly in the cytosoli c fraction. We have shown that immunoprecipitation of the rat immunore active protein from rat islet homogenates significantly decreases the total dithiothreitol/beta-mercaptoethanol-insensitive PLA(2) activity by 56.4 +/- 7%. This provides further evidence that the immunoreactive rat protein is a type IV cPLA(2) and is responsible for a large compo nent of the PLA(2) activity in rat islets of Langerhans. It is possibl e that, in the rat beta-cell, type IV cPLA(2) couples the increase in intracellular Ca2+ brought about by insulin secretagogues, to the libe ration of AA and the subsequent release of insulin.