A cytosolic 84-kDa group VIA
phospholipase A(2) (
iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential
iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating
lysophosphatidylcholine acceptors for
arachidonic acid incorporation into P388D1 cell
phosphatidylcholine (PC). Proposals for
iPLA(2)beta function rest in part on effects of inhibiting
iPLA(2)beta activity with a
bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB
phospholipase A(2). Manipulation of
iPLA(2)beta expression by molecular
biologic means is an alternative approach to study
iPLA(2)beta functions, and we have used a retroviral construct containing
iPLA(2)beta
cDNA to prepare two INS-1
insulinoma cell clonal lines that stably overexpress
iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified
insulin secretory responses to
glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of
arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of
iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with
antibodies directed against
iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that
iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for
iPLA(2)beta in
insulin-secreting beta-cells.