Blockade by lipoxygenase inhibitors of Ca 2+-dependent insulin secretion from permeabilized rat islets : A molecular mechanism distinct from that of α2-adrenergic agonists

Abstract

To evaluate the regulation and effects of pancreatic islet lipoxygenase, adult rat islets were permeabilized, using digitonin or staphylococcal α-toxin, and then were studied in a medium simulating an intracellular milieu at fixed ambient concentrations of Ca 2+. Permeabilized islets retained 12-lipoxygenase activity, as indicated by conversion of tritiated arachidonic acid to a predominant peak of [ 3H]12-hydroxy-eicosatetraenoic acid (12-HETE); this activity was inhibited (89–98%) by the lipoxygenase blockers nor-dihydroguaiaretic acid (35 μM), BW755c (250 μM) or ETYA (35 μM). Lesser amounts of compounds coeluting with 15- and 11-HETE (but little or no 5-HETE) were formed; however, 11-HETE (and possibly some 15-HETE) was probably synthesized (at least in part) via cyclooxygenase, as suggested by the partial synthesis blockade induced by 50 μM ibuprofen. The production of 12-HETE did not require the presence of Ca 2+, Mg 2+ or ATP; it also was not stimulated by addition of cyclic AMP, a phorbol ester, or calmodulin. However, it was augmented modestly by provision of a basal cytosolic free Ca 2+ concentration of 60–80 nM, with no further increase at physiologically elevated levels of 260–530 nM. Elevations in cytosolic free Ca 2+ concentrations induced insulin release which was inhibited by cooling, epinephrine or protein kinase inhibitors and, therefore, was exocytotic in nature. Lipoxygenase inhibitors blocked this insulinotropic effect of calcium at submaximal or saturating Ca 2+ concentrations (with or without its potentiation by 12- O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C) by 53–82%. However, they did not reduce the Ca 2+-independent secretory effects (at subnanomolar Ca 2+ concentrations) of the phorbol ester alone. Similar results were seen using dibutyryl cyclic AMP to activate protein kinase A. The α 2-adrenergic agonists epinephrine or clonidine inhibited Ca 2+-, TPA- or cyclic AMP-induced insulin release without reducing HETE formation. We conclude that (1) islet lipoxygenase is constitutively expressed and is not physiologically regulated by α 2-adrenergic agonism, Ca 2+ or protein kinases; (2) lipoxygenase modulates insulin release; HETE production is not merely an epiphenomenon reflecting the activation (or inhibition) of exocytotic secretion; (3) islet lipoxygenase inhibitors reduce insulin secretion, at least in part, by blocking the direct effects of Ca 2+ on exocytosis and/or its synergism with Ca 2+-binding proteins such as protein kinase C; and (4) these same inhibitors do not directly poison protein kinase C or A, or the exocytotic apparatus. Furthermore, the retention of significant activity and biological effects of lipoxygenase in leaky islets suggests that this enzyme may be associated with, and act upon, cell membranes in islets.