Lipoxygenase inhibitors reduce insulin secretion without impairing calcium mobilization

Inhibitors of pancreatic islet lipoxygenase (LPX) impair nutrient-induced insulin (I) release. To define the mechanism of action of these inhibitors, studies were carried out at subthreshold glucose concentrations (0-1.7 mM) in order to minimize any effects of LPX blockade on the potentiating effect of extracellular fuels. Barium chloride (Ba2+; 2 mM) increased 45Ca2+ release from prelabeled islets in Ca2+-free medium and, thus, is a model for the mobilization of intracellular Ca2+ stores. Inhibition of LPX (using nordihydroguaiaretic acid, BW755c [3-amino-1-(trifluomethyl-phenyl)2-pyrazoline] or butylated hydroxytoluene) did not have any consistent effect on the influx of Ba2+ (as assessed by 133Ba uptake) or on the consequent release of cellular Ca2+ stores; however, each LPX inhibitor vitiated Ba2+-induced I release. The LPX inhibitors were not merely acting as nonspecific antioxidants, since two inhibitors which do not act by scavenging hydroperoxides (5,8,11,14-eicosatetraynoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid) also impeded the effect of Ba2+ on I secretion; furthermore, a series of hydroxyl radical scavengers, reducing agents, or agents that block nonenzymatic and/or NADPH-activated lipid peroxidation did not inhibit I secretion. LPX inhibitors also blocked the residual I response to 16.7 mM glucose in Ca2+-free medium. Additionally, they reduced secretion induced by 46 mM K+ or 1 mM isobutylmethylxanthine (provided in the presence of extracellular Ca2+), without inhibiting K+- or isobutylmethylxanthine-induced Ca2+ fluxes. Stimuli sensitive to LPX blockade were also antagonized by antimycin A (an inhibitor of energy flux) or TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride; which appeared to deplete critical intracellular Ca2+ stores]. In contrast, the effects of exogenous phospholipase C (and several other Ca2+-dependent membrane-active agonists) were resistant to the LPX inhibitors, TMB-8, and antimycin A; thus, LPX inhibitors are not nonspecific global poisons of all Ca2+-dependent exocytotic hormone release. We conclude that LPX (or a very similar enzyme) may modulate the effects (or redistribution) of an ATP-dependent trigger pool of Ca2+ at a site distal to and independent of its mobilization by primary islet agonists. LPX inhibitors also blocked secretion induced by 12-O-tetradecanoyl-phorbol-13-acetate; this effect may reflect an effect of LPX on the activation of protein kinase C or a modulation of its synergism with the same trigger Ca2+ pool(s).