Arachidonic acid mobilizes Ca2+ from the endoplasmic reticulum and an acidic store in rat pancreatic ß cells.

In rat pancreatic ß cells, arachidonic acid (AA) triggered intracellular Ca(2+) release. This effect could be mimicked by eicosatetraynoic acid, indicating that AA metabolism is not required. The AA-mediated Ca(2+) signal was not affected by inhibition of ryanodine receptors or emptying of ryanodine-sensitive store but was reduced by ∼70% following the disruption of acidic stores (treatment with bafilomycin A1 or glycyl-phenylalanyl-ß-naphthylamide (GPN)). The action of AA did not involve TRPM2 channels or NAADP receptors because intracellular dialysis of adenosine diphosphoribose (ADPR; an activator of TRPM2 channels) or NAADP did not affect the AA response. In contrast, stimulation of IP(3) receptors via intracellular dialysis of adenophostin A, or exogenous application of ATP largely abolished the AA-mediated Ca(2+) signal. Intracellular dialysis of heparin abolished the ATP-mediated Ca(2+) signal but not the AA response, suggesting that the action of AA did not involve the IP(3)-binding site. Treatment with the SERCA pump inhibitor, thapsigargin, reduced the amplitude of the AA-mediated Ca(2+) signal by ∼70%. Overall, our finding suggests that AA mobilizes Ca(2+) from the endoplasmic reticulum as well as an acidic store and both stores could be depleted by IP(3) receptor agonist. The possibility of secretory granules as targets of AA is discussed.

Cholesterol elevation impairs glucose-stimulated Ca(2+) signaling in mouse pancreatic ß-cells.

Recent studies have demonstrated that cholesterol elevation in pancreatic islets is associated with a reduction in glucose-stimulated insulin secretion, but the underlying cellular mechanisms remain elusive. Here, we show that cholesterol enrichment dramatically reduced the proportion of mouse ß-cells that exhibited a Ca(2+) signal when stimulated by high glucose. When cholesterol-enriched ß-cells were challenged with tolbutamide, there was a decrease in the amplitude of the Ca(2+) signal, and it was associated with a reduction in the cell current density of voltage-gated Ca(2+) channels (VGCC). Although the cell current densities of the ATP-dependent K(+) channels and the delayed rectifier K(+) channels were also reduced in the cholesterol-enriched ß-cells, glucose evoked only a small depolarization in these cells. In cholesterol-enriched cells, the glucose-mediated increase in cellular ATP content was dramatically reduced, and this was related to a decrease in glucose uptake via glucose transporter 2 and an impairment of mitochondrial metabolism. Thus, cholesterol enrichment impaired glucose-stimulated Ca(2+) signaling in ß-cells via two mechanisms: a decrease in the current density of VGCC and a reduction in glucose-stimulated mitochondrial ATP production, which in turn led to a smaller glucose-evoked depolarization. The decrease in VGCC-mediated extracellular Ca(2+) influx in cholesterol-enriched ß-cells was associated with a reduction in the amount of exocytosis. Our findings suggest that defect in glucose-stimulated Ca(2+) signaling is an important mechanism underlying the impairment of glucose-stimulated insulin secretion in islets with elevated cholesterol level.

Arachidonic acid stimulates extracellular Ca(2+) entry in rat pancreatic beta cells via activation of the noncapacitative arachidonate-regulated Ca(2+) (ARC) channels.

Arachidonic acid (AA) is generated in the pancreatic islets during glucose stimulation. We investigated whether AA activated extracellular Ca(2+) entry in rat pancreatic beta cells via a pathway that was independent of the activation of voltage-gated Ca(2+) channels. The AA triggered [Ca(2+)](i) rise did not involve activation of GPR40 receptors or AA metabolism. When cells were voltage clamped at -70mV, the AA-mediated intracellular Ca(2+) release was accompanied by extracellular Ca(2+) entry. AA accelerated the rate of Mn(2+) quench of indo-1 fluorescence (near the Ca(2+)-independent wavelength of indo-1), reflecting the activation of a Ca(2+)-permeable pathway. The AA-mediated acceleration of Mn(2+) quench was inhibited by La(3+) but not by 2-APB (a blocker of capacitative Ca(2+) entry), suggesting the involvement of arachidonate-regulated Ca(2+) (ARC) channels. Consistent with this, intracellular application of the charged membrane-impermeant analog of AA, arachidonyl-coenzyme A (ACoA) triggered extracellular Ca(2+) entry, as well as the activation of a La(3+)-sensitive small inward current (1.7pA/pF) at -70mV. Our results indicate that the activation of ARC channels by intracellular AA triggers extracellular Ca(2+) entry. This action may contribute to the effects of AA on Ca(2+) signals and insulin secretion in rat beta cells.