Regulation of the kinetics of intracellular Ca(2+) signals with a novel, membrane-penetrable, inositol 1,4,5-trisphosphate (InsP(3)) receptor/Ca(2+) channel modulator, 2-amino-ethoxydiphenyl borate (2APB), has been investigated using patch-clamp, whole-cell recording to monitor Ca(2+)-activated Cl(-) currents in single isolated pancreatic acinar cells. 2APB itself fails to evoke a detectable current response but it dramatically changes the kinetics of agonist-induced Ca(2+) release from pulsatile spikes to long-lasting, huge Ca(2+) waves, suggesting that 2APB coordinates local Ca(2+) release to generate global Ca(2+) signals. The regulation by 2APB can be elicited by internal perfusion of InsP(3) in a concentration-dependent manner, indicating that this regulation is not mediated through membrane receptors or G protein signal transduction. The InsP(3) receptor blocker heparin, but not the ryanodine-sensitive receptor blockers ruthenium red or ryanodine, abolishes 2APB-mediated regulation of Ca(2+) release. This results also suggest that 2APB effects are mediated through InsP(3) receptors. 2APB substantially modifies single inward Cl(-) current pulse evoked by the photolytic release of caged InsP(3) but not by caged Ca(2+). These data indicate that 2APB-induced regulation is mediated neither by Ca(2+)-induced Ca(2+) release nor by affecting Cl(-) channel activity directly. We conclude that 2APB regulates the kinetics of intracellular Ca(2+) signals, represented as the change in the Ca(2+) oscillation patterns from brief pulsatile spikes to huge, long-lasting Ca(2+) waves. Moreover, this regulation seems to be mediated through InsP(3)-sensitive Ca(2+) pools. 2APB may act as a novel, useful pharmacological tool to study the genesis of intracellular Ca(2+) signals.

The NADH shuttle system is composed of the glycerol phosphate and malate-aspartate shuttles. We generated mice that lack mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), a rate-limiting enzyme of the glycerol phosphate shuttle. Application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle, to mGPDH-deficient islets demonstrated that the NADH shuttle system was essential for coupling glycolysis with activation of mitochondrial ATP generation to trigger glucose-induced insulin secretion.The present study revealed that blocking the NADH shuttle system severely suppressed closure of the ATPsensitive potassium (K ATP ) channel and depolarization of the plasma membrane in response to glucose in ␤ cells, although properties of the K ATP channel on the excised ␤ cell membrane were unaffected. In mGPDHdeficient islets treated with aminooxyacetate, Ca 2؉ influx through the plasma membrane induced by a depolarizing concentration of KCl in the presence of the K ATP channel opener diazoxide restored insulin secretion. However, the level of the secretion was only ϳ40% of wild-type controls. Thus, glucose metabolism through the NADH shuttle system leading to efficient ATP generation is pivotal to activation of both the K ATP channeldependent pathway and steps distal to an elevation of cytosolic Ca 2؉ concentration in glucose-induced insulin secretion.In pancreatic ␤ cells, glucose metabolism in glycolysis and in mitochondria is pivotal to glucose-induced insulin secretion. Thus, mutations in the glucokinase gene (1, 2) and mitochondrial DNA (3) can cause type 2 diabetes mellitus, which is characterized by insufficient insulin secretion in response to glucose. Moreover, abrogation of ␤ cell-specific glucokinase in mice (4, 5) and mitochondrial DNA in insulinoma cell lines (6 -9) was associated with defective insulin secretion in response to glucose. The following cascade has been generally accepted as the glucose-induced insulin secretory pathway (10 -13). Glucose-stimulated increase in cytosolic ATP or in the ratio of ATP to ADP closes ATP-sensitive potassium (K ATP ) 1 channels, which depolarizes the plasma membrane potential above a threshold, leading to Ca 2ϩ entry into the cytosol through activation of voltage-dependent Ca 2ϩ channels (VDCCs). The rise in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] c ) is thought to finally trigger exocytosis of insulin from secretory vesicles.It has been known that pyruvate, an end product of aerobic glycolysis, cannot stimulate insulin secretion although it is oxidized as efficiently as glucose in ␤ cells (14,15). Studies with an inhibitor of pyruvate transport into mitochondria or an inhibitor of the tricarboxylic acid cycle suggested that metabolism of glucose-derived pyruvate in mitochondria or in the tricarboxylic acid cycle was not well correlated with glucoseinduced insulin secretion (16,17). These results suggested that glycolysis-derived factor(s) other than pyruvate are required for activation of mitochondrial metabolism and for generation of the metabolic ...

Intracellular cAMP-dependent modulation of L-type Ca2+ channel activation in cultured rat islet beta-cells has been investigated using the patch-clamp whole-cell current recording mode. The L-type voltage-dependent Ca2+ current (ICa) showed a fast activation followed by a slow inactivation, and was sensitive to Ca2+ channel blockers, for example nifedipine. Application of a cAMP analogue, dibutyryl cyclic AMP (db-cAMP), increased the magnitude of the peak ICa in a concentration-dependent manner. Values of the half-activation potentials (V1/2), taken from activation curves for ICa, were -16.7 +/- 1.8 and -21.9 +/- 3.4 mV (P < 0.05) before and after application of db-cAMP, respectively, with no change of the slope factor (k) or the reversal potential. Pretreatment with a specific protein kinase A antagonist, Rp-cAMP, prevented the potentiating effect of db-cAMP. These results indicate that in rat islet beta-cells, phosphorylation of cAMP-dependent kinase potentiates the voltage-dependent activation of L-type Ca2+ channels.