Phorbol ester impairs electrical excitation of rat pancreatic beta-cells through PKC-independent activation of K(ATP) channels

BACKGROUND: Phorbol 12-myristate 13-acetate (PMA) is often used as an activating phorbol ester of protein kinase C (PKC) to investigate the roles of the kinase in cellular functions. Accumulating lines of evidence indicate that in addition to activating PKC, PMA also produces some regulatory effects in a PKC-independent manner. In this study, we investigated the non-PKC effects of PMA on electrical excitability of rat pancreatic β-cells by using patch-clamp techniques. RESULTS: In current-clamp recording, PMA (80 nM) reversibly inhibited 15 mM glucose-induced action potential spikes superimposed on a slow membrane depolarization and this inhibition can not be prevented by pre-treatment of the cell with a specific PKC inhibitor, bisindolylmaleimide (BIM, 1 μM). In the presence of a subthreshold concentration (5.5 mM) of glucose, PMA hyperpolarized β-cells in a concentration-dependent manner (0.8–240 nM), even in the presence of BIM. Based on cell-attached single channel recordings, PMA increased ATP-sensitive K(+) channel (K(ATP)) activity. Based on inside-out patch-clamp recordings, PMA had little effect on K(ATP) activity if no ATP was in the bath, while PMA restored K(ATP) activity that was suppressed by 10 μM ATP in the bath. In voltage-clamp recording, PMA enhanced tolbutamide-sensitive membrane currents elicited by repetitive ramp pulses from -90 to -50 mV in a concentration-dependent manner, and this potentiation could not be prevented by pre-treatment of cell with BIM. 4α-phorbol 12,13-didecanoate (4α-PDD), a non-PKC-activating phorbol ester, mimicked the effect of PMA on both current-clamp and voltage-clamp recording configurations. With either 5.5 or 16.6 mM glucose in the extracellular solution, PMA (80 nM) increased insulin secretion from rat islets. However, in islets pretreated with BIM (1 μM), PMA did not increase, but rather reduced insulin secretion. CONCLUSION: In rat pancreatic β-cells, PMA modulates insulin secretion through a mixed mechanism: increases insulin secretion by activation of PKC, and meanwhile decrease insulin secretion by impairing β-cell excitability in a PKC-independent manner. The enhancement of K(ATP) activity by reducing sensitivity of K(ATP) to ATP seems to underlie the PMA-induced impairment of β-cells electrical excitation in response to glucose stimulation

2-Aminoethoxydiphenyl Borate Inhibits Agonist-Induced Ca2+ Signals By Blocking Inositol Trisphosphate Formation In Acutely Dissociated Mouse Pancreatic Acinar Cells

Evidence suggests that 2-aminoethoxydiphenyl borate (2-APB) modulates intracellular Ca2+ signals in a complex manner. 2-APB inhibits or potentiates intracellular Ca2+ signals in different cell types, perhaps through different mechanisms. Here, we report a novel mechanism underlying 2-APB-induced inhibition of agonist-activated Ca2+ oscillations in mouse pancreatic acinar cells, using patch-clamp and biochemical techniques. Pre-treatment of the cells with 100 μM 2-APB completely abolished ACh-but not inositol trisphosphate (InsP3)-induced Ca 2+ oscillations, suggesting that the mechanism of inhibition occurs between cytoplasmic receptors and InsP3 receptor activation. In addition, 100 μM 2-APB significantly inhibited ACh-induced phospholipase C (PLC) activation. These findings indicate that, in mouse pancreatic acinar cells, in addition to modulating InsP3 receptors and blocking the store-operated Ca2+ pathway, high concentrations of 2-APB also inhibit agonist-induced Ca2+ signals by reducing InsP3 formation

2-Aminoethoxydiphenyl Borate Modulates Kinetics Of Intracellular Ca 2+ Signals Mediated By Inositol 145-Trisphosphate-Sensitive Ca 2+ Stores In Single Pancreatic Acinar Cells Of Mouse

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 patchclamp, 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

Closure by iptakalim, a cardiovascular KATP channel opener, of rat pancreatic β-cell KATP channels and its molecular basis

Diabetes mellitus is a group of diseases characterized by high levels of blood glucose resulting from defects in insulin production, insulin action, or both. Diabetes patients usually have accompanying cardiovascular disorders. Sulfonylureas have been the leading oral antihyperglycemic agents for type 2 diabetes treatment, which currently still constitute the most popular anti-diabetic drugs. Nevertheless, concern has arisen over the side eff ects of sulfonylureas on the cardiovascular system. Here we report that iptakalim, a novel cardiovascular ATP-sensitive potassium（ KATP） channel opener, closed rat pancreatic β-cell KATP channels and increased insulin release. Using whole-cell patch-clamp recordings, iptakalim depolarized β-cells, induced action potential fi ring and reduced pancreatic KATP channel currents. Using single-channel recordings, iptakalim reduced KATP channel open-probability independently of intracellular ATP concentrations. We demonstrated that iptakalim elevated intracellular Ca2+ concentrations and increased insulin release as revealed by fl uorescence imaging（ fura-2） and biochemical measurements, respectively. In addition, iptakalim signifi cantly inhibited the open-probability of recombinant Kir6.2/SUR1 and Kir6.2FL4A（ a traffi cking mutant of the Kir6.2） channels expressed in transfected human embryonic kidney （HEK） 293 cells. Collectively, iptakalim, a cardiovascular KATP channel opener, closes rat pancreatic β-cell KATP channels, which may result from direct inhibition of the Kir6.2 subunit. Therefore, iptakalim bi-directionally regulates KATP channels in cardiovascular and pancreatic tissues, and this unique pharmacological property suggests iptakalim could be used as a new therapeutic strategy for the treatment of type 2 diabetes with the potential benefi t in alleviating cardiac and/or vascular disorders frequently associated with diabetes

Hydrogen Peroxide Mobilizes Ca2+ Through Two Distinct Mechanisms In Rat Hepatocytes

Aim:Hydrogen peroxide (H\ 2O\ 2) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca2+ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.Methods:Hepatocytes were extracted from rats. Intracellular Ca2+ concentrations ([Ca2+](i)), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP\ 2. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.Results:H\ 2O\ 2 increased intracellular Ca 2+ concentrations ([Ca2+](i)) across two kinetic phases. A low concentration (400 μmol/L) of H\ 2O\ 2 induced a sustained elevation of [Ca2+](i) that was reversed by removing extracellular Ca2+. H\ 2O\ 2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H\ 2O\ 2-induced membrane current increases and [Ca2+](i) elevation. A high concentration (1 mmol/L) of H\ 2O\ 2 induced an additional transient elevation of [Ca 2+](i), which was abolished by the specific PLC blocker U73122 but was not eliminated by removal of extracellular Ca2+. PLC activity was increased by 1 mmol/L H\ 2O\ 2 but not by 400 μmol/L H\ 2O\ 2.Conclusion:H\ 2O\ 2 mobilizes Ca2+ through two distinct mechanisms. In one, 400 μmol/L H\ 2O\ 2-induced sustained [Ca2+](i) elevation is mediated via a Ca2+ influx mechanism, under which H\ 2O\ 2 impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca2+ influx. In contrast, 1 mmol/L H\ 2O\ 2-induced transient elevation of [Ca 2+](i) is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca2+ from intracellular Ca 2+ stores. © 2009 CPS and SIMM