Hyung Seo Park

The Type 2 Inositol (1,4,5)-Trisphosphate (InsP3) Receptor Determines the Sensitivity of InsP3-induced Ca2+ Release to ATP in Pancreatic Acinar Cells

Calcium release through inositol (1,4,5)-trisphosphate receptors (InsP3R) is the primary signal driving digestive enzyme and fluid secretion from pancreatic acinar cells. The type 2 (InsP3R2) and type 3 (InsP3R3) InsP3R are the predominant isoforms expressed in acinar cells and are required for proper exocrine gland function. Both InsP3R2 and InsP3R3 are positively regulated by cytosolic ATP, but InsP3R2 is 10-fold more sensitive than InsP3R3 to this form of modulation. In this study, we examined the role of InsP3R2 in setting the sensitivity of InsP3-induced Ca2+ release (IICR) to ATP in pancreatic acinar cells. IICR was measured in permeabilized acinar cells from wild-type (WT) and InsP3R2 knock-out (KO) mice. ATP augmented IICR from WT pancreatic cells with an EC50 of 38 μm. However, the EC50 was 10-fold higher in acinar cells isolated from InsP3R2-KO mice, indicating a role for InsP3R2 in setting the sensitivity of IICR to ATP. Consistent with this idea, heterologous expression of InsP3R2 in RinM5F cells, which natively express predominately InsP3R3, increased the sensitivity of IICR to ATP. Depletion of ATP attenuated agonist-induced Ca2+ signaling in WT pancreatic acinar cells. This effect was more profound in acinar cells prepared from InsP3R2-KO mice. These data suggest that the sensitivity of IICR to ATP depletion is regulated by the particular complement of InsP3R expressed in an individual cell. The effects of metabolic stress on intracellular Ca2+ signals can therefore be determined by the relative amount of InsP3R2 expressed in cells.

ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs

ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

Ca2+-induced Ca2+ Release from Internal Stores in INS-1 Rat Insulinoma Cells

The secretion of insulin from pancreatic β-cells is triggered by the influx of Ca(2+) through voltage-dependent Ca(2+) channels. The resulting elevation of intracellular calcium ([Ca(2+)](i)) triggers additional Ca(2+) release from internal stores. Less well understood are the mechanisms involved in Ca(2+) mobilization from internal stores after activation of Ca(2+) influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic β-cell line, INS-1 cells. To measure cytosolic and stored Ca(2+), respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. [Ca(2+)](i) was repetitively increased by caffeine stimulation in normal Ca(2+) buffer. However, peak [Ca(2+)](i) was only observed after the first caffeine stimulation in Ca(2+) free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in [Ca(2+)](i) were reduced by pretreatment with ruthenium red, as well as by depletion of internal Ca(2+) stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced Ca(2+) mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells, Ca(2+) release from internal stores was activated by caffeine, Ca(2+), or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in permeabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic β-cells.

Caffeine and 2-Aminoethoxydiphenyl Borate (2-APB) Have Different Ability to Inhibit Intracellular Calcium Mobilization in Pancreatic Acinar Cell

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) modulate Ca(2+) release from intracellular Ca(2+) store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block InsP(3)Rs, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) entry through store-operated Ca(2+) (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the Ca(2+) entry, but significantly inhibited carbamylcholine (CCh)-induced Ca(2+) release. In contrast, 2-APB did not block CCh-induced Ca(2+) release, but remarkably blocked SOC-mediated Ca(2+) entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP(3)-induced Ca(2+) release, but 2-APB at lower concentration, which effectively blocked Ca(2+) entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of InsP(3)-induced Ca(2+) release and direct stimulation of Ca(2+) release. Based on the results, we concluded that caffeine is useful as an inhibitor of InsP(3)R, and 2-APB at lower concentration is considered a blocker of Ca(2+) entry through SOC channels in the pancreatic acinar cell.

Altered Purkinje cell responses and calmodulin expression in the spontaneously ataxic mouse, Pogo

Ataxia is often associated with altered cerebellar motor control, a process in which Purkinje cells (PCs) play a principal role. Pogo mice display severe motor deficits characterized by an ataxic gait accompanying hindlimb hyperextension. Here, using whole‐cell patch‐clamp recordings, we show that parallel fiber (PF)‐excitatory post‐synaptic currents (PF‐EPSCs) are reduced, paired‐pulse facilitation (PPF) is increased and PF‐PC long‐term depression (LTD) is impaired in Pogo mice; in contrast, climbing‐fiber EPSCs are preserved. In control mice, treatment with the calmodulin antagonist calmidazolium (5 μm) impaired PPF and LTD. Notably, cerebellar calmodulin expression was significantly reduced in Pogo mice compared with control mice. Control PCs predominantly exhibited a tonic firing pattern, whereas the firing pattern in Pogo PCs was mainly a complex burst type. These results implicate alterations in PC responses and calmodulin content in the abnormal cerebellar function of Pogo mice.

Vasorelaxing Mechanism of Crude Saponin of Korea Red Ginseng in the Resistance-sized Mesenteric Artery of Rat

It has been well known that Korea red ginseng has an antihypertensive effect. The antihypertensive effect may be due to its ability to change the peripheral resistance. Change of vascular tone in the resistance-sized artery contribute to the peripheral resistance, thereby regulate the blood pressure. Therefore, we investigated to clarify the vasorelaxing mechanism induced by crude saponin of Korea red ginseng in the resistance-sized mesenteric artery of rats. The resistance-sized mesenteric artery was isolated and cut into a ring. The ring segment was immersed in HEPES-buffered solution and its isometric tension was measured using myograph force-displacement transducer. Crude saponin of ginseng relaxed the mesenmetric arterial rings precontracted with norepinephrine (3M) in dose-dependent manner (0.01 mg/㎖ -1 mg/㎖. The relaxation by crude saponin was smaller in endothelium-intact preparation than that in endothelium-denuded preparation. The contraction induced by A23187 or phorbol 12,13-dibutyrate was not affected by crude saponin of ginseng. The vasorelaxing effect of crude saponin of ginseng was significantly attenuated by the increase of the extracellular K+/ concentration. Crude saponin-induced vasorelaxation was not affected by tetraethylammonium (1 mM), glybenclamide (10M), and 4-aminopyridine (0.1 mM) in these preparations. Ba2+/(10M ∼100M) markedly reduced the crude saponin-induced vasorelakation dose-dependently. From the above results, we suggest that crude saponin of ginseng may stimulate K+/ efflux and hyperpolarize the membrane, thereby cause the vasorelaxation in the resistance-sized mesenteric artery of rats.

Propofol effects on cerebellar long-term depression

Propofol is an intravenously administered anesthetic that induces γ-aminobutyric acid-mediated inhibition in the central nervous system. It has been implicated in prolonged movement disorders. Since the cerebellum is important for motor coordination and learning, we investigated the potential effects of propofol on cerebellar circuitry. Using the whole-cell patch-clamp technique in Wister rat cerebellar slices, we demonstrated that propofol administration impaired long-term depression from the parallel fiber (PF) to Purkinje cell (PC) synapses (PF-LTD). Also, propofol reduced metabotropic glutamate receptor 1 (mGluR1)-mediated and group I mGluR agonist-induced slow currents in PCs. These results suggest that the propofol-induced PF-LTD impairment may be related to an alteration in mGluR1 signaling, which is essential to motor learning.

Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

Intracellular calcium (Ca2+) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H2O2) on intracellular Ca2+ accumulation in mouse pancreatic acinar cells. Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels and termination of oscillatory Ca2+ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca2+. Antioxidants, catalase or DTT, completely prevented H2O2-induced additional Ca2+ increase and termination of Ca2+ oscillation. In Ca2+-free medium, H2O2 still enhanced CCh-induced intracellular Ca2+ levels and thapsigargin (TG) mimicked H2O2-induced cytosolic Ca2+ increase. Furthermore, H2O2-induced elevation of intracellular Ca2+ levels was abolished under sarco/endoplasmic reticulum Ca2+ ATPase-inactivated condition by TG pretreatment with CCh. H2O2 at 300 µM failed to affect store-operated Ca2+ entry or Ca2+ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca2+ uniporter blocker, failed to attenuate H2O2-induced intracellular Ca2+ elevation. These results provide evidence that excessive generation of H2O2 in pathological conditions could accumulate intracellular Ca2+ by attenuating refilling of internal Ca2+ stores rather than by inhibiting Ca2+ extrusion to extracellular fluid or enhancing Ca2+ mobilization from extracellular medium in mouse pancreatic acinar cells.

Alteration of Ryanodine-receptors in Cultured Rat Aortic Smooth Muscle Cells

Vascular smooth muscle cells can obtain a proliferative function in environments such as atherosclerosis in vivo or primary culture in vitro. Proliferation of vascular smooth muscle cells is accompanied by changes in ryanodine receptors (RyRs). In several studies, the cytosolic Ca2+ response to caffeine is decreased during smooth muscle cell culture. Although caffeine is commonly used to investigate RyR function because it is difficult to measure Ca2+ release from the sarcoplasmic reticulum (SR) directly, caffeine has additional off-target effects, including blocking inositol trisphosphate receptors and store-operated Ca2+ entry. Using freshly dissociated rat aortic smooth muscle cells (RASMCs) and cultured RASMCs, we sought to provide direct evidence for the operation of RyRs through the Ca2+- induced Ca2+-release pathway by directly measuring Ca2+ release from SR in permeabilized cells. An additional goal was to elucidate alterations of RyRs that occurred during culture. Perfusion of permeabilized, freshly dissociated RASMCs with Ca2+ stimulated Ca2+ release from the SR. Caffeine and ryanodine also induced Ca2+ release from the SR in dissociated RASMCs. In contrast, ryanodine, caffeine and Ca2+ failed to trigger Ca2+ release in cultured RASMCs. These results are consistent with results obtained by immunocytochemistry, which showed that RyRs were expressed in dissociated RASMCs, but not in cultured RASMCs. This study is the first to demonstrate Ca2+ release from the SR by cytosolic Ca2+ elevation in vascular smooth muscle cells, and also supports previous studies on the alterations of RyRs in vascular smooth muscle cells associated with culture.

Hydrogen peroxide inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in mouse parotid acinar cells

Intracellular Ca2+ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H2O2) on cytosolic Ca2+ accumulation in mouse parotid acinar cells. Intracellular Ca2+ levels were slowly elevated when 1 mM H2O2 was perfused in the presence of normal extracellular Ca2+. In a Ca2+-free medium, 1 mM H2O2 still enhanced the intracellular Ca2+ level. Ca2+ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H2O2. On the other hand, 10 mM H2O2 induced more rapid Ca2+ accumulation and facilitated Ca2+ entry from extracellular fluid. Ca2+ refill into intracellular Ca2+ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca2+ release from Ca2+ store was not affected by 1 mM H2O2 in permeabilized cells. Ca2+ efflux through plasma membrane Ca2+-ATPase (PMCA) was markedly blocked by 1 mM H2O2 in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H2O2-induced Ca2+ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H2O2 under pathological conditions may lead to cytosolic Ca2+ accumulation and that the primary mechanism of H2O2-induced Ca2+ accumulation is likely to inhibit Ca2+ efflux through PMCA rather than mobilize Ca2+ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.