Ca2+ entry through reverse Na+/Ca2+ exchanger in NCI-H716, glucagon-like peptide-1 secreting cells.

Glucagon like peptide-1 (GLP-1) released from enteroendocine L-cells in the intestine has incretin effects due to its ability to amplify glucose-dependent insulin secretion. Promotion of an endogenous release of GLP-1 is one of therapeutic targets for type 2 diabetes mellitus. Although the secretion of GLP-1 in response to nutrient or neural stimuli can be triggered by cytosolic Ca2+ elevation, the stimulus-secretion pathway is not completely understood yet. Therefore, the aim of this study was to investigate the role of reverse Na+/Ca2+ exchanger (rNCX) in Ca2+ entry induced by muscarinic stimulation in NCI-H716 cells, a human enteroendocrine GLP-1 secreting cell line. Intracellular Ca2+ was repetitively oscillated by the perfusion of carbamylcholine (CCh), a muscarinic agonist. The oscillation of cytosolic Ca2+ was ceased by substituting extracellular Na+ with Li+ or NMG+. KB-R7943, a specific rNCX blocker, completely diminished CCh-induced cytosolic Ca2+ oscillation. Type 1 Na+/Ca2+ exchanger (NCX1) proteins were expressed in NCI-H716 cells. These results suggest that rNCX might play a crucial role in Ca2+ entry induced by cholinergic stimulation in NCI-H716 cells, a GLP-1 secreting cell line.

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.

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.

Monoisotopic mass determination algorithm for selenocysteine-containing polypeptides from mass spectrometric data based on theoretical modeling of isotopic peak intensity ratios.

Selenoproteins, containing selenocysteine (Sec, U) as the 21st amino acid in the genetic code, are well conserved from bacteria to human, except yeast and higher plants that miss the Sec insertion machinery. Determination of Sec association is important to find substrates and to understand redox action of selenoproteins. While mass spectrometry (MS) has become a common and powerful tool to determine an amino acid sequence of a protein, identification of a protein sequence containing Sec was not easy using MS because of the limited stability of Sec in selenoproteins. Se has six naturally occurring isotopes, 74Se, 76Se, 77Se, 78Se, 8°Se, and 8²Se, and 8°Se is the most abundant isotope. These characteristics provide a good indicator for selenopeptides but make it difficult to detect selenopeptides using software analysis tools developed for common peptides. Thus, previous reports verified MS scans of selenopeptides by manual inspection. None of the fully automated algorithms have taken into account the isotopes of Se, leading to the wrong interpretation for selenopeptides. In this paper, we present an algorithm to determine monoisotopic masses of selenocysteine-containing polypeptides. Our algorithm is based on a theoretical model for an isotopic distribution of a selenopeptide, which regards peak intensities in an isotopic distribution as the natural abundances of C, H, N, O, S, and Se. Our algorithm uses two kinds of isotopic peak intensity ratios: one for two adjacent peaks and another for two distant peaks. It is shown that our algorithm for selenopeptides performs accurately, which was demonstrated with two LC-MS/MS data sets. Using this algorithm, we have successfully identified the Sec-Cys and Sec-Sec cross-linking of glutaredoxin 1 (GRX1) from mass spectra obtained by UPLC-ESI-q-TOF instrument.

Regulation of Ca²âº release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells.

Secretagogue-stimulated intracellular Ca(2+) signals are fundamentally important for initiating the secretion of the fluid and ion component of saliva from parotid acinar cells. The Ca(2+) signals have characteristic spatial and temporal characteristics, which are defined by the specific properties of Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptors (InsP(3)R). In this study we have investigated the role of adenine nucleotides in modulating Ca(2+) release in mouse parotid acinar cells. In permeabilized cells, the Ca(2+) release rate induced by submaximal [InsP(3)] was increased by 5 mM ATP. Enhanced Ca(2+) release was not observed at saturating [InsP(3)]. The EC(50) for the augmented Ca(2+) release was ∼8 µM ATP. The effect was mimicked by nonhydrolysable ATP analogs. ADP and AMP also potentiated Ca(2+) release but were less potent than ATP. In acini isolated from InsP(3)R-2-null transgenic animals, the rate of Ca(2+) release was decreased under all conditions but now enhanced by ATP at all [InsP(3)]. In addition the EC(50) for ATP potentiation increased to ∼500 µM. These characteristics are consistent with the properties of the InsP(3)R-2 dominating the overall features of InsP(3)R-induced Ca(2+) release despite the expression of all isoforms. Finally, Ca(2+) signals were measured in intact parotid lobules by multiphoton microscopy. Consistent with the release data, carbachol-stimulated Ca(2+) signals were reduced in lobules exposed to experimental hypoxia compared with control lobules only at submaximal concentrations. Adenine nucleotide modulation of InsP(3)R in parotid acinar cells likely contributes to the properties of Ca(2+) signals in physiological and pathological conditions.

Exendin-4 Protects Against Sulfonylurea-Induced ß-Cell Apoptosis.

Sulfonylurea is one of the commonly used anti-diabetic drugs that stimulate insulin secretion from ß-cells. Despite their glucose lowering effects in type 2 diabetes mellitus, long-term treatment brought on secondary failure characterized by ß-cell exhaustion and apoptosis. ER stress induced by Ca2+ depletion in endoplasmic reticulum (ER) is speculated be one of the causes of secondary failure, but it remains unclear. Glucagon like peptide-1 (GLP-1) has anti-apoptotic effects in ß-cells after the induction of oxidative and ER stress. In this study, we examined the antiapoptotic action of a GLP-1 analogue in ß-cell lines and islets against ER stress induced by chronic treatment of sulfonylurea. HIT-T15 and dispersed islet cells were exposed to glibenclamide for 48 h, and apoptosis was evaluated using Annexin/PI flow cytometry. Expression of the ER stress-related molecules and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2/3 was determined by real-time PCR and western blot analysis. Chronic exposure to glibenclamide increased apoptosis by depletion of ER Ca2+ concentration through reduced expression of SERCA 2/3. Pretreatment with Exendin-4 had an anti-apoptotic role through ER stress modulation and ER Ca2+ replenishing by SERCA restoration. These findings will further the understanding of one cause of glibenclamide-induced ß-cell loss and therapeutic availability of GLP-1-based drugs in secondary failure by sulfonylurea during treatment of diabetes.

Exendin-4 protects against sulfonylurea-induced ß-cell apoptosis.

Sulfonylurea is one of the commonly used anti-diabetic drugs that stimulate insulin secretion from ß-cells. Despite their glucose lowering effects in type 2 diabetes mellitus, long-term treatment brought on secondary failure characterized by ß-cell exhaustion and apoptosis. ER stress induced by Ca(2+) depletion in endoplasmic reticulum (ER) is speculated be one of the causes of secondary failure, but it remains unclear. Glucagon like peptide-1 (GLP-1) has anti-apoptotic effects in ß-cells after the induction of oxidative and ER stress. In this study, we examined the anti-apoptotic action of a GLP-1 analogue in ß-cell lines and islets against ER stress induced by chronic treatment of sulfonylurea. HIT-T15 and dispersed islet cells were exposed to glibenclamide for 48 h, and apoptosis was evaluated using Annexin/PI flow cytometry. Expression of the ER stress-related molecules and sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2/3 was determined by real-time PCR and western blot analysis. Chronic exposure to glibenclamide increased apoptosis by depletion of ER Ca(2+) concentration through reduced expression of SERCA 2/3. Pretreatment with Exendin-4 had an anti-apoptotic role through ER stress modulation and ER Ca(2+) replenishing by SERCA restoration. These findings will further the understanding of one cause of glibenclamide-induced ß-cell loss and therapeutic availability of GLP-1-based drugs in secondary failure by sulfonylurea during treatment of diabetes.

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.

Comparison of the Autism Diagnostic Observation Schedule and Childhood Autism Rating Scale in the Diagnosis of Autism Spectrum Disorder: A Preliminary Study.

OBJECTIVES: We examined the agreement between the Autism Diagnostic Observation Schedule (ADOS) and the Childhood Autism Rating Scale (CARS) in the diagnosis of autism spectrum disorder. METHODS: The ADOS and CARS scores of 78 children were retrospectively collected from a chart review. A correlation analysis was performed to examine the concurrent validity between the two measures. Using the receiver operating characteristic (ROC) curve, we determined the optimal cut-off score of the CARS for identifying autism spectrum disorder. RESULTS: The CARS score was significantly correlated with the ADOS score (r=0.808, p<0.001). Taking ADOS as the ideal standard, the optimal cut-off scores of CARS for identifying autism and autism spectrum were 30 and 24.5, respectively. CONCLUSION: We determined the optimal cut-off scores of CARS for screening and diagnosing autism spectrum disorder.

Intravenous Anesthetic, Propofol Affects Synaptic Responses in Cerebellar Purkinje Cells.

OBJECTIVE: Propofol is an intravenously administered anesthetic that enhances γ-aminobutyric acid-mediated inhibition in the central nerve system. Other mechanisms may also be involved in general anesthesia. Propofol has been implicated in movement disorders. The cerebellum is important for motor coordination and motor learning. The aim of the present study was to investigate the propofol effect on excitatory synaptic transmissions in cerebellar cortex. METHODS: Excitatory postsynaptic currents by parallel fiber stimulation and complex spikes by climbing fiber stimulation were monitored in Purkinje cells of Wister rat cerebellar slice using whole-cell patch-clamp techniques. RESULTS: Decay time, rise time and amplitude of excitatory postsynaptic currents at parallel fiber Purkinje cell synapses and area of complex spikes at climbing fiber Purkinje cell synapses were significantly increased by propofol administration. CONCLUSION: The detected changes of glutamatergic synaptic transmission in cerebellar Purkinje cell, which determine cerebellar motor output, could explain cerebellar mechanism of motor deficits induced by propofol.

Ethanol suppresses carbamylcholine-induced intracellular calcium oscillation in mouse pancreatic acinar cells.

Oscillation of intracellular calcium levels is closely linked to initiating secretion of digestive enzymes from pancreatic acinar cells. Excessive alcohol consumption is known to relate to a variety of disorders in the digestive system, including the exocrine pancreas. In this study, we have investigated the role and mechanism of ethanol on carbamylcholine (CCh)-induced intracellular calcium oscillation in murine pancreatic acinar cells. Ethanol at concentrations of 30 and 100 mM reversibly suppressed CCh-induced Ca2+ oscillation in a dose-dependent manner. Pretreatment of ethanol has no effect on the store-operated calcium entry induced by 10 µM of CCh. Ethanol significantly reduced the initial calcium peak induced by low concentrations of CCh and therefore, the CCh-induced dose-response curve of the initial calcium peak was shifted to the right by ethanol pretreatment. Furthermore, ethanol significantly dose-dependently reduced inositol 1,4,5-trisphosphate-induced calcium release from the internal stores in permeabilized acinar cells. These results provide evidence that excessive alcohol intake could impair cytosolic calcium oscillation through inhibiting calcium release from intracellular stores in mouse pancreatic acinar cells.

Gamma-aminobutyric acid secreted from islet beta-cells modulates exocrine secretion in rat pancreas.

AIM: To investigate the role of endogenous gamma-amino-butyric acid (GABA) in pancreatic exocrine secretion. METHODS: The isolated, vascularly perfused rat pancreas was employed in this study to eliminate the possible influences of extrinsic nerves and hormones. Cholecystokinin (CCK; 10 pmol/L) was intra-arterially given to stimulate exocrine secretion of the pancreas. RESULTS: Glutamine, a major precursor of GABA, which was given intra-arterially at concentrations of 1, 4 and 10 mmol/L, dose-dependently elevated the CCK-stimulated secretions of fluid and amylase in the normal pancreas. Bicuculline (10 micromol/L), a GABA(A) receptor antagonist, blocked the enhancing effect of glutamine (4 mmol/L) on the CCK-stimulated exocrine secretions. Glutamine, at concentrations of 1, 4 and 10 mmol/L, dose-dependently increased the GABA concentration in portal effluent of the normal pancreas. The effects of glutamine on the CCK-stimulated exocrine secretion as well as the GABA secretion were markedly reduced in the streptozotocin-treated pancreas. CONCLUSION: GABA could be secreted from beta-cells into the islet-acinar portal system after administration of glutainine, and could enhance the CCK-stimulated exocrine secretion through GABA(A) receptors. Thus, GABA in islet beta-cells is a hormone modulating pancreatic exocrine secretion.

Pancreatic exocrine response to long-term high-fat diets in rats.

CONTEXT: Although the synthesis and secretion of pancreatic enzymes are dependent on the composition of diet, little is known about the long-term adaptation of the exocrine pancreas to the chronic intake of high-fat, low-carbohydrate diets. OBJECTIVE: The effects of long-term (48-weeks) high-fat, low-carbohydrate feeding on the intracellular activity as well as on the secretory activities of the pancreas regarding the secretion of amylase and lipase in isolated pancreatic lobules of rats were investigated. ANIMALS: Twenty-six male Sprague-Dawley rats were randomly divided into two groups. DESIGN: The high-fat diet group was fed a diet containing 12.3% Kcal of carbohydrates, 76.0% Kcal of fats, and 11.7% Kcal of proteins for 48 weeks. In the control group, the rats were fed a standard diet (50.3% Kcal of carbohydrates, 33.2% Kcal of fats, and 16.5% Kcal of proteins) for the same period of time. MAIN OUTCOME MEASURES: The intracellular activity of alpha-amylase and lipase were defined as enzyme activity per 1 mg protein in the pancreatic homogenate. The secretory activity of the pancreatic enzymes was defined as enzyme activity per 1 mg protein or as the mean percentage of released enzyme activity in the medium to that initially retained in the lobules over the entire 90 minute incubation period. RESULTS: The intracellular activity as well as the secretory activities of pancreatic amylase in the basal and in the cholecystokinin (10 pmol/L)-stimulated states of high-fat, low-carbohydrate diet fed rats were significantly lower than that in the control rats. Nevertheless, those of lipase were not significantly different between the two groups. However, when the secretory enzyme activity was illustrated as the percentage released, basal as well as CCK-stimulated amylase and lipase activity were not significantly different between the two groups. CONCLUSIONS: The reduction of the intracellular amylase activity and the secretory amylase activity may be a long-term adaptive response of the pancreas to the reduced carbohydrate composition of the diets. Moreover, the reduction of the secretory amylase activity may be due to reduced intracellular amylase activity rather than to the altered sensitivity of the acinar cells to cholecystokinin. However, we failed to observe a long-term adaptive response of pancreatic lipase to increased fat composition of diets.

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.

Endogenous somatostatin inhibits interaction of insulin and cholecystokinin on exocrine secretion of isolated, perfused rat pancreas.

INTRODUCTION: Although somatostatin inhibits pancreatic exocrine secretion, the inhibitory mechanism of endogenous somatostatin is not clearly understood. AIM: To investigate the effect of endogenous somatostatin on the interaction between endogenous insulin and exogenous cholecystokinin (CCK) in exocrine secretion of the totally isolated, perfused rat pancreas. METHODOLOGY: Endogenous releases of somatostatin and insulin were induced by 18 mM glucose. Streptozotocin (75 mg/kg) or cysteamine (300 mg/kg) was injected into rats 24 hours before the experiment to deplete insulin or somatostatin in the pancreas. RESULTS: Glucose (18 mM) enhanced CCK (10 pM)-stimulated secretions of fluid and amylase in the normal pancreas, which was further elevated by a somatostatin antagonist. Exogenous insulin (100 nM) also enhanced CCK-stimulated secretions in the streptozotocin-treated pancreas, which was also markedly increased by the somatostatin antagonist. The glucose (18 mM)-enhanced CCK-stimulated secretions were much higher in the cysteamine-treated pancreas than in the normal pancreas, which was dose-dependently reduced by exogenous somatostatin (30, 100 pM). However, endogenous or exogenous somatostatin did not modify the pancreatic responses to CCK alone. CONCLUSION: Endogenous somatostatin inhibits the interaction of endogenous insulin and CCK on pancreatic exocrine secretion in the rat rather than reducing the action of CCK alone or endogenous release of insulin.

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 Ca(2+) 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 Ca(2+) release from the sarcoplasmic reticulum (SR) directly, caffeine has additional off-target effects, including blocking inositol trisphosphate receptors and store-operated Ca(2+) 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 Ca(2+)- induced Ca(2+)-release pathway by directly measuring Ca(2+) 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 Ca(2+) stimulated Ca(2+) release from the SR. Caffeine and ryanodine also induced Ca(2+) release from the SR in dissociated RASMCs. In contrast, ryanodine, caffeine and Ca(2+) failed to trigger Ca(2+) 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 Ca(2+) release from the SR by cytosolic Ca(2+) elevation in vascular smooth muscle cells, and also supports previous studies on the alterations of RyRs in vascular smooth muscle cells associated with culture.

Ca-induced Ca Release from Internal Stores in INS-1 Rat Insulinoma Cells.

The secretion of insulin from pancreatic ß-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 ß-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 ß-cells.

Endothelial dysfunction in the smokers can be improved with oral cilostazol treatment.

BACKGROUND: Smoking is one of well known environmental factors causing endothelial dysfunction and plays important role in the atherosclerosis. We investigated the effect of cilostazol could improve the endothelial dysfunction in smokers with the measurement of flow-mediated dilatation (FMD). METHODS: We enrolled 10 normal healthy male persons and 20 male smokers without any known cardiovascular diseases. After measurement of baseline FMD, the participants were medicated with oral cilostazol 100 mg bid for two weeks. We checked the follow up FMD after two weeks and compared these values between two groups. RESULTS: There was no statistical difference of baseline characteristics including age, body mass index, serum cholesterol profiles, serum glucose and high sensitive C-reactive protein between two groups. However, the control group showed significantly higher baseline endothelium-dependent dilatation (EDD) after reactive hyperemia (12.0 ± 4.5% in the control group vs. 8.0 ± 2.1% in the smoker group, p = 0.001). However, endothelium-independent dilatation (EID) after sublingual administration of nitroglycerin was similar between the two groups (13.6 ± 4.5% in the control group vs. 11.9 ± 4.9% in the smoker group, p = 0.681). Two of the smoker group were dropped out due to severe headache. After two weeks of cilostazol therapy, follow-up EDD were significantly increased in two groups (12.0 ± 4.5% to 16.1 ± 3.7%, p = 0.034 in the control group and 8.0 ± 2.1% to 12.2 ± 5.1%, p = 0.003 in the smoker group, respectively). However, follow up EID value was not significantly increased compared with baseline value in both groups (13.6 ± 4.5% to 16.1 ± 3.7%, p = 0.182 in the control group and 11.9 ± 4.9% to 13.7 ± 4.3%, p = 0.430 in the smoker group, respectively). CONCLUSION: Oral cilostazol treatment significantly increased the vasodilatory response to reactive hyperemia in two groups. It can be used to improve endothelial function in the patients with endothelial dysfunction caused by cigarette smoking.

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.

Tumor Suppressor Serine/Threonine Kinase LKB1 Expression, Not Kinase Activity, Increased in the Vascular Smooth Muscle Cells and Neointima in the Rat Carotid Artery Injury Model.

BACKGROUND AND OBJECTIVES: Vascular smooth muscle cell (VSMC) proliferation is responsible for the restenosis of previously inserted coronary stents. Angiotensin II (Ang II) is known to regulate VSMC proliferation. LKB1, a serine/threonine kinase, interacts with the p53 pathway and acts as a tumor suppressor. MATERIALS AND METHODS: We assessed the association of Ang II and the expression of LKB1 in primary cultured murine VSMCs and neointima of the Sprague Dawley rat carotid artery injury model. We created carotid balloon injuries and harvested the injured carotid arteries 14 days after the procedure. RESULTS: Ang II increased LKB1 expression in a time-dependent manner and peaked at an Ang II concentration of 10(-7) mole/L in VSMCs. In the animal experiment, neointima was markedly increased after balloon injury compared to the control group. Immunohistochemical studies showed that LKB1 expression increased according to neointima thickness. Ang II augmented LKB1 expression after the injury. Western blot analysis of LKB1 with carotid artery lysate revealed the same pattern as LKB1 immunohistochemistry. Increased LKB1 expression started at 5 days after the balloon injury, and peaked at 14 days after the injury. Although LKB1 expression was increased after the injury, LKB1 kinase activity was not increased. Ang II or balloon-injury increased the expression of LKB1 although the LKB1 activity was reduced. CONCLUSION: Ang II increased LKB1 expression in VSMCs and neointima. These findings were not kinase dependant.