Unexpected Relationship of Extracellular ATP with Intracellular Ca Concentration in HepG2 Cells

The aim of our study is to examine the effect of extracellular ATP whether it is correlated with intracellular Ca concentrations ([Ca2+]i) on human liver hepatocellular cells (HepG2). The extracellular ATP is responsible for regulating both cells signaling and cell functions. ATP maintains these effects through purinergic P2 receptors. The extracellular ATP promotes the release of Ca2+ from the Ca2+ stores to the cytoplasm in the cell and increases [Ca2+] I in the cell. In our study, various concentrations of ATP (10-3M-10-7M) were applied to HepG2 cells and incubated for 24 hours, 48 hours, and 72 hours. At these concentrations, the proliferation of cells and apoptosis of the cells was examined for 24 hours, 48 hours, and 72 hours. Similarly, cells with different ATP concentrations incubated for 24 hours and 48 hours were loaded with Indo 1FF AM calcium indicator to measure [Ca2+]i. Surprising results were obtained, 10-6M-10-7M extracellular ATP was found to be more toxic than 10-3 M-10-4 M extracellular ATP, (p<0.05). Low concentrations of ATP also reduced [Ca2+]i for 24 hours and 48 hours of incubations (p<0.01). As a result, low concentration extracellular ATP is more toxic in HepG2 cells. At the same time, the extracellular ATP correlates with [Ca2+]i.

Role of intracellular Ca2+ in regulating NHE3 expression in Caco-2 cells during rotavirus infection / 中华微生物学和免疫学杂志

Objective To investigate the effects of rotavirus ( RV) on the expression and bioactiv-ity of Na+-H+ exchanger 3 ( NHE3 ) in Caco-2 cells and the possible regulatory mechanism. Methods Caco-2 cells expressing NHE3 were constructed and divided into four groups as follows: control ( CTL ) group, RV group, BAPTA-AM ( a Ca2+ chelator) group and BAPTA-AM+RV group. Na+-H+ exchanger ac-tivity and NHE3 expression on cell surface were determined using BCECF-AM and biotinylation assay, re-spectively. Expression of Cdc42 at protein level was measured by Western blot. Results Compared with the control group, RV infection significantly decreased the activity of NHE3 and its expression on cell surface. BATPA-AM antagonized the inhibitory effects on NHE3. Moreover, the expression of Cdc42 at protein level was increased following RV infection, which was also antagonized by BATPA-AM. Conclusions Intracellu-lar Ca2+-mediated Cdc42-dependent endocytosis pathway might be involved in regulating the expression and bioactivity of NHE3 during RV infection.

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.

ROS mediates regulation of intracellular Ca2 +induced by angiotensin II in primarily cultured medullary neurons / 中国病理生理杂志

AIM:To investigate the role of reactive oxygen species (ROS) in the regulation of intracellular Ca2+induced by angiotensin II ( Ang II) in the primarily cultured medullary neurons .METHODS:Primarily cultured me-dullary neurons were prepared from 14-day-old embryos of Sprague-Dawley rats in the study .The identification of medullary neurons was assessed by double-labeling immunofluorescence .To explore the role of ROS , mainly the superoxide ( O2 ·-) , the O2 ·-generation was measured using the fluorogenic probe dihydroethidium ( DHE) .To determine intracellular free cal-cium concentration ( [ Ca2+] i ) , the neurons were loaded with the Ca 2+-specific dye Fura-2/AM.The cell viability after adding Ang II was also examined using CCK-8 assay.RESULTS:Most of the cultured cells were medullary neurons , more than 80%of which were glutamate positive neurons .Ang II (5 μmol/L) increased the level of ROS within 10 min in the medullary neurons .Ang II at 5μmol/L induced a significant [ Ca2+] i increase in the medullary neurons , and the effect of Ang II occurred rapidly and reached a peak within 20 min after administration.The level of [Ca2+]i started to decline after washout .The Ca2+elevation induced by Ang II was significantly decreased by apocynin or TEMPOL .No significant differ-ence in the cell viability between control group and 5μmol/L Ang II treatment group was observed .CONCLUSION:ROS is involved in the regulation of [Ca2+]i induced by Ang II in the primarily cultured medullary neurons , suggesting a poten-tial intracellular signaling mechanism involved in the Ang II-mediated oxidant regulation of central neural control of blood pressure.

Activation of acetylcholine receptor elicits intracellular Ca2+ mobilization, transient cytotoxicity, and induction of RANKL expression

Acetylcholine receptors (AChR) including muscarinic and nicotinic AChR are widely expressed and mediate a variety of physiological cellular responses in neuronal and non-neuronal cells. Notably, a functional cholinergic system exists in oral epithelial cells, and nicotinic AChR (nAChR) mediates cholinergic anti-inflammatory responses. However, the pathophysiological roles of AChR in periodontitis are unclear. Here, we show that activation of AChR elicits increased cytosolic Ca²⁺ ([Ca²⁺]ᵢ), transient cytotoxicity, and induction of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. Intracellular Ca²⁺ mobilization in human gingival fibroblast-1 (hGF-1) cells was measured using the fluorescent Ca²⁺ indicator, fura-2/AM. Cytotoxicity and induction of gene expression were evaluated by measuring the release of glucose-6-phosphate dehydrogenase and RT-PCR. Activation of AChR in hGF-1 cells by carbachol (Cch) induced [Ca²⁺]ᵢ increase in a dose-dependent manner. Treatment with a high concentration of Cch on hGF-1 cells caused transient cytotoxicity. Notably, treatment of hGF-1 cells with Cch resulted in upregulated RANKL expression. The findings may indicate potential roles of AChR in gingival fibroblast cells in bone remodeling.

Inhibition of Hepatic Stellate Cell Proliferation and Extracellular Ca2+ Influx by Leptin-siRNA / 华中科技大学学报(医学版)

Objective To evaluate the effect of silencing leptin by small interfering RNA(siRNA)on the expression of lep‐tin ,and apoptosis ,proliferation and intracellular Ca2+ concentration([Ca2+ ]i )of hepatic stellate cells(HSCs)and to provide evi‐dence for liver fibrosis gene therapy.Methods HSCs were divided into normal control group ,blank vector group ,siRNA nega‐tive control group and leptin‐siRNA group.After transfection of the leptin‐siRNAs into HSCs ,cell proliferation was measured by MTT assay.Cell cycle and apoptosis were measured by flow cytometry.Expression of leptin was detected by immunocyto‐chemistry and Western blot. [Ca2+ ]i was measured by Fura‐2/AM loading.Results Compared with the normal control group , the blank vector group and the siRNA control group ,the protein expression of leptin and the cell growth were significantly in‐hibited in the leptin‐siRNA group(P<0.05). The proliferation rate of HSCs was significantly different at different time points (24 ,48 and 72 h)(P<0.05).The cell apoptosis rate was increased significantly in the leptin‐siRNA group(P<0.01).At the same time ,Leptin‐siRNA‐induced [Ca2+ ]i was also significantly reduced(P<0.05).Conclusion The leptin gene may play an important role in liver fibrosis progression and is potentially a novel predictive and prognostic marker for liver fibrosis.

Hesperetin Stimulates Cholecystokinin Secretion in Enteroendocrine STC-1 Cells

Hesperetin (3',5,7-trihydroxy 4'-methoxyflavanone) and its glycoside hesperidin (hesperetin 7-rhamnoglucoside) in oranges have been reported to possess pharmacological effects related to anti-obesity. However, hesperetin and hesperidin have not been studied on suppressive effects on appetite. This study examined that hesperetin and hesperidin can stimulate the release of cholecystokinin (CCK), one of appetite-regulating hormones, from the enteroendocrine STC-1 cells, and then examined the mechanisms involved in the CCK release. Hesperetin significantly and dose-dependently stimulated CCK secretion with an EC50 of 0.050 mM and increased the intracellular Ca2+ concentrations ([Ca2+]i) compared to the untreated control. The stimulatory effect by hesperetin was mediated via the entry of extracellular Ca2+ and the activation of TRP channels including TRPA1. These results suggest that hesperetin can be a candidate biomolecule for the suppression of appetite and eventually for the therapeutics of obesity.

Effect of n-butanol Extract from Potentilla anserina on Hypoxiainduced Calcium Overload and SERCA2 Expression of Rat Cardiomyocytes / 中草药·英文版

ObjectiveTo investigate the effect of n-butanol extract from Potentilla anserina (NP) intervention on hypoxia-induced Ca2+ overload and SERCA2 expression of rat cardiomyocytes.MethodsPrimary cultured myocardial cell from SD neonatal rat (1-3 d) was used in the establishment of hypoxia model.After hypoxia for 3 h,the Ca2+ concentration of myocardial cells was measured with fura-2/AM fluorescent probe,and the biochemical indicator intracellular Ca2+-ATPase was examined and the mRNA and its protective protein levels of the sarcoplasmic reticulum (SR) Ca2+-ATPases (SERCA2) were assayed with RT-PCR,Western-blotting,and immune-cytochemical staining in each group.ResultsThe results showed that NP decreased Ca2+ concentration,increased the activity of Ca2+-ATPase,and improved the mRNA and protein expression of SERCA2 in hypoxia-injured myocardial cells as compared with the model group.ConclusionThese results indicate that NP could attenuate the Ca2+ overload.The mechanism might be explained as that NP could elevate the SERCA2 level,increase the activity of myocardium in rats,and further enhance the capacity of SR Ca2+ re-uptake.

STIM1/Orai1-mediated store-operated Ca2+ entry: the tip of the iceberg

Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

Protective Effect of Wenxin Granula on Heart from Myocardial Infarction through Regulating Intracellular Ca2+ / 中草药·英文版

Objective To assess the anti-arrhythmic activity and cardioprotective effects of Wenxin Granula, a traditional Chinese formula (consisting of Salviae Miltiorrhizae Radix, Polygonati Rhizoma, Notoginseng Radix et Rhizoma, Nardostachyos Radix et Rhizoma, Angelicae Sinensis Radix, and Succinum), on heart in ischemic-induced myocardial infarction (MI) rats and compare with those of Amiodarone which have been demonstrated in clinic. Methods Rats were randomly divided into Sham-operated (control), Ml + Amiodarone [5 mg/(kg·d)] (MI), and MI + Wenxin Granula [10 mg/(kg·d)] groups and left anterior descending coronary artery was occluded in each group. After left anterior descending for 12 h, standard lead Ⅱ of administration electrocardiogram was recorded in order to analyze the occurrence of arrhythmia. After one month, the size of the infarct area of heart was evaluated by TTC staining method and haemodynamic function was assessed to detect the heart function. Laser scanning confocal microscope and the technique of patch clamp were used to detect the intracellular Ca2+ ([Ca2+]j) and L-type calcium current (ICa-L), respectively. Results Both Wenxin Granula [10 mg/(kg·d)] and Amiodarone [5 mg/(kg·d)] could markedly decrease the incidence of arrhythmia in heart of rats which were subjected to ischemic injury. After one month, Wenxin Granula could significantly decrease mortality to 22.22% and reduce the infarct area (P < 0.05), but Amiodarone did not. The mechanism may involve that Wenxin Granula attenuated [Ca2+]j decreasing in MI rats. Additionally, Wenxin Granula could obviously ameliorate the impaired heart function of MI rats by decreasing the elevated left ventricular end-diastolic pressure and increasing the attenuated maximum change velocity of left ventricular pressure in the isovolumic contraction or relaxation period. On the other hand, electrophysiological experiment results revealed that Wenxin Granula administration one month later also increased the reduced ICa-L density in rat ventricular myocytes in MI rats. The results of LSCM showed that Wenxin Granula could recover the amplitude of [Ca2+]j decreased by heart failure during long term. Conclusion Wenxin Granula could not only inhibit the incidence of arrhythmia but also decrease the mortality, which was accompanied by recovering the amplitude of [Ca2+]j. This protective effect of Wenxin Granula may partially be mediated through changing ICa-L.as well as increasing [Ca2+]j.

Influence of coal-arsenic exposure on human T cells proliferation and its mechanism / 中国地方病学杂志

Objective To explore the influence of coal-arsenic exposure on human T cells proliferation and its mechanism.Methods Blood samples colleoted from individuals which lived in arsenism area of coal-burning type and non-arsenism area in Guizhou Province were divided into exposed group(17),mild(35),moderate(38) and severe arsenism group(19)and control group(35)according to Diagnosis Smndard for Endemic Arsenism (WS/T 211-2001).T cell stimulation index wag determined by methyl thiazolyl tetrazolium(MTT)colorimetric method.The intracellular Ca2+ exponential(IECa2+)in peripheral blood mononuclear cell(PBMC)was analyzed by Fho-3/AM dye and flow cytometry.DNA binding activity of actively T cells nuclear factor(NF-AT)in PBMC was evaluated by electrophoretie mobility shift assay(EMSA).Results Concanavalin A(ConA)stimulation decreased the T cells stimulation indexes in exposed group,mild,moderate and severe arsenism groups(1.315±0.962, 1.611±1.224,1.114±0.545,1.289±0.875)compared with control group(2.322±1.241),all the differences being statistically significant(P＜0.01).After stimulated by anti-CD3 monoclonal antibody(McAb),the T cells stimulation index in exposed group,mild,moderate and severe arsenism group(0.997±0.177,1.103±0.291,1.007±0.221, 0.957±0.205) were lower than that of control group(1.842±0.429,P ＜ 0.01 ). IECa2+ of PBMC after treated by anti-CD3 McAb in mild,moderate and severe arsenism group( 110.130±49.637,92.429±31.191,77.640± 35.372) were lower compared with control group(145.986±59.450,P ＜0.01 ). Moreover,IECa2+ in moderat and severe arsenism group were lower than exposed group(121.337±46.410,P ＜ 0.05). DNA binding activity of PBMC NF-AT in mild,moderate and severe arsenism group(1.354±0.446,1.290±0.291,1.159±0.411 ) were lowered than that of control group(1.722±0.291,P ＜ 0.01) and exposed group(1.611±0.294,P ＜ 0.05). Conclusions The coal-arsenic exposure can reduce the human T cells stimulation indexes,IECa2+ in PBMC and the DNA binding activity of NF-AT. It suggest that arsenic may suppress the proliferation ability of human T cells,which may be partly related to the influence of arsenic on T cell receptor(TCR)/CD3 signal transduetion pathway.

Nicotine-evoked cytosolic Ca2+ increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla

Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca2+ dependent exocitosis of vasoactive compounds. Using the Ca2+ indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs) respond to acetylcholine (ACh) with a cytosolic Ca2+ increase and depolarization of the membrane potential (20.3±0.9 mV; n=23). The increase in cytosolic Ca2+ induced by 10µM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 µM of hexamethonium. On the other hand, the increase in cytosolic Ca2+ could be depressed by nifedipine (0.01 -100 µM) or withdrawal of extracellular Ca2+. Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal gland's microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca2+ channels, influx of external Ca2+ and liberation of vasoactive compounds.

Serotonin (5-HT) Receptor Subtypes Mediate Regulation of Neuromodulin Secretion in Rat Hypothalamic Neurons

Serotonin (5-HT), the endogenous nonselective 5-HT receptor agonist, activates the inositol -1,4,5- triphosphate / calcium (InsP3/Ca2+) signaling pathway and exerts both stimulatory and inhibitory actions on cAMP production and neuromodulin secretion in rat hypothalamic neurons. Specific mRNA transcripts for 5-HT1A, 5-HT2C and 5-HT4 were identified in rat hypothalamic neurons. These experiments were supported by combined techniques such as cAMP and a Ca2+ assays in order to elucidate the associated receptors and signaling pathways. The cAMP production and neuromodulin release were profoundly inhibited during the activation of the Gi-coupled 5-HT1A receptor. Treatment with a selective agonist to activate the Gq-coupled 5-HT2C receptor stimulated InsP3 production and caused Ca2+ release from the sarcoplasmic reticulum. Selective activation of the Gs-coupled 5-HT4 receptor also stimulated cAMP production, and caused an increase in neuromodulin secretion. These findings demonstrate the ability of 5-HT receptor subtypes expressed in neurons to induce neuromodulin production. This leads to the activation of single or multiple G-proteins which regulate the InsP3/Ca2+/PLC-gamma and adenyl cyclase / cAMP signaling pathways.

La testosterona inhibe las respuestas contráctiles del agonista adrenérgico alpha1, fenilefrina, asociadas con la liberación de calcio intracelular en la aorta de rata / Testosterone inhibits the contractile responses to phenylephrine associated with the release of intracellular calcium in rat aorta

Utilizando segmentos de aorta de rata sin endotelio inmersos en solución sin Ca2+, evaluamos la capacidad de la testosterona para modificar el efecto contráctil del agonista adrenérgico fenilefrina, así como el incremento en el tono de reposo (ITR) asociado con la entrada capacitativa de calcio por el sarcoplasma. La testosterona [10-5–10- 4 M] inhibió significativamente la contracción activada por la fenilefrina [10-6 M] y el ITR. Estos efectos no fueron modificados con cicloheximida [10-5 M] (inhibidor de la síntesis protéica), flutamida [10-5 M] (antagonista de receptores androgénicos), o aminoglutetimida [10-5 M] (inhibidor de la citocromo P450 aromatasa). La testosterona también inhibió las respuestas contráctiles de la serotonina [10-5 M], pero no de la cafeína [10-2 M]. Además, la testosterona inhibió las contracciones del ácido ciclopiazónico [10-6 M] y de la ryanodina [10- 5 M] asociadas con el ingreso capacitativo de Ca2+ mediante canales de Ca2+ tipo no L. Estos datos sugieren que la testosterona interfiere con la vía de transducción de los receptores acoplados a proteínas Gq- 11, e inhibe la entrada capacitativa de Ca2+ a través de canales de Ca2+ tipo L y tipo no L; los efectos son no genómicos, independientes de receptores androgénicos, y de la conversión testosterona en estrógenos.

Using endothelium-denuded rat aortic rings incubated in Ca2+ -free solution, we assessed the ability of testosterone to influence the contractile effect of phenylephrine, and the increase in resting tone (IRT) associated with Ca2+ ability to cross the plasma membrane. The addition of testosterone [10(-5)-10(-4) 5 min before phenylephrine [10(-6) M], inhibited both phenylephrine-induced contraction and IRT. These changes were not affected by cycloheximide (10(-5) M; a protein synthesis inhibitor of), flutamide (10(-5) M; an androgenic receptor antagonist), or by adding aminoglutethimide (10(-5) M; an aromatase inhibitor). Testosterone also blocked the contractile response to serotonin [10(-5) M] but not to caffeine [10(-2) M]. On the other hand, testosterone inhibited the contractile responses to cyclopiazonic acid (10(-6) M; a selective Ca2+ -ATPase inhibitor) or ryanodine (10(-5 M; an activator of sarcoplasmic reticulum Ca2+ -release channels) associated with capacitative Ca2+ influx through non-L-type Ca2+ channels. These data suggest that by acting on the cellular membrane, testosterone interferes with the signal transduction pathway of G(q-11) protein-coupled receptors, and inhibits capacitative Ca2+ influx through both L-type and non-L-type Ca2+ channels. These effects are non-genomic, non-mediated by the intracellular androgen receptor, and not due to the conversion of testosterone to estrogens.

Toosendanin Modifies K~+-and Ca~(2+)-Channel Activity and Intracellular Ca~(2+) Concentration / 生物化学与生物物理进展

Usage of the fruit and bark of a Melia-family plant as a digestive tract-parasiticide and agricultural insecticide was recorded about two thousant years ago in ancient China. Toosendanin (TSN), a triterpenoid, is an effectual ingredient extracted from the plant. Studies have demonstrated that TSN selectively affects neurotransmitter release, effectively antagonizes botulism, induces cell differentiation and apoptosis and inhibits proliferation of various human cancer cells, inhibits feeding and dovelopment in insects and modifies K+- and Ca2+-channel activity. The research data to demonstrate that TSN inhibits K+-channel and facilitates L-type Ca2+-channel are summarized, and the mechanism of action of TSN is discussed.

Buffering Contribution of Mitochondria to the Ca2+i Increase by Ca2+ Influx through Background Nonselective Cation Channels in Rabbit Aortic Endothelial Cells

To prove the buffering contribution of mitochondria to the increase of intracellular Ca2+ level ([Ca2+]i) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial Ca2+ entry and Ca2+ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM (R340/380) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh (10microM) increased R340/380 by 1.1+/-0.15 (mean+/-S.E., n=6). When the external Na+ was totally replaced by NMDG+, R340/380 was increased by 1.19+/-0.17 in a reversible manner (n=27). NMDG+-induced [Ca2+]i increase was followed by oscillatory decay after [Ca2+]i reached the peak level. The increase of [Ca2+]i by NMDG+ was completely suppressed by replacement with Cs+. When 1microM CCCP was applied to bath solution, the ratio of [Ca2+]i was increased by 0.4+/-0.06 (n=31). When 1microM CCCP was used for pretreatment before application of NMDG+, oscillatory decay of [Ca2+]i by NMDG+ was significantly inhibited compared to the control (p < 0.05). In addition, NMDG+-induced increase of [Ca2+]i was highly enhanced by pretreatment with 2microM CCCP by 320+/-93.7%, compared to the control (mean+/-S.E., n=12). From these results, it is concluded that mitochondria might have buffering contribution to the [Ca2+]i increase through regulation of the background NSCC in RAECs.

Effects of bupivacaine on intracellular Ca~(2+) in rat ventricular myocytes / 中国临床药理学与治疗学

0.05).Intracellular Ca2+ FI in rat ventricular myocytes induced by KCl was inhibited significantly in group B2 and B3 compared with that in group C(P

Differential effects of Ach and ATP on [Ca2+]c changes in mouse pancreatic β-cells / 中国康复理论与实践

@#ObjectiveTo identify the differences between acetylcholine(Ach)-induced increase and adenosine triphosphate(ATP)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]c) in mouse pancreatic β-cells. MethodsMouse pancreatic β-cells were primarily cultured and divided into two groups,one group was stimulated by Ach and another by ATP.[Ca2+]c was recorded with Fura-2 in normal condition, chelation of extracellular Ca2+ by EGTA and depletion of intracellular Ca2+ stores by Thapsigargin.ResultsAch induced a transient peak increase and sustained increase in [Ca2+]c. ATP induced a transient peak increase and no sustained increase. Chelation of extracellular Ca2+ by EGTA eliminated the sustained increase induced by Ach, and did not eliminate the transient peak increase induced by Ach and ATP. Depletion of intracellular Ca2+ stores by Thapsigargin eliminated the transient peak increase induced by Ach and ATP and the sustained increase induced by Ach. ConclusionsAch induces intracellular Ca2+ release and the following Ca2+ release-activated Ca2+ influx, and ATP induces intracellular Ca2+ release, but blocks the following Ca2+ release-activated Ca2+ influx.

Testosterone Causes Simultaneous Decrease of Ca2+i and Tension in Rabbit Coronary Arteries: by Opening Voltage Dependent Potassium Channels

The relationship between the level of testosterone and the incidence of coronary heart disease is still controversial in the view of the results of clinical and epidemiologic studies. This uncertainty might be partly due to relatively small number of experimental studies undertaken to investigate the cellular mechanism underlying the vascular responses to testosterone. To further investigate the cellular mechanisms of testosterone with respect to vascular response, we investigated the effect of testosterone on contractility and intracellular Ca2+ regulation in a rabbit coronary artery and evaluated the underlying mechanism of testosterone-induced changes of coronary vascular tone by using various pharmacological blockers. Testosterone was found to relax rabbit coronary arteries in a dose-dependent manner, and no significant difference was found in the relaxation response to testosterone with or without endothelium. Similar results were obtained in male and non-pregnant female rabbit coronary arteries. The relaxation response of rabbit coronary arteries to testosterone was greater for PGF2alpha-contracted rings than for KCl contracted rings, which suggest the involvement of K+ channels. Furthermore, the relaxation response to testosterone was significantly reduced by 4-aminopyridine, a sensitive blocker of voltage dependent K+ channels, but not by low doses of tetraethylammonium or iberiotoxin, a Ca2+ activated K+ channel blocker. Testosterone simultaneously reduced the intracellular Ca2+ concentration ([Ca2+]i) and tension, and 4-AP effectively antagonized the testosterone-induced change of [Ca2+]i and tension. Therefore, it may be concluded that the stimulation of voltage dependent K channels is responsible, at least in part, for the testosterone-induced relaxation of rabbit coronary arteries.

Subcellular Ca2+ Mobilization in Gastric Smooth Muscle Contraction / 대한마취과학회지

BACKGROUND: The stomach can be generally classified anatomically into three parts; fundus, corpus, and antrum. It has not been well demonstrated how the three regions contribute to specified gastric motility. In the present study, the regional differences on contractile response and intracellular Ca2+ levels ([Ca2+]i) were investigated in a mouse gastric muscle. METHODS: An isometrical contraction was measured with a computerized physiograph, and [Ca2+]i was measured with fura-PE3/AM, a fluorescent Ca2+ indicator in gastric smooth muscle from mice. RESULTS: Carbachol (CCh), a potent muscarinic receptor agonist, generated rhythmic contractions in a dose dependent manner, superimposed on tonic components in the antral muscle. Whereas similar contractile responses to CCh was obtained in the antrum, CCh evoked tonic components predominantly. CCh increased [Ca2+]i in a dose dependent manner in both the antral and fundic smooth muscle. However, the increment of [Ca2+]i in the fundus was greater than that of the antrum. Verapamil (10nM), a l-type Ca2+ channel blocker, inhibited completely the contraction and [Ca2+]i induced by CCh in the antral strips, whereas the responses in the fundus showed a resistance to verapamil. CONCLUSIONS: These results suggest that muscarinic stimulation has a regional difference on muscle contractility and [Ca2+]i, which is mediated by differences of Ca2+ movement in mouse gastric muscle.