1.Role of store-operated Ca2+ channels in ethanol-induced intracellular Ca2+ increase in HepG2 cells.
Hui-min LIU ; Li-hui YAN ; Zheng LUO ; Xiao-meng SUN ; Rui-bing CUI ; Xue-hui LI ; Ming YAN
Chinese Journal of Hepatology 2013;21(12):949-954
OBJECTIVETo investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs).
METHODSHepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting.
RESULTSThe ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment.
CONCLUSIONEthanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.
Calcium ; metabolism ; Calcium Channels ; metabolism ; Ethanol ; adverse effects ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; Humans
2.Effects of Dantrolene on the Interaction of Calcium and Erythrocyte Membrane.
Yonsei Medical Journal 1987;28(4):249-254
Dantrolene sodium in vitro inhibited the ATP-dependent efflux of calcium from human Fed cells, the Ca++-ATPase activity of red blood cell membrane fragments (RBCMF) and passive calcium binding to RBCMF. These effects were obtained With concentrations of dantrolene sodium between 2.5 and 20 uM. However the passive influx of Ca++ was measured at 37 degrees C in cells pretreated to abolish Ca++ pumping and was not influenced by dantrolene sodium. From these results, it was concluded that dantrolene sodium inhibits an active Ca++ extrusion across the red cell membrane by inhibiting Ca++-ATPase activity which is intimately involved with the Ca++ transport mechanism in the red cell membrane.
Calcium/metabolism*
;
Dantrolene/pharmacology*
;
Erythrocyte Membrane/drug effects*
;
Erythrocyte Membrane/metabolism
;
Human
;
Ion Channels/drug effects*
;
Ion Channels/metabolism
3.New insights into the activation mechanism of store-operated calcium channels: roles of STIM and Orai.
Journal of Zhejiang University. Science. B 2008;9(8):591-601
The activation of Ca2+ entry through store-operated channels by agonists that deplete Ca2+ from the endoplasmic reticulum (ER) is a ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past two decades. Store-operated Ca2+-release-activated Ca2+ (CRAC) channels constitute the sole pathway for Ca2+ entry following antigen-receptor engagement. In a set of breakthrough studies over the past two years, stromal interaction molecule 1 (STIM1, the ER Ca2+ sensor) and Orai1 (a pore-forming subunit of the CRAC channel) have been identified. Here we review these recent studies and the insights they provide into the mechanism of store-operated Ca2+ channels (SOCCs).
Animals
;
Calcium
;
metabolism
;
Calcium Channels
;
metabolism
;
Calcium Signaling
;
drug effects
;
Humans
;
Membrane Proteins
;
antagonists & inhibitors
;
metabolism
;
Protein Binding
4.Different effects of acetylcholine on the action potential and force contraction in guinea pig atrial and ventricular myocardium.
Ping FANG ; Wei-Jin ZANG ; Xiao-Jiang YU ; Qiang SUN ; Yi-Min ZANG ; Jun LU
Acta Physiologica Sinica 2002;54(4):311-316
The purpose of this study was to investigate the different effects of ACh on the action potential and force contraction in guinea pig atrial and ventricular myocardium by using standard microelectrodes and force transducer. The results showed that the duration of the action potential (APD) of atrial myocardium was shortened from 208.57+/-36.05 to 101.78+/-14.41 ms (n=6, P<0.01), and the APD of the ventricular myocardium was shortened from 286.73+/-36.11 to 265.16+/-30.06 ms (n=6, P<0.01).The amplitude of the action potential (APA) of the atrial myocardium was decreased from 88.00+/-9.35 to 62.62+/-20.50 mV (n=6, P<0.01), while the APA of the ventricular myocardium did not change significantly.The force contraction of atrial myocardium was inhibited completely (n=6, P<0.01), while the force contraction of ventricular myocardium was inhibited by 37.57+/-2.58% (n=6, P<0.01). The ACh effects correlated with its concentration. The K(D) of the APD shortening effects in the atrial and ventricular myocardium were 0.275 and 0.575 micromol/L. The K(D) of the negative inotropic in the atrial and ventricular myocardium were 0.135 and 0.676 micromol/L, respectively. The corresponding data points were compared using t test between the atrial and ventricular myocardium, and the differences were significant when the ACh concentration was above 10 nmol/L. Furthermore, atropine (10 micromol/L) and CsCl (20 mmol/L) blocked the effects of 10 micromol/L ACh on the APD of ventricular myocardium, while CdCl2 (0.1 mmol/L) had no influence on these effects. In conclusion, ACh could shorten the action potential duration and inhibit the force contraction of atrial and ventricular myocardium in a concentration-dependent manner. There are differences in the effects of ACh on the atrial and ventricular myocardium. The atrial myocardium is more sensitive to ACh than the ventricular myocardium. It is probable that the muscarinic receptor and the potassium channel, but not the calcium channel, are involved in the ACh-induced shortening of the ventricular APD.
Acetylcholine
;
pharmacology
;
Action Potentials
;
drug effects
;
Animals
;
Calcium Channels
;
metabolism
;
Guinea Pigs
;
Heart Atria
;
drug effects
;
Heart Ventricles
;
drug effects
;
Microelectrodes
;
Potassium Channels
;
metabolism
;
Receptors, Muscarinic
;
metabolism
5.Calcium channel mechanism by which betaine promotes proliferation of lymphocytes in mice.
Yubin JI ; Shiyong GAO ; Xiaoyan FENG ; Liwei HE
China Journal of Chinese Materia Medica 2009;34(15):1959-1963
OBJECTIVETo study how the way in which betaine promotes the proliferation of mouse spleen lymphocytes is related to calcium channels.
METHODBALB/c mice were used for this experiment. Mouse spleen lymphocytes were obtained through in vitro cultivation after they had been separated, and were divided into a negative control group, a Con A group, and 0.04, 0.4, 4, and 20 mmol x L(-1) betaine groups. MTT was used to observe the effect of betaine on the proliferation of mouse spleen lymphocytes; flow cytometry was used to measure the changes in the cell cycle of mouse spleen lymphocytes; and laser confocal scanning microscopy was used to observe the changes in the intracellular [Ca2+]i of mouse spleen lymphocytes after betaine or different calcium channel blockers were applied.
RESULTBetaine was found to promote the proliferation of mouse spleen lymphocytes 12 h after it had been applied in vitro in concentrations of 4 and 20 mmol x L(-1). It was also found to promote the proliferation of mouse spleen lymphocytes 24 h and 48 h after it had been applied in vitro in concentrations of 0.04, 0.4, 4, and 20 mmol x L(-1), with the effect being most marked for the 4 mmol x L(-1) group 24 h after its application. It was found to facilitate the entry of mouse spleen lymphocytes from the G0/G1 to the S phase 4, 6, 18, and 24 h after it had been applied to mouse spleen lymphocytes in a concentration of 4 mmol x L(-1), with the effect being most marked at 18 h after its application. Intracellular [Ca2+]i in mouse spleen lymphocytes increased significantly (P < 0.01) 6, 12, 18 h after 4 mmol x L(-1) betaine had acted on the lymphocytes, with the effect being most marked at 6 h. The calcium channel blockers nifidipine, diltiazem, mibefradil, and genistein had no effect on the increase of the intracellular [Ca2+]i in mouse spleen lymphocytes due to the application of betaine, while verapamil, mycifradin, heparin, and procaine could block such increase.
CONCLUSIONBetaine facilitates the entry of mouse spleen lymphocytes from the G0/G1 into the S phase by raising the intracellular [Ca2+]i in these cells, thus promoting their proliferation. Intracellular [Ca2+]i increases mainly in two ways: (1) By affecting the alpha1 subunit of the L-type voltage-gated calcium channel with mediation by G proteins and thus leading to an efflux of intracellular calcium: (2) By affecting the IP3R and RyR calcium channels of the intracellular calcium stores and thus leading to the release of intracellular calcium.
Animals ; Betaine ; pharmacology ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Female ; Lymphocytes ; cytology ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred BALB C
6.Modulation by nicotine on the genes expression of brain potassium, sodium and calcium channels.
Xiu-Lan SUN ; Yue LIU ; Gang HU ; Hai WANG
Chinese Journal of Applied Physiology 2004;20(4):359-362
AIMUsing GeneChip to analyze the changes in genes expression of brain potassium, sodium and calcium channels after chronic treatment with nicotine.
METHODSAnimals were treated with nicotine at the doses of 2.4 mg/kg sc. twice a day for 14 days. RNA was extracted from the whole brain samples and converted to double-stranded cDNA and then to biotinylated cRNA. The biotinylated cRNA was fragmented, and hybridized to GeneChip (Affymetrix Rat Neurobiology U34). The chips were scanned with a probe array scanner, and the data were analyzed with the Affymetrix Microarray Analysis Suite (MAS). The GeneChip data were confirmed u sing RT-PCR.
RESULTSAfter treatment with chronic nicotine, transcripts of potassium, sodium and calcium channels showed altered expression. K+ channel: outward rectifier K+ channel and Ca2(+)-activated K+ channel were down-regulated, other voltage-dependent K+ channel including Kv2.3r were up-regulated. Voltage-dependent Na+ channel: beta2 subunit was increased, alpha subunit and beta1 subunit were decreased. Beta3 subunit of Ca2+ channel was up-regulated.
CONCLUSIONChronic exposure to nicotine not only desensitized nicotinic receptors, but also effected genes expression, of important ion channels, such as sodium channels, potassium channels and calcium channels.
Animals ; Brain ; drug effects ; metabolism ; Calcium Channels ; drug effects ; metabolism ; Gene Expression ; Male ; Nicotine ; pharmacology ; Potassium Channels ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium Channels ; drug effects ; metabolism
7.Androgen correlates with expressions of ryanodine receptor 1 and voltage-gated calcium channel 1.3 in rat corpus cavernosum smooth muscle.
Hua LUO ; Hai-fan YANG ; Rui JIANG
National Journal of Andrology 2009;15(10):895-900
OBJECTIVETo study the expressions of ryanodine receptor 1 (RyR1) and voltage-gated calcium channel 1.3 (CaV1.3) in the corpus cavernosum smooth muscle of castrated rats and to investigate their role in androgen deficiency-related erectile dysfunction.
METHODSForty 8-week-old SD rats were equally randomized into Groups A (2-week sham-operation), B (4-week sham-operation), C (2-week castration), and D (4-week castration). After surgery, the levels of serum testosterone in different groups of rats were determined, and the expressions of RyR1 and CaV1.3 in the corpus cavernosum were detected by immunohistochemical staining and RT-PCR.
RESULTSThe levels of serum testosterone were significantly decreased in Groups C ([15.97 +/- 5.67] nmol/L) and D ([2.03 +/- 1.57] nmol/L) as compared with A ([90.54 +/- 20.13] nmol/L) and B ([120.35 +/- 30.32] nmol/L) (P < 0.05). RyR1 and CaV1.3 expressed in all the groups. RyR1 mRNA, CaV1.3 mRNA and their proteins were remarkably reduced in Groups C (0.51 +/- 0.24, 0.50 +/- 0.12, 120.36 +/- 25.78, 103.37 +/- 39.52, respectively) and D (0.33 +/- 0.15, 0.32 +/- 0.07, 67.39 +/- 30.54, 67.56 +/- 20.12, respectively) in comparison with A (1.53 +/- 0.25, 1.33 +/- 0.05, 300.96 +/- 135.12, 298.68 +/- 126.35, respectively) and B (1.37 +/- 0.23, 1.25 +/- 0.03, 330.38 +/- 128.59, 327.35 +/- 117.37, respectively) (P < 0.05). The androgen level was positively correlated with the expressions of RyR1 and CaV1.3.
CONCLUSIONAndrogen can regulate erectile function via RyR1 and CaV1.3.
Androgens ; pharmacology ; Animals ; Calcium Channels ; metabolism ; Male ; Muscle, Smooth ; drug effects ; metabolism ; Penis ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel ; metabolism
8.Resveratrol reduces intracellular free calcium concentration in rat ventricular myocytes.
Zheng LIU ; Li-Ping ZHANG ; Hui-Jie MA ; Chuan WANG ; Ming LI ; Qing-Shan WANG
Acta Physiologica Sinica 2005;57(5):599-604
Resveratrol (trans-3, 4', 5-trihydroxy stilbene), a phytoalexin found in grape skins and red wine, has been reported to have a wide range of biological and pharmacological properties. It has been speculated that resveratrol may have cardioprotective activity. The objective of our study was to investigate the effects of resveratrol on intracellular calcium concentration ([Ca(2+)](i)) in rat ventricular myocytes. [Ca(2+)](i) was detected by laser scanning confocal microscopy. The results showed that resveratrol (15~60 mumol/L) reduced [Ca(2+)](i) in normal and Ca(2+)-free Tyrode's solution in a concentration-dependent manner. The effects of resveratrol on [Ca(2+)](i) in normal Tyrode's solution was partially inhibited by pretreatment with sodium orthovanadate (Na3VO4, 1.0 mmol/L, P<0.01), an inhibitor of protein tyrosine phosphatase, or L-type Ca(2+) channel agonist Bay K8644 (10 mumol/L, P<0.05), but could not be antagonized by NO synthase inhibitor L-NAME (1.0 mmol/L). Resveratrol also markedly inhibited the ryanodine-induced [Ca(2+)](i) increase in Ca(2+)-free Tyrode's solution (P<0.01). When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, resveratrol (60 mumol/L) could reduce the velocity and duration of propagating waves, and block the propagating waves of elevated [Ca(2+)](i). These results suggest that resveratrol may reduce the [Ca(2+)](i) in isolated rat ventricular myocytes. The inhibition of voltage-dependent Ca(2+) channel and tyrosine kinase, and alleviation of Ca(2+) release from sarcoplasmic reticulum (SR) are possibly involved in the effects of resveratrol on rat ventricular myocytes. These findings could help explain the protective activity of resveratrol against cardiovascular disease.
Animals
;
Calcium
;
metabolism
;
Calcium Channels
;
drug effects
;
Heart Ventricles
;
cytology
;
metabolism
;
Intracellular Fluid
;
drug effects
;
metabolism
;
Male
;
Myocytes, Cardiac
;
drug effects
;
metabolism
;
Protein-Tyrosine Kinases
;
drug effects
;
Rats
;
Rats, Sprague-Dawley
;
Sarcoplasmic Reticulum
;
drug effects
;
metabolism
;
Stilbenes
;
pharmacology
9.Effect of Hg2+ on voltage-dependent calcium channels and intracellular free calcium in trigeminal ganglion neurons of rats.
Xiao YIN ; Jing-zhi SUN ; Yong MEI ; Xiang GUO ; Song-lin CHEN ; Zheng-lun WANG ; Lei YANG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2008;26(9):542-545
OBJECTIVETo investigate the effects of Hg2+ on voltage-dependent calcium channels and intracellular free calcium in trigeminal ganglion neurons of rats and explore the toxicity mechanism of Hg2+ on these neurons.
METHODSWhole cell patch-clamp technique was used to determine ICa of voltage-dependent calcium channels in trigeminal ganglion neurons of rats. Intracellular free calcium was measured to explore [Ca2+]i dynamic changes from a single cell level by laser scanning confocal microscopy and fluorescence probe techniques.
RESULTS0.01, 0.10, 1.00 and 10.00 micromol/L Hg2+ could reduce voltage-dependent calcium channel currents ICa by (1.80+/-0.32)%, (23.04+/-9.46)%, (58.20+/-7.90)% and (82.00+/-5.77)% in trigeminal ganglion neurons. The inhibiting effects reached their maximum in 5 minutes and could not be reversed significantly during wash with Hg2+-free solution. Also, 0.01, 0.10 and 1.00 micromol/L Hg2+ increased intracellular free calcium concentrations by (2.50+/-0.83)%, (82.81+/-35.36)% and (222.70+/-62.48)% in trigeminal ganglion neurons. Pre-administrated trigeminal ganglion neurons with nifedipine for 10 minutes could decrease the effects and delay the effecting time.
CONCLUSIONThe inhibition of Hg2+ on the voltage-dependent calcium channel currents ICa depends on voltage-dependent calcium channels. And the increase of intracellular free calcium concentration in trigeminal ganglion neurons induced by Hg2+ is related to the release of intracellular stored calcium. However, the relationship between them needs further investigation.
Animals ; Calcium ; metabolism ; Calcium Channels ; drug effects ; metabolism ; physiology ; Cells, Cultured ; Female ; Male ; Mercury ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Trigeminal Ganglion ; cytology ; drug effects ; metabolism ; physiology
10.Effect of shenmai injection on L-type calcium current of diaphragmatic muscle in rats.
Limin ZHAO ; Shengdao XIONG ; Ruji NIU ; Yongjian XU ; Zhengxiang ZHANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2004;24(4):376-378
In this study, whole cell patch clamp recording technique was employed to investigate the effect of Shenmai Injection (SMI) on L-type calcium current of diaphragmatic muscle in rats. The result showed that when the diaphragmatic muscle cell was held at -80 mV and depolarized to +60 mV, 10 microl/ml, 50 microl/ml and 100 microl/ml SMI enhanced the inner peak L-type calcium current from -(6.8 +/- 0.7) pA/pF (n=7) to -(7.3 +/- 0.8) pA/pF (P>0.05, n=7), -(8.6 +/- 1.0) pA/pF (P<0.05, n=7) and -(9.4 +/- 1.2) pA/pF (P<0.05, n=7), respectively, The rates of L-type calcium current were increased by (7.34 +/- 2.37)%, (25.72 +/- 5.94)%, and (38.16 +/- 7.33)%, respectively. However, it had no significant effect on maximal activation potential and reversal potential. Our results suggested that SMI could activate the calcium channel of the diaphragmatic fibers of the rats, increase the influx of Ca2+, and enhance the contractility of diaphragmatic muscles.
Animals
;
Calcium
;
metabolism
;
Calcium Channels, L-Type
;
drug effects
;
Diaphragm
;
drug effects
;
metabolism
;
Drug Combinations
;
Drugs, Chinese Herbal
;
Male
;
Patch-Clamp Techniques
;
Plant Extracts
;
pharmacology
;
Rats
;
Rats, Wistar