BACKGROUNDThe contractility of hepatic stellate cells (HSCs) may play an important role in the pathogenesis of cirrhosis with portal hypertension. The aim of this study was to research the effects of octreotide, an analogue of somatostatin, on intracellular Ca2+ and on the expression of L-type voltage-operated calcium channels (L-VOCCs) in activated HSCs, and to try to survey the use of octreotide in treatment and prevention of cirrhosis with portal hypertension complications.

METHODSHSC-T6, an activated HSCs line, was plated on small glass coverslips in 35-mm culture dishes at a density of 1 x 10(5)/ml, and incubated in DMEM media for 24 hours. After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured by Laser Scanning Confocal Microscopy (LSCM). The dynamic changes in activated HSCs of intracellular Ca2+, stimulated by octreotide, endothelin-1, and KCl, respectively, were also determined by LSCM. Each experiment was repeated six times. L-VOCC expression in HSCs was estimated by immunocytochemistry.

RESULTSAfter octreotide stimulation, a significant decrease in the intracellular Ca2+ of activated HSCs was observed. However, octreotide did not inhibit the increases in intracellular Ca2+ after stimulation by KCl and endothelin-1. Moreover, octreotide did not significantly affect L-VOCC expression. These results suggest that neither L-VOCC nor endothelin-1 receptors in activated HSCs are inhibited by octreotide.

CONCLUSIONSOctreotide may decrease portal hypertension and intrahepatic vascular tension by inhibiting activated HSCs contractility through decreases in intracellular Ca2+. The somatostatin receptors in activated HSCs may be inhibited by octreotide.

Calcium ; analysis ; Calcium Channels, L-Type ; analysis ; Cells, Cultured ; Hepatocytes ; chemistry ; cytology ; drug effects ; Microscopy, Confocal ; Octreotide ; pharmacology

The aim of this study was to investigate the inhibitory effect and the underlying mechanism of ethacrynic acid (EA) on the contraction in mice. BL-420S force measuring system was used to measure the tension of mouse tracheal rings. The whole cell patch clamp technique was utilized to record the channel currents of airway smooth muscle (ASM) cells. The calcium imaging system was used to determine the intracellular Ca concentration ([Ca]) in ASM cells. The results showed that EA significantly inhibited the high K (80 mmol/L) and acetylcholine (ACh, 100 µmol/L)-induced contraction of mouse tracheal rings in a dose-dependent manner. The maximal relaxation percentages were (97.02 ± 1.56)% and (85.21 ± 0.03)%, and the median effective concentrations were (40.28 ± 2.20) μmol/L and (56.22 ± 7.62) μmol/L, respectively. EA decreased the K and ACh-induced elevation of [Ca] from 0.40 ± 0.04 to 0.16 ± 0.01 and from 0.50 ± 0.01 to 0.39 ± 0.01, respectively. In addition, EA inhibited L-type voltage-dependent calcium channel (LVDCC) and store-operated calcium channel (SOCC) currents in ASM cells, and Ca influx. Moreover, EA decreased the resistance of the respiratory system (Rrs) in vivo in mice. These results indicated that EA inhibits LVDCC and SOCC, which results in termination of Ca influx and decreases of [Ca], leading to relaxation of ASM. Taken together, EA might be a potential bronchodilator.
Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; Enzyme Inhibitors ; pharmacology ; Ethacrynic Acid ; pharmacology ; Mice ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; Respiratory System ; cytology ; drug effects

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

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.
Calcium/metabolism ; Calcium Channels, L-Type/*drug effects ; Diaphragm/drug effects ; Diaphragm/*metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; Patch-Clamp Techniques ; Plant Extracts/*pharmacology ; Rats, Wistar

Action Potentials ; drug effects ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Biphenyl Compounds ; pharmacology ; Calcium Channels, L-Type ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; Myocytes, Cardiac ; drug effects ; physiology ; Potassium Channels ; drug effects ; physiology ; Rabbits ; Tetrazoles ; pharmacology

BACKGROUNDRecent studies have revealed that pretreatment with statin is effective in preventing arrhythmia, but its electrophysiological mechanism is unclear. This study was conducted to investigate the cardioprotective effects of simvastatin on reversing electrical remodeling in left ventricular myocytes of rabbit heart undergoing ischemia-reperfusion, so as to explore the ionic mechanism responsible for the anti-arrhythmic effect of statin.

METHODSForty-five rabbits were randomly divided into three groups: ischemic-reperfusion group (I-R), simvastatin intervention group (Statin) and sham-operated control group (CON). Anesthetized rabbits were subjected to 30-minute ischemia by ligation of the left anterior descending coronary artery and a 60-minute reperfusion after a 3-day administration of oral simvastatin of 5 mg x kg(-1) x d(-1) in the Statin group or a placebo in the I-R group. Single ventricular myocytes were isolated enzymatically from the epicardial zone of the infracted region derived from the hearts in the I-R and Statin group and the same anatomical region in the CON animals. The whole cell patch-clamp technique was used to record membrane ionic currents, including sodium current (I(Na)), L-type calcium current (I(Ca-L)) and transient outward potassium current (I(to)). Simultaneously, the level of serum cholesterol was examined.

RESULTSThere was no significant difference in the serum cholesterol concentration among the three groups. The peak I(Na) current density (at -30 mV) was significantly decreased in I-R ((-22.46+/-5.32) pA/pF, n=12) compared with CON ((-42.78+/-5.48) pA/pF, n=16, P<0.01) and Statin ((-40.66+/-5.89) pA/pF, n=15, P<0.01), while the peak I(Na) current density in the Statin group was not different from CON (P>0.05). The peak I(Ca-L) current density (at 0 mV) was significantly increased in I-R ((-4.34+/-0.92) pA/pF, n=15) compared with CON ((-3.13+/-1.22) pA/pF, n=13, P<0.05) and Statin ((-3.46+/-0.85) pA/pF, n=16, P<0.05), while the Peak I(Ca-L) current density in Statin was not different from CON (P>0.05). The I(to) current density (at +60 mV) was significantly decreased in I-R ((9.49+/-1.91) pA/pF, n=11) compared with CON ((17.41+/-3.13) pA/pF, n=15, P<0.01) and Statin ((14.54+/-2.41) pA/pF, n=11, P<0.01), although there was a slight reduction in the Statin group compared with CON (P<0.05).

CONCLUSIONSIt is implied that ischemia-reperfusion induces significant down-regulation of I(Na) and I(to) and up-regulation of I(Ca-L), which may underlie the altered electrical activity and long abnormal transmembrane action potential duration of the surviving ventricular myocytes, thus contributing to ventricular arrhythmias during acute ischemia-reperfusion period. Pretreatment with simvastatin could attenuate these changes and reverse this electrical remodeling without lowering the serum cholesterol level, contributing to the ionic mechanism of statin in treatment of arrhythmia independent of a decrease in cholesterol.

Animals ; Calcium Channels, L-Type ; drug effects ; Cholesterol ; blood ; Female ; Heart ; drug effects ; physiopathology ; Male ; Myocardial Ischemia ; physiopathology ; Myocardial Reperfusion Injury ; prevention & control ; Potassium Channels ; drug effects ; Rabbits ; Simvastatin ; pharmacology ; Sodium Channels ; drug effects

AIMTo study the effect of ginkgolide B from Ginkgo leave on action potential (AP), L-type calcium current (I(Ca) - L) and delayed rectifier potassium current (I(K)) in normal and ischemic guinea pig ventricular myocytes.

METHODSWith the standard microelectrode technique to record action potential and whole-cell variant patch-clamp technique to record calcium and potassium current.

RESULTS(1) Under normal condition, ginkgolide B shortened APD and had no effect on RP, AP and V(max). Ginkgolide B also increased I(K) in a concentration dependent manner and had no significant effect on I(Ca) - L (2) Under ischemia condition, it was observed that shortening of APD, APA, decrease V(max) and depolarization of RP was induced by ischemia, but ginkgolide B could attenuate above--mentioned changes. (3) Under ischemia condition, I(Ca) - L and I(K) were inhibited, perfusion with ischemia solution containing ginkgolide B could reverse the decrease of I(Ca) - L and I(K).

CONCLUSIONGinkgolide B had protective effect on ischemic myocardium to prevent ischemic arrhythmia.

Action Potentials ; drug effects ; Animals ; Calcium Channels, L-Type ; drug effects ; Delayed Rectifier Potassium Channels ; drug effects ; Ginkgolides ; pharmacology ; Guinea Pigs ; Heart Ventricles ; drug effects ; Lactones ; pharmacology ; Myocardial Ischemia ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Patch-Clamp Techniques

Neuropeptide Y (NPY) co-exists with norepinephrine (NE) in sympathetic terminals, and is the most abundant neuropeptide in myocardium. Many studies have focused on the effects of NE on ion channels in cardiac myocytes and its physiological significance has been elucidated relatively profoundly. There have been few investigations, however, on the physiological significance of NPY in myocardium. The effects of NPY on L-type Ca2+ channel currents (I(Ca-L)) were evaluated in some studies and different results were presented, which might be attributed to the different species of animal tested and different methods used. It is necessary, therefore, to study the effects of NPY on ion channels in cardiac myocytes systematically and further to discuss the biological significance of their coexistence with NE in sympathetic terminals. The single ventricular myocytes from adult rat or guinea pig (only for measuring I(K)) were prepared using enzymatic dispersion. I(Ca-L), I(to), I(Na/Ca), I(Na) and I(K) in the cellular membrane were observed using whole cell voltage-clamp recording. In the present study, NPY from 1.0 to 100 nmol/L dose-dependently inhibited I(Ca-L) (P<0.01, n=5). The maximal rate of inhibition in this study reached 39% and IC(50) was 1.86 nmol/L. NPY had no effect on the voltage-dependence of calcium current amplitude and on the voltage-dependence of the steady-state gating variables. I(Ca-L) was activated at -30 mV, reaching the maximum at 0 mV. When both NE and NPY were applied with a concentration ratio of 500:1, 10 nmol/L NPY inhibited I(Ca-L) that had been increased by 5 mumol/L NE, which was consistent with the effect of NPY only on I(Ca-L). NPY also inhibited I(Na/Ca). At a concentration of 10 nmol/L, NPY inhibited inward and outward I(Na/Ca) from (0.27+/-0.11) pA/pF and (0.45+/-0.12) pA/pF to (0.06+/-0.01) pA/pF and (0.27+/-0.09) pA/pF, respectively (P<0.05, n=4). NPY at 10 nmol/L increased I(to) from (12.5+/-0.70) pA/pF to (14.7+/-0.59) pA/pF(P<0.05, n=4). NPY at 10 nmol/L did not affect I(Na) in rat myocytes and I(K) in guinea pig myocytes. NPY increased the speed of action potential depolarization and reduced action potential duration of I(Ca-L), I(Na/Ca) and I(to), which contributed to the reduction of contraction. These results indicate that the effects of NPY are opposite to the effects of NE on ion channels of cardiac myocytes.
Animals ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; drug effects ; Female ; Guinea Pigs ; Heart Ventricles ; cytology ; Ion Channels ; drug effects ; Male ; Myocytes, Cardiac ; metabolism ; Neuropeptide Y ; pharmacology ; Norepinephrine ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Sodium-Calcium Exchanger ; antagonists & inhibitors

Using whole cell patch clamp, the effects of angiotensin II (Ang II) on the current L-type calcium channel (I(Ca.L)) were observed in guinea pig isolated ventricular myocytes under simulated ischemia condition, which was realized through hypoxia, glucose deficiency, high lactic acid and acidosis. The results showed that, under the condition of simulated ischemia, the peak of I(Ca.L) was reduced with maximal activation potential at 0 mV. Administration of Ang II (100 nmol/L) enhanced the peak of I(Ca.L) during ischemia and shifted the maximal activation potential to -10 mV. The possible mechanism of these effects is discussed.
Angiotensin II ; pharmacology ; Animals ; Calcium Channels, L-Type ; drug effects ; Cells, Cultured ; Guinea Pigs ; Heart Ventricles ; cytology ; Myocytes, Cardiac ; cytology ; drug effects ; Patch-Clamp Techniques

OBJECTIVETo investigate the effects of microRNA-133a on isoproterenol (ISO)-induced neonatal rat cardiomyocyte hypertrophy and related molecular mechanism focusing on the changes of L-type calcium channel α1C subunit.

METHODSNeonatal rat cardiomyocytes were cultured, cardiomyocyte hypertrophy was induced by isoproterenol (ISO, 10 µmol/L). The cell surface area was measured by phase contrast microscope and Leica image analysis system. The mRNA expressions of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), miR-133a and the α1C were detected by qRT-PCR. The protein expression of α1C was evaluated by Western blot. MiR-133a mimic was transfected into cardiomyocytes to investigate the effects of miR-133a on ISO-induced cardiomyocyte hypertrophy. The targets of miR-133a were predicted by online database Targetscan. The 3' untranslated region sequence of α1C was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells. The luciferase activities of samples were measured to verify the expression of luciferase reporter vector. The expression level of α1C was inhibited by RNAi to determine the effects of α1C on cardiomyocyte hypertrophy. Intracellular Ca(2+) content was measured by confocal laser microscope.

RESULTS(1) The expression of miR-133a was significantly reduced in ISO-induced cardiomyocyte hypertrophy (P < 0.01) . Upregulating miR-133a level could suppress the increase of cell surface area, the mRNA expression of ANP and β-MHC (P < 0.01) . (2) α1C was the one of potential target of miR-133a by prediction using online database Targetscan. The luciferase activities of HEK293 cells with the plasmid containing wide type α1C 3'UTR sequence were significantly decreased compared with control group (P < 0.01) . Upregulation of the miR-133a level by miR-133a mimic transfection could suppress the protein expression of α1C (P < 0.05) . (3) The expression of α1C was significantly increased in ISO treated cardiomyocytes (P < 0.05) . Downregulation of α1C by RNAi could markedly inhibit the increase of cell surface area, the mRNA expression of ANP and β-MHC (P < 0.01, P < 0.05, P < 0.05). (4) Downregulation of α1C expression by RNAi or upregulation of miR-133a level by miR-133a mimic transfection significantly inhibited intracellular Ca(2+) content (P < 0.01) .

CONCLUSIONSOur data confirms that α1C is the target of miR-133a. MiR-133a can negatively regulate the expression of L-type calcium α1C subunit, resulting in the decrease of intracellular Ca(2+) content and the attenuation of ISO-induced cardiomyocyte hypertrophy.

Animals ; Calcium Channels, L-Type ; metabolism ; Cell Enlargement ; drug effects ; Cells, Cultured ; Isoproterenol ; pharmacology ; MicroRNAs ; genetics ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Transfection