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

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

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

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

OBJECTIVETo investigate the relationship between the changes of the L-type calcium current (I(Ca, L)) and the calcium-activated transient outward chloride current (I(Cl, Ca)), and the repolarization characteristic of action potential in phase 1 under isoprenaline (ISO) stimulation in atrium myocytes of rabbit.

METHODSAtrium myocytes were obtained by enzymatic dissociation from a section of atrial free wall. The membrane currents and action potential were recorded by the whole-cell patch-clamp technique.

RESULTSAfter recording I(Ca, L), atrium myocytes were perfused with ISO (1 micromol/L) immediately. Five minutes later, a transient outward current (I(to)) was significantly induced, and the peak of I(to) was gradually increased while I(Ca, L) gradually decreased with increasing in clamp voltage. The I(to) was resistant to 4-AP (3 mmol/L) but sensitive to DIDS (150 micromol/L, Cl(-) channel blocker). This current was blocked by CdCl(2) (200 micromol/L, Ca(2+) channel blocker). The elicited rate of I(to) was 91.67% (P < 0.05). (2) The shape of AP was like an inverse triangle with no plateau in Phase 2 after ISO (1 micromol/L) perfusion. Moreover, compared to the parameters of control group, APD(50) and APD(90) were significantly shortened from (65.4 +/- 4.2) ms and (95.8 +/- 3.8) ms to (12.8 +/- 3.8) ms and (27.0 +/- 4.7) ms, and reduced to 80.46% and 71.87%, respectively (P < 0.01, n = 12). 4-AP (3 mmol/L) had on obvious effect on the shape of AP, however, the plateau of AP in phase 2 was recovered by DIDS (150 micromol/L) perfusion, APD(50) and APD(90) were (41.1 +/- 4.5) ms and (79.6 +/- 3.4) ms respectively. Compared to the parameters of control group, there were no significant differences (P > 0.05, n = 12). These results indicated that ionic transport were changed by ISO perfusion in atrium myocytes and I(to) played an important role in the phase 1 repolarization of AP.

CONCLUSIONSBefore ISO administration, we could only observe I(Ca, L) in atrium myocytes of rabbit. After isoproterenol intervention, certain intracellular ionic consistency and membrane ionic channels were changed. Calcium activated chloride channel and I(to2) revealed obvious predominance which shorten APD significantly. Action potential showed a triangle with no plateau, suggesting an electrical remodeling in atrium myocytes. The remodeling of ionic channel is related possibly with the opening of Ca(2+)-activated Cl(-) current, which maybe the electrophysiological base of reentrant atrial tachycardia.

Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; metabolism ; Calcium Signaling ; Cells, Cultured ; Chloride Channels ; metabolism ; Heart Atria ; cytology ; metabolism ; Ion Transport ; Isoproterenol ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; Patch-Clamp Techniques ; Rabbits

The aim of the present study was to investigate the effects of quercetin on colon contractility and voltage-dependent Ca(2+) channels in the single smooth muscle cell isolated from the proximal colon of guinea-pig and to clarify whether its effect on L-type Ca(2+) current (I(Ca,L)) would be related to its myorelaxing properties. Colon smooth muscle strips were used to take contractile tension recordings. Smooth muscle cells were freshly isolated from the proximal colon of guinea-pig by means of papain treatment. I(Ba,L) (barium instead of calcium as current carrier) was measured by using whole-cell patch-clamp techniques. The results showed that quercetin relaxed colon muscle strips in a concentration-dependent manner and antagonized the contractile effect of acetylcholine and neostigmine. Preincubation with indomethcin [cyclooxygenase (COX) inhibitor] and methylene blue [guanylate cyclase (GC) inhibitor] significantly attenuated the relaxing effect of quercetin, respectively. Quercetin increased I(Ba,L) in a concentration- [EC(50)= (7.59+/-0.38) mumol/L] and voltage-dependent pattern, and shifted the maximum of the current-voltage curve by 10 mV in the depolarizing direction without modifying the threshold potential for Ca(2+) influx. Quercetin shifted the steady-state inactivation curve toward more positive potentials by approximately 3.75 mV without affecting the slope of activation and inactivation curve. H-89 (PKA inhibitor) abolished quercetin-induced I(Ba,L) increase, while cAMP enhanced the quercetin-induced I(Ba,L) increase. The patch-clamp results proved that quercetin increased I(Ba,L) via PKA pathway. It is therefore suggested that the relaxing effect of quercetin attributes to the interaction of GC and COX stimulation, as well as the antagonism effect on acetylcholine, which hierarchically prevails over the increase in the Ca(2+) influx to be expected from I(Ca,L) stimulation.
Animals ; Calcium Channels, L-Type ; metabolism ; Cells, Cultured ; Colon ; drug effects ; Guinea Pigs ; Muscle Contraction ; Myocytes, Smooth Muscle ; drug effects ; Patch-Clamp Techniques ; Quercetin ; pharmacology

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

The effects of adrenomedullin (ADM) on the L-type calcium currents (I(Ca,L)) and the mechanism of the signal transduction process were studied. Enzymatically isolated guinea-pig ventricular myocytes were used to measure ICa,L with whole-cell patch-clamp techniques. ADM at the concentrations of 1-100 nmol/L decreased ICa,L in a dose-dependent manner (P<0.05). ADM22-52) (100 nmol/L), a specific ADM-receptor antagonist, completely abolished the ADM-induced inhibition of ICa,L. Pretreatment of the cells with H-89 (10 micromol/L), a specific PKA inhibitor, did not attenuate the effects of ADM. Intracellular application of 10 micromol/L PKC19-36), a specific PKC inhibitor, prevented the ADM-induced inhibition of the ICa,L, while the specific PKC activator PMA could mimic the effects of ADM on the ICa,L. PMA (1 micromol/L) decreased the ICa,L by 32.26+/-4.20%(P<0.05). These findings indicate that ADM can inhibit the ICa,L in guinea-pig ventricular myocytes, and the inhibition is mediated by the specific ADM-receptor and an activation of protein kinase C.
Adrenomedullin ; pharmacology ; Animals ; Calcium Channels, L-Type ; metabolism ; Guinea Pigs ; Heart Ventricles ; cytology ; Myocytes, Cardiac ; drug effects ; metabolism ; Patch-Clamp Techniques ; Protein Kinase C ; metabolism

OBJECTIVETo investigate cardiac effects of arsenic trioxide (As(2)O(3)) at conventional dosage in acute promyelocytic leukemia (APL) patients.

METHODSThe basical heart rate, electrocardiograph, plasma As(2)O(3) concentration of APL patients were dynamically monitored. The action potential duration and current of I(Ca-L) in guinea pig cardiac ventricular myocytes were assayed by patch clamp technique, and the elevated cytosolic [Ca(2+)]i of guinea pig ventricular myocytes induced by As(2)O(3) by laser confocal microscopy.

RESULTSApproximately 52.5% - 35% of 40 APL patients manifested poor cardiac effects of different degree when As(2)O(3) intravenous infused at conventional doses in the initial 1 or 2 weeks with fast heart rate or prolonged QT interval. As(2)O(3) at concentration of 1, 2, 5 micro mol/L prolonged action potential duration from (563.0 +/- 55.8) ms to (737.7 +/- 131.7), (842.4 +/- 115.6) and (1103.2 +/- 96.3) ms respectively (P < 0.05, P < 0.01, P < 0.01), and increased I(Ca-L) of guinea pig cardiac ventricular myocytes as well as the respectively cytosolic [Ca(2+)]i. Calcium channel blocking agent can cut-out the effect.

CONCLUSIONAs(2)O(3) intravenous infusion at conventional doses can cause tachycardia and prolong QT interval. The probable mechanism might be that As(2)O(3) affects the ion channels and cytosolic calcium.

Adult ; Animals ; Antineoplastic Agents ; adverse effects ; Arsenicals ; adverse effects ; blood ; Calcium ; metabolism ; Calcium Channels, L-Type ; drug effects ; Cricetinae ; Electrocardiography ; drug effects ; Female ; Heart ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; Male ; Oxides ; adverse effects ; blood