Effects of tetrandrine on calcium and potassium currents in isolated rat hepatocytes

AIM: To study the effects of tetrandrine (Tet) on calciumrelease-activated calcium current (ICRAC), delayed rectifierpotassium current (IK), and inward rectifier potassiumcurrents (IK1) in isolated rat hepatocytes.METHODS: Hepatocytes of rat were isolated by usingperfusion method. Whole cell patch-clamp techniques wereused in our experiment.RESULTS: The peak amplitude.of ICRAC was -508±115 pA(n＝15), its reversal potential of ICRAC was about 0 mV. At thepotential of -100 mV, Tet inhibited the peak amplitude ofICRAC from -521±95 pA to -338±85 pA (P＜0.01 vs control,n＝5), with the inhibitory rate of 35 % at 10 μmol/L andfrom -504±87 pA to -247±82 pA (P＜0.01 vscontrol, n＝5),with the inhibitory rate of 49 % at 100 μmol/L, withoutaffecting its reversal potential. The amplitude of ICRAC wasdependent on extracellular Ca2+ concentration. The peakamplitude of ICRAC was -205±105 pA (n＝3) in tyrode's solutionwith Ca2+ 1.8 mmol/L (P＜0.01 vs the peak amplitude ofICRAC in external solution with Ca2+ 10 mmol/L). Tet at theconcentration of 10 and 100 μmol/L did not markedly changethe peak amplitude of delayed rectifier potassium currentand inward rectifier potassium current (P＞0.05 vs control).CONCLUSION: Tet protects hepatocytes by inhibiting ICRAC,which is not related to I K and IK1.     

Inhibitory effects of berberine on ion channels of rat hepatocytes

AIM: To examine the effects of berberine, an isoquinoline alkaloid with a long history used as a tonic remedy for liver and heart, on ion channels of isolated rat hepatocytes.METHODS: Tight-seal whole-cell patch-clamp techniques were performed to investigate the effects of berberine on the delayed outward potassium currents (Ik), inward rectifier potassium currents (Ik1) and Ca^2+ release-activated Ca^+ currents (ICRAC) in enzymatically isolated rat hepatocytes.RESULTS: Berbenne 1-300 μmol/L reduced/K in a concentration-dependent manner with EC50 of 38.86=1=5.37 μmol/L and nH of 0.82±0.05 (n = 8). When the bath solution was changed to tetraethylammonium (TEA) 8 retool/L,IK was inhibited.Berberine 30 μmol/L reduced/K at all examined membranepote ntials, especially at potentials positive to +60 mV (n = 8,P<0.05 or P<0.01 vs control). Berberine had mild inhibitory effects on IK1 in rat hepatocytes. Berberine 1-300 μmol/L also inhibited ICRAC in a concentration-dependent fashion. The fitting parameters were EC50 = 47.20±10.86 μmol/L,nH= 0.71±0.09 (n = 8). The peak value of/CRAC in the I-Vrelationship was decreased by berberine 30 μmol/L at potentialnegative to -80 mV (n = 8, P<0.05 vscontrol). But the reverse potential of/CRAC occurred at voltage 0 mV in all cells.CONCLUSION: Berberine has inhibitory effects on potassium and calcium currents in isolated rat hepatocytes, which may be involved in hepatoprotection.     

Electrophysiological effects of anthopleurin-Q on rat hepatocytes

AIM: To study the effects of AP-Q on CCl4-induced acute liver injury, delayed outward potassium current (Iκ), inward rectifier potassium current (Iκ1) and calcium release-activated calcium current (ICRAC) in isolated rat hepatocytes. METHODS: A single dose of CCl4 (10 μg/mL, ip) was injected to induce acute liver injury in rats. Serum aminotransferase activities were determined. Whole cell patch-clamp techniques were used to investigate the effects of AP-Q on delayed outward potassium current (Iκ), inward rectifier potassium current (IKI) and calcium release-activated calcium current (ICRAC). RESULTS: AP-Q (3.5 and 7 μg/kg) pretreatment significantly reduced ALT and AST activities. AP-Q 0.1-100 nM produced a concentration-dependent increase of Iκ with EC50 value of 5.55±1.8 nM (n=6). AP-Q 30 nM shifted the I-V curve of Iκ leftward and upward. CCl4 4 mM decreased Iκ current 28.6±0.5% at 140 mV. After exposure to CCl4 for 5 rain, AP-Q 30 nM attenuated the decrease of Iκ induced by CCl4 close to normal amplitude. AP-Q 0.01-100 nM had no significant effect on either inward or outward components of Iκ1 at any membrane potential examined. AP-Q 0.1-100 nM had no significant influence on the peak amplitude of ICRAC, either,and did not affect the shape of its current voltage curve. CONCLUSION: AP-Q has a protective effect on CCl4-induced liver injury, probably through selectively increased Iκ in hepatocytes.     

The slow component of the delayed rectifier potassium current in undiseased human ventricular myocytes

OBJECTIVE: The purpose of this study was to investigate the properties of the slow component of the delayed rectifier potassium current (I(Ks)) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 58 left ventricular myocytes from 15 hearts at 37 degrees C. Nisoldipine (1 microM) was used to block inward calcium current (I(Ca)) and E-4031 (1-5 microM) was applied to inhibit the rapid component of the delayed rectifier potassium current (I(Kr)). RESULTS: In 31 myocytes, an E-4031 insensitive, but L-735,821 and chromanol 293B sensitive, tail current was identified which was attributed to the slow component of I(K) (I(Ks)). Activation of I(Ks) was slow (tau=903+/-101 ms at 50 mV, n=14), but deactivation of the current was relatively rapid (tau=122.4+/-11.7 ms at -40 mV, n=19). The activation of I(Ks) was voltage independent but its deactivation showed clear voltage dependence. The deactivation was faster at negative voltages (about 100 ms at -50 mV) and slower at depolarized potentials (about 300 ms at 0 mV). In six cells, the reversal potential was -81.6+/-2.8 mV on an average which is close to the K(+) equilibrium potential suggesting K(+) as the main charge carrier. CONCLUSION: In undiseased human ventricular myocytes, I(Ks) exhibits slow activation and fast deactivation kinetics. Therefore, in humans I(Ks) differs from that reported in guinea pig, and it best resembles I(Ks) described in dog and rabbit ventricular myocytes.     

Decreased ATP-sensitive K(+) current density during chronic human atrial fibrillation

Chronic atrial fibrillation (AF) is associated with shortening of action potential duration (APD), which involves modified activity of atrial ion currents. However, little is known about the activity of ATP-sensitive K(+) channels (I(K,ATP)) during chronic AF. An AF-related increase in the activity of I(K,ATP) would reduce APD and could contribute to initiation and/or perpetuation of AF. Here, we studied the activity of I(K,ATP) in atrial myocytes from patients with sinus rhythm (SR) and chronic AF. Human atrial myocytes were isolated from atrial tissue obtained from patients undergoing open-heart surgery. Inward rectifier currents were measured with the whole-cell patch-clamp technique by applying a depolarizing ramp pulse (1245 ms) from -100 to +40 mV (0.5 Hz). I(K,ATP) was activated with the I(K,ATP) channel opener rilmakalim. The inward rectifier I(K1) and I(K,ATP) were identified by their sensitivity to 1 mM Ba(2+). Density of I(K1) did not differ between cells from patients with AF (at -100 mV: -14.8 +/- 1.3 pA/pF, n = 38/10 (cells/patients)) and SR (-13.8 +/- 1.5 pA/pF, n = 33/16). In both types of cells, rilmakalim stimulated I(K,ATP) (defined as rilmakalim-inducible current) in a concentration-dependent manner (0.3-10 microM). However, maximum activation of I(K,ATP) with 10 microM rilmakalim was smaller in AF than in SR cells (at -100 mV: -5.3 +/- 0.8 pA/pF, n = 22/7 vs. -11.2 +/- 2.9 pA/pF, n = 19/9; at +40 mV: +9.6 +/- 2.1 pA/pF, n = 22/7 vs. +23.7 +/- 3.4 pA/pF, n = 19/9 for AF and SR, respectively; P < 0.05). Only aortic valve disease and pulmonary hypertension were found to be independent contributors to I(K,ATP) current density. We provide evidence that chronic AF is associated with a downregulation of ATP-sensitive K(+) currents. These changes may provide an additional molecular mechanism for electrical remodeling in chronic AF.     

Effect of actin microfilament on potassium current in guinea pig gastric myocytes

AIM: To investigate the effect of actin microfilament on potassium current and hyposmotic membrane stretch-induced increase of potassium current in gastric antral circular myocytes of guinea pig. METHODS: Whole-cell patch clamp technique was used to record potassium current in isolated gastric myocyes. RESULTS: When the membrane potential was clamped at -60mV, an actin microfilament disruptor, cytochanlasin-B (Cyt-B, 20μmol/L in pipette) increased calcium-activated potassium current (IK（Ca）) and delayed rectifier potassium current (IK（V）)to 138.4&#177;14.3% and 142.1&#177;13.1%respectively at +60mV. In the same condition, an actin microfilament stabilizer phalloidin (20μmol/L in pipette)inhibited IK（Ca） and IK（V） to 74.2&#177;7.1% and 75.4&#177;9.9% respectively. At the holding potential of -60mV, hyposmotic membrane stretch increased IK（Ca） and IK（V） by 50.6&#177;9.7% and 24.9&#177;3.3% at +60mV respectively. In the presence of cytochalasin-B and phalloidin (20μmol/L, in the pipette)condition, hyposmotic membrane stretch also increased IK（Ca） by 44.5&#177;7.9% and 55.7&#177;9.8% at +60mV respectively. In the same condition, cytochalasin-B and phalloidin also increased IK（V） by 23.0&#177;5.5% and 30.3&#177;4.5% respectively. However, Cyt-B and phalloidin did not affect the amplitude of hyposmotic membrane stretch-induced increase of IK（Ca） and IK（V）. CONCLUSION: Actin microfilaments regulate the activities of potassium channels, but they are not involved in the process of hyposmotic membrane stretch-induced increase of potassium currents in gastric antral circular myocytes of guinea pig.     

An inward rectifier and a voltage-dependent K+ current in single, cultured pericytes from bovine heart

OBJECTIVE: The purpose of this study was to describe passive electrical properties and major membrane currents in coronary pericytes. METHODS: 78 single, cultured bovine pericytes were studied with the patch-clamp technique in the whole-cell mode. RESULTS: The membrane potential of the cells was -48.9+/-9.6 mV (mean+/-S.D.) with 5 mM and -23.2+/-2.2 mV with 60 mM extracellular K+. The membrane capacitance was 150.2+/-123.2 pF. The current-voltage relation of the pericytes was dominated by an inward current at hyperpolarized potentials and an outward current at depolarized potentials. Increasing extracellular K+ from 5 to 60 mM led to an increase of the inward current and to a shift of this current to more depolarized potentials. The inward current was very sensitive to extracellular barium (50 microM). The maximum slope conductance of the cells at hyperpolarized potentials was 2.9+/-2.8 nS. Inward rectification of whole-cell currents was steep (slope factor = 6.8 mV). With elevated external K+ the outward current reversed near the potassium equilibrium potential. Onset of the outward current was sigmoid and inactivation of this current was monoexponential, slow (time constant = 12.8 s) and incomplete. Voltage-dependence of outward current steady-state activation was steep (slope factor = 4.6 mV). The outward current was very sensitive to 4-aminopyridine (dissociation constant = 0.1 mM). The maximum slope conductance at depolarized potentials was 16.6+/-15.6 nS. CONCLUSION: We report for the first time, patch-clamp recordings from coronary pericytes. An inward rectifier and a voltage-dependent K+ current were identified and characterized. Regulation of these currents may influence coronary blood flow.     

溴苄基四氢小檗碱对豚鼠心室肌离子通道的影响

探讨溴苄基四氢小檗碱 (CPU86 0 35 )对豚鼠心室肌细胞钾离子通道及L 型钙离子通道的作用 ,探讨其抗心律失常的离子通道机制。取 2 0只健康豚鼠 ,雌雄不拘 ,体重 2 5 0～ 30 0g ,双酶法酶解获取单个豚鼠心室肌细胞 ,采取用药前后自身对照及全细胞膜片钳记录方法观察CPU86 0 35对正常豚鼠心室肌细胞延迟整流钾离子流 (IK)、内向整流钾离子流 (IK1)及L 型钙离子流 (ICa L)的影响。结果 :CPU86 0 35对豚鼠单个心室肌细胞IK 呈浓度依赖性抑制作用 ,半数抑制浓度 (ID50 )为 5 6 μmol/L ;CPU86 0 35对IK1无明显影响。CPU86 0 35呈剂量依赖性抑制ICa L,ID50 为 6 5μmol/L。CPU86 0 35对ICa L的抑制依赖于保持电位 (HP) ,HP =- 4 0mV和 - 80mV时 ,75 μmol/LCPU86 0 35对ICa L的抑制率分别为 0 .4 92 2± 0 .0 2 1和 0 .6 377± 0 .0 36 6。结论 :CPU86 0 35对正常豚鼠心室肌细胞IK、及ICa L均有阻断作用 ,这种多离子通道的阻断剂可有效抗快速心律失常 ,并且不会引起动作电位时程和有效不应期的过度延长 ,从而减少药物的致心律失常作用。     

Pacing-induced heart failure causes a reduction of delayed rectifier potassium currents along with decreases in calcium and transient outward currents in rabbit ventricle

OBJECTIVE: Heart failure in patients and in animal models is associated with action potential prolongation of the ventricular myocytes. Changes in several membrane currents have been already demonstrated to underlie this prolongation. However, information on the two components (I(Kr) and I(Ks)) of the delayed rectifier potassium current (I(K)) in rapid pacing induced heart failure is lacking. METHODS AND RESULTS: Action potentials and whole-cell currents, I(K), I(to1), I(K1), and I(Ca-L) were recorded in apical myocytes of left ventricle from 10 rabbits subjected to left ventricular pacing at 350-380 beats/min for 3-4 weeks and 10 controls with sham operation. Action potential duration at 90% repolarization (APD(90)) was prolonged in myocytes from failing hearts compared to controls at both cycle lengths of 333 and 1000 ms. Both E-4031-sensitive and -resistant components of I(K) (I(Kr), I(Ks)) in myocytes from failing hearts were significantly less than those of control hearts; tail current densities of I(Kr) and I(Ks) following depolarization to +50 mV were 0.62+/-0.05 vs. 0.96+/-0.12 pA/pF (P<0.05), and 0.27+/-0.08 vs. 0.52+/-0.08 pA/pF (P<0.05), respectively. There was no significant difference between control and failing myocytes in the voltage- and time-dependence of activation of total I(K), I(Kr) and I(Ks). The peak of L-type Ca(2+) current (I(Ca-L)) was significantly reduced in myocytes from failing hearts (at +10 mV, -9.29+/-0.52 vs. -12.28+/-1.63 pA/pF, P<0.05), as was the Ca(2+)-independent transient outward current (I(to1); at +40 mV, 4.8+/-0.9 vs. 9.6+/-1.3 pA/pF, P<0.05). Steady state I-V curve for I(K1) was similar in myocytes from failing and control hearts. CONCLUSIONS: Decrease of I(K) (both I(Kr) and I(Ks)) in addition to reduced I(to1), may underly action potential prolongation at physiological cycle length and thereby contribute to arrhythmogenesis in heart failure.     

Inhibition of ultra-rapid delayed rectifier K+ current by verapamil in human atrial myocytes

Verapamil is a widely used Ca(2+) channel antagonist in the treatment of cardiovascular disorders including atrial arrhythmias. However, it is unknown whether the drug would inhibit the repolarization currents transient outward K(+) current (I(to1)) and ultra-rapid delayed rectifier K(+) current (I(Kur)) in human atrium. With whole-cell patch configuration, we evaluated effects of verapamil on I(to1) and I(Kur) in isolated human atrial myocytes. It was found that verapamil did not decrease I(to1) at 1-50 microM. However, verapamil reversibly inhibited I(Kur) in a concentration-dependent manner (IC(50) = 3.2 microM). At test potential of +50 mV, 5 microM verapamil decreased I(Kur) by 61.3 +/- 7.5%. Verapamil significantly accelerated inactivation of I(Kur), suggesting an open channel block mechanism. The results indicate that verapamil significantly blocks the repolarization K(+) current I(Kur), but not I(to1), in human atrial atrium, which may account at least in part for the atrial effect of the drug.     

Effects of trimetazidine on action potentials and membrane currents of guinea-pig ventricular myocytes

The effects of trimetazidine on membrane potentials and membrane currents of enzymatically isolated guinea-pig ventricular cells were studied with the use of giga-seal suction pipettes for patch clamp. Trimetazidine (3 X 10(-5) M) decreased the action potential duration from 433 +/- 179 ms (mean and S.D., n = 9) to 319 +/- 156 ms within 8 mins. In voltage clamp experiment, trimetazidine at a concentration of 1.5 X 10(-4) M decreased the peak amplitude of calcium current by 40% (0.92 +/- 0.46 nA to 0.55 +/- 0.19 nA, mean +/- S.D., n = 5). The effect on calcium current was rate-dependent, e.g., at 1 Hz, trimetazidine blocked a larger fraction of the calcium current than at 0.2 Hz. The drug decreased the conductance of potassium current which flows via inward rectifier potassium channel from 28 +/- 11 nS to 19 +/- 10 nS, n = 5, P less than 0.05). Trimetazidine shifted the steady state current-voltage relationship outward at potentials positive to -20 mV. This shift was not due to the enhanced time- and voltage-dependent outward current (Ik). From these findings, it was concluded that trimetazidine shortens action potential duration by blocking the calcium channels with increases in steady state outward current or a possible blockade of non-inactivated component of the calcium current, at the plateau potentials. The reduction of calcium current and of inward rectifier potassium current may protect the cardiac cells from accumulation of calcium ions and from loss of potassium ions, in the presence of ischemia.     

Ionic mechanisms of cardiotropic action of a new class III antiarrhythmic drug RG-2

A perforated patch-clamp analysis of the effect of a novel class III antiarrhythmic agent RG-2, on voltage-dependent currents was made in rat ventricular myocytes. In these cells, RG-2 decreased delayed rectifier outward K(+) current, I(k), in concentration dependent manner with threshold concentration 0.1 microM/l. In contrast, the drug did not have significant effects on the transient outward and inward rectifier K(+) current. RG-2 in concentration dependent manner decreased Ca(2+) current (I(Ca,L)) with threshold concentration 1 microM/l, tenfold higher than threshold concentration for I(k). We can conclude that decreasing of I(k) may explain prolongation of cardiac repolarization induced by RG-2, and contribute to its antiarrhythmic action.     

Indapamide blocks the rapid component of the delayed rectifier current in atrial tumor cells (AT-1 cells)

We studied the effects of a well known blocker (indapamide) of the slow component (I(ks)) of the delayed rectifier (I(k)) on K(+) currents in atrial tumor myocytes derived from transgenic mice (AT-1 cells) using one electrode voltage clamp method. These cells have been shown to express mRNAs encoding cardiac K(+) channels and display a cardiac electrophysiological phenotype. The major K(+) current is the rapid component (I(kr)) of the delayed rectifier current (I(k)). The purpose of this study was to show that a diuretic agent, indapamide, which was shown to be a selective blocker of the slow component (I(ks)) of delayed rectifier, also blocks I(kr) in a dose dependent manner. The steady state current at the end of a 1s pulse (I(1s), step to +40 mV from a holding potential of -40 mV) was 1070.4+/-202.2 pA (n=5) and the tail current (I(tail)) was 416.3+/-112.9 pA. Indapamide (750 microM) reduced I(1s) and I(tail) to 254.5+/-62.3 pA and 42.2+/-37.7 pA respectively. Indapamide induced block was partially reversible for higher concentrations (> or =750 microM).     

Effects of chromanol 293B on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes

It is unclear whether chromanol 293B, a selective inhibitor of slow component of delayed rectifier K(+) current (I(Ks)), may affect other K(+) currents in human atrium. With whole-cell patch configuration, we evaluated effects of 293B on transient outward K(+) current (I(to1)) and ultra-rapid delayed rectifier K(+) current (I(Kur)) in isolated human atrial myocytes. It was found that 293B inhibited I(to1) and I(Kur) in a concentration-dependent manner. At 10 microM 293B suppressed I(to1) to 3.4 +/- 0.4 from 5.1 +/- 0.3 pA/pF (P < 0.01), and I(Kur) to 1.5 +/- 0.2 from 2.1 +/- 0.3 pA/pF (P < 0.01) at +50 mV. The inhibition of I(to1) and I(Kur) was independent of depolarizing voltage, and the concentration of 50% inhibition was 31.2 microM for I(to1), and 30.9 microM for I(Kur). 293B blocked I(to1) and I(Kur) with the same concentration range, and the significant effect was observed from the concentration of 1 microM. The maximum inhibitive effect was 88% for I(to1) and 96% for I(Kur) at 250 microM. Voltage dependence of activation and inactivation, and time-dependent recovery from inactivation of I(to1) were not altered by 293B; however, time to peak and time-dependent inactivation of I(to1) was significantly accelerated. The results indicate that 293B significantly inhibits the major repolarization K(+) currents I(to1) and I(Kur) in human atrial myocytes.     

Protein kinase C activation enhances the delayed rectifier potassium current in guinea-pig heart cells

The possible involvement of protein kinase C in modulating membrane currents was investigated in isolated guinea-pig ventricular cells. In a Na(+)-and K(+)-free external solution, the delayed rectifier K+ current (IK) was increased by the activator of protein kinase C (PKC), 12-O-tetradecanoylphorbol-13-acetate (TPA). The amplitude of the IK tail elicited by a return from a depolarizing pulse for 3 s at + 50 mV to a holding potential of -30 mV was increased by 32 +/- 4% (mean +/- S.E., (n = 6) after the external application of 1 nM TPA, and by 60 +/- 17% (n = 5) after 10 nM. The increase in IK produced by 1 nM TPA was abolished by the inhibitor of PKC, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 microM). In addition, the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG, 125 microM) also increased IK (58 +/- 9%, n = 3). PKC purified from bovine brain remarkably increased IK (151 +/- 101%, n = 5) in the presence of 1 nM TPA when it was internally applied using the cell dialysis method. The concentration-response curve of IK for the intracellular concentration of Ca2+ was shifted to the left by 1 nM TPA, suggesting a Ca2(+)-dependent action of PKC and/or altered Ca2(+)-sensitivity of IK channels by phosphorylation. On the other hand, 1 nM TPA had no substantial influence on the Ca2+ current (decreased by 7 +/- 4%, n = 5) or the inward-rectifier K+ current (decreased by 5 +/- 5% in outward component, and 3 +/- 8% in inward component, n = 6). Therefore, the action of PKC was to specifically increase IK without affecting the other two currents.     

Evidence for a differential cellular distribution of inward rectifier K channels in the rat isolated mesenteric artery

The distribution of functionally active, inwardly rectifying K (K(IR)) channels was investigated in the rat small mesenteric artery using both freshly isolated smooth muscle and endothelial cells and small arterial segments. In Ca(2+)-free solution, endothelial cells displayed a K(IR) current with a maximum amplitude of 190 +/- 16 pA at -150 mV and sensitivity to block with 30 microM Ba(2+) (n = 7). In smooth muscle cells, outward K current was activated at around -47 +/- 3 mV, but there was no evidence of K(IR) current (n = 6). Furthermore, raising extracellular [K(+)] to either 60 or 140 mM, or applying the alpha(1)-adrenoceptor agonist phenylephrine (PE; 30 microM), failed to reveal an inwardly rectifying current in the smooth muscle cells, although PE did stimulate an iberiotoxin-sensitive outward K current (n = 4). Exogenous K(+) (10.8-16.8 mM) both relaxed and repolarized endothelium-denuded segments of the mesenteric artery contracted with PE. These effects were depressed by 100 microM ouabain but unaffected by either 30 microM BaCl(2) or 3 microM glibenclamide. These data suggest that functional, inwardly rectifying Ba(2+)-sensitive channels are restricted to the endothelial cell layer in the rat small mesenteric artery.     

Demonstration of calcium-activated transient outward chloride current and delayed rectifier potassium currents in Swine atrial myocytes

Cellular electrophysiology is not fully understood in the atrium of pig heart. The objective of the present study was to determine whether transient outward current (I(to)), ultra-rapid delayed rectifier potassium current (I(Kur)), and rapid and slow delayed rectifier K(+) currents (I(Kr) and I(Ks)) were present in pig atrium. The whole-cell patch technique was applied to record membrane currents and action potentials in myocytes isolated from pig atrium. It was found that an I(to) was activated upon depolarization voltage steps to between -10 and +60 mV from -50 mV in pig atrial cells, and the I(to) was sensitive to the inhibition by the blockade of L-type calcium (Ca(2+)) current, showed a "bell-shaped" I-V relationship, typical of I(to2) (i.e. I(Cl.Ca)). The I(to2) was inhibited by the chloride (Cl(-)) channel blocker anthracene-9-carboxylic acid (9-AC, 200 micromol/l) or 4,4'-diisothiocyanostilben-2,2'disulfonic acid (200 micromol/l), and by Cl(-) substitution in the superfusate. I(Kur) was found in pig atrial myocytes, and the current showed properties of weak inward rectification and use- and frequency-dependent reduction. I(Kur) was resistant to tetraethylammonium, but sensitive to inhibition by 4-aminopyridine (4-AP) (IC(50) = 71.7 +/- 3.5 micromol/l). In addition, E-4031-sensitive I(Kr) and chromanol 293B-sensitive I(Ks) were observed in pig atrial myocytes. Blockade of I(to2), I(Kur), I(Kr) or I(Ks) with corresponding blockers significantly prolonged atrial action potentials. These results indicate that Ca(2+)-activated I(to2), 4-AP-sensitive I(Kur), E-4031-sensitive I(Kr), and 293B-sensitive I(Ks) are present in pig atrial myocytes, and these currents play important roles in action potential repolarization of pig atria.     

Functional expression of the Na+/Ca2+ exchanger in the embryonic mouse heart

The Na(+)/Ca(2+) exchanger (NCX) is one of the earliest functional genes and is currently assumed to compensate at least in part for the rudimentary sarcoplasmic reticulum in the developing mouse heart. However, to date little is known about the functional expression of NCX during development. This prompted us to investigate the NCX current (I(NCX)) in very early (embryonic day E8.5-E9.5 post coitum), early (E10.5-E11.5), middle (E13.5) and late (E16.5) stage mouse embryonic cardiomyocytes. For standard I(NCX) measurements, [Ca(2+)](i) was buffered to 150 nmol/l and voltage ramps were applied from +60 mV to -120 mV. At very early stages of development, we observed a prominent role of the I(NCX) Ca(2+) inward mode in elevating the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Accordingly, a high I(NCX) density was observed (+60 mV: 4.6+/-0.7 pA/pF, n=14). Likewise, we found a strong Ca(2+) outward mode of I(NCX) (-120 mV: -3.9+/-0.7 pA/pF, n=14). At later stages, however, I(NCX) Ca(2+) inward mode was reduced by 54+/-6% (n=15, p<0.0001) in ventricular and 68+/-10% (n=9, p<0.0006) in atrial cells. For the outward mode, a reduction by 43+/-10% (n=15, p<0.01) in ventricular and 62+/-11% (n=9, p<0.004) in atrial cardiomyocytes was observed. By contrast, NCX isoform expression and the reversal potential did not significantly change during development. Thus, NCX displays a prominent Ca(2+) inward and outward mode during early embryonic heart development pointing to its important contribution to maintain [Ca(2+)](i) homeostasis. The functional and protein expression of NCX declines during further development.     

Role of the 293b-sensitive, slowly activating delayed rectifier potassium current, i(Ks), in pacemaker activity of rabbit isolated sino-atrial node cells.

OBJECTIVES: (i) to characterize the electrophysiological properties of the slowly activating delayed rectifier potassium current, i(Ks), defined as the 293b-sensitive current, during the action potential (AP) of rabbit sino-atrial node (SAN) pacemaker cells; (ii) to evaluate the contribution of i(Ks) to the pacemaker AP under physiological conditions and during beta-adrenergic stimulation. METHODS: Rabbit SAN pacemaker cells were studied using the perforated patch clamp technique in voltage-, AP- and current-clamp modes. RESULTS: Voltage-clamp findings. Block of i(Ks) by 293b is dose-dependent, with an IC(50) (half block) in rabbit SAN cells of 1.35 microM and an IC(80) (sub-maximal block) of 5 microM. Sub-maximal concentrations of 293b have no significant effects on long-lasting and transient inward calcium currents, i(Ca,L) and i(Ca,T), inward hyperpolarization activated current, i(f), and transient outward current, i(to). AP-clamp experiments. The 293b-sensitive current activates near the peak of the SAN pacemaker action potential, reaches a mean maximal current density of 1.0+/-0.3 pA/pF (n=8, cell capacitances 27 to 62 pF, mean 35+/-4.0 pF) during late repolarization, and inactivates towards the end of repolarization. Additionally, in two smaller cells (cell capacitances 15 and 23 pF), no discernible 293b-sensitive current component was detected. Current-clamp data. In spontaneously beating SAN cells under control conditions, sub-maximal block of i(Ks) by 5 microM 293b has negligible effects on action potential characteristics and does not change average cycle length (n=11). In contrast, after pre-treatment with 10 nM isoprenaline to mimic beta-adrenergic stimulation, cells showed a 293b-induced depolarization of maximum diastolic potential by 2.2+/-1%, a decrease in diastolic depolarization rate by 9.9+/-4%, and a slowing of late action potential repolarization by 28.7+/-10.2%, resulting in a prolongation of spontaneous cycle length by 9.8+/-3.0% (P<0.05, n=10; for all parameters). CONCLUSION: Our findings suggest that in rabbit SAN: (i) i(Ks) is activated during the normal pacemaker AP; (ii) the contribution of i(Ks) to beating rate is small under control conditions; and (iii) i(Ks) contributes significantly to spontaneous pacemaker rate during beta-adrenergic stimulation.     

Voltage-gated currents of tilapia prolactin cells

The first recordings of neuron-like electrical activity from endocrine cells were made from fish pituitary cells. However, patch-clamping studies have predominantly utilized mammalian preparations. This study used whole-cell patch-clamping to characterize voltage-gated ionic currents of anterior pituitary cells of Oreochromis mossambicus in primary culture. Due to their importance for control of hormone secretion we emphasize analysis of calcium currents (I(Ca)), including using peptide toxins diagnostic for mammalian neuronal Ca(2+) channel types. These appear not to have been previously tested on fish endocrine cells. In balanced salines, inward currents consisted of a rapid TTX-sensitive sodium current and a smaller, slower I(Ca); there followed outward potassium currents dominated by delayed, sustained TEA-sensitive K(+) current. About half of cells tested from a holding potential (V(h)) of -90 mV showed early transient K(+) current; most cells showed a small Ca(2+)-mediated outward current. I-V plots of isolated I(Ca) with 15 mM [Ca(2+)](o) showed peak currents (up to 20 pA/pF from V(h) -90 mV) at approximately +10 mV, with approximately 60% I(Ca) for V(h) -50 mV and approximately 30% remaining at V(h) -30 mV. Plots of normalized conductance vs. voltage at several V(h)s were nearly superimposable. Well-sustained I(Ca) with predominantly Ca(2+)-dependent inactivation and inhibition of approximately 30% of total I(Ca) by nifedipine or nimodipine suggests participation of L-type channels. Each of the peptide toxins (omega-conotoxin GVIA, omega-agatoxin IVA, SNX482) alone blocked 36-54% of I(Ca). Inhibition by any of these toxins was additive to inhibition by nifedipine. Combinations of the toxins failed to produce additive effects. I(Ca) of up to 30% of total remained with any combination of inhibitors, but 0.1mM cadmium blocked all I(Ca) rapidly and reversibly. We did not find differences among cells of differing size and hormone content. Thus, I(Ca) is carried by high voltage-activated Ca(2+) channels of at least three types, but the molecular types may differ from those characterized from mammalian neurons.