Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as 'Danshen' in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca(2+) current (I(Ca,L)). The inhibition was use-dependent and voltage-dependent (the IC(50) value of MLB was 30 microM and 393 microM, respectively, at the holding potential of -50 mV and -100 mV). In the presence of 100 microM MLB, both the activation and steady-state inactivation curves of I(Ca,L) were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I(Ca,L) from inactivation was not altered. MLB up to 300 microM had no significant effect on the fast-inactivating Na(+) current (I(Na)), delayed rectifier K(+) current (I(K)) and inward rectifier K(+) current (I(K1)). The results suggest that the voltage-dependent Ca(2+) antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.     

Voltage-dependent modulation of TRPA1 currents by diphenhydramine

Diphenhydramine (DPH) has been broadly used to treat allergy. When used as a topical medicine, DPH temporarily relieves itching and pain. Although transient receptor potential type A1 (TRPA1) channel is known to play roles in both acute and chronic itch and pain, whether DPH affects the activities of TRPA1 remains unclear. Using whole-cell patch clamp recordings, we demonstrated that DPH modulates the voltage-dependence of TRPA1. When co-applied with a TRPA1 agonist, DPH significantly enhanced the inward currents while suppressing the outward currents of TRPA1, converting the channel from outwardly rectifying to inwardly rectifying. This effect of DPH occurred no matter TRPA1 was activated by an electrophilic or non-electrophilic agonist and for both mouse and human TRPA1. The modulation of TRPA1 by DPH was maintained in the L906C mutant, which by itself also causes inward rectification of TRPA1, indicating that additional acting sites are present for the modulation of TRPA1 currents by DPH. Our recordings also revealed that DPH partially blocked capsaicin evoked TRPV1 currents. These data suggest that DPH may exert its therapeutic effects on itch and pain, through modulation of TRPA1 in a voltage-dependent fashion.     

肾上腺髓质素对豚鼠心室肌细胞L-型钙通道的调制

应用全细胞膜片钳技术研究了肾上腺髓质素 (ADM )对豚鼠心室肌细胞L 型钙电流 (ICa ,L)的影响及其信号传导机制。结果发现 :ADM ( 1～ 10 0nmol/L)浓度依赖性抑制ICa,L(P <0 0 5 ) ,并可被ADM特异受体阻断剂ADM2 2 52 ( 10 0nmol/L)完全阻断。用蛋白激酶A特异拮抗剂H 89( 10 μmol/L)预处理 ,对ADM抑制ICa ,L的作用无影响。但用蛋白激酶C (PKC)特异性拮抗剂PKC19 36 预处理 ,可完全阻断ADM的抑制效应 ;而PKC特异性激动剂PMA则可以模仿ADM的抑制效应 (P <0 0 5 )。上述结果提示 :ADM作用于特异性ADM受体可浓度依赖性地抑制豚鼠心室肌细胞ICa ,L,而此作用可能是PKC介导的。     

Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).     

Antisense oligonucleotide against the Ca channel beta subunit decreases L-type Ca current in rat ventricular myocytes

The role of endogenous beta subunits of the L-type Ca channel in native cardiac ventricular myocytes is unclear. We have therefore investigated the effect of inhibiting beta subunit expression in rat myocytes, by culturing isolated myocytes for 24 h with either antisense oligonucleotide against the beta subunit or with scrambled oligonucleotide (control). Alpha1 subunit expression and distribution were then determined by immunolabeling, and L-type Ca current measured using the whole cell patch-clamp technique. Cells treated with antisense showed increased perinuclear staining for alpha1, decreased Ca current amplitude and a small rightward shift of the activation curve and the I-V relation, with no significant effect on inactivation. These data suggest that endogenous beta subunits in native cardiac myocytes help to traffic the alpha1 subunit to the cell membrane and thus play a major role in determining Ca current amplitude.     

Regulation of calcium currents and secretion by magnesium in crustacean peptidergic neurons

The effect of varying the external Mg²⁺ concentration on Ca²⁺ currents through voltage-operated Ca²⁺ channels has been examined with the patch-clamp technique in acutely isolated neuronal somata from the X-organ-sinus gland (XOSG) of the crab,Cardisoma carnifex. Neurons from this neurosecretory system were selected for morphology associated with crustacean hyperglycemic hormone (CHH) content. In parallel, the effects of Mg²⁺ concentration on K⁺-evoked secretion of CHH from isolated, intact XOSGs have been assayed by ELISA. At physiological Ca²⁺ levels the high-voltage-activated Ca²⁺ currents were attenuated with increasing Mg²⁺ concentration, with 50% inhibition at 75 mM. Mg²⁺ block was voltage-dependent, relief from block occurring with increasing depolarization. Thus, in 24 mM Mg²⁺ inhibition of the Ca²⁺ current was 55% at –10 mV and 30% at +20 mV. Secretion of CHH varied almost linearly with the log of Mg²⁺ concentration; in 2.4 mM Mg²⁺ it was double that in 24 mM Mg²⁺ and almost completely inhibited in 100 mM. Thus, Mg²⁺ produces a parallel inhibition of Ca²⁺ currents and CHH secretion and may play a role as a physiological modulator of neuronal activity and secretion in the XOSG of these crabs.     

Effects of N-n-butyl haloperidol iodide on L-type calcium channels and intracellular free calcium in rat ventricular myocytes.

The ability of N-n-butyl haloperidol iodide (F2) to cause vasodilation, and thereby produce a cardioprotective effect, has been well documented. The aim of this study was to investigate whether F2 might act as a Ca2+ antagonist. Myocytes were obtained from rat heart, and the whole-cell patch-clamp technique was used to record Ca2+ current. Laser scanning confocal microscopy was used to measure intracellular free calcium ([Ca2+]i). The results obtained from this study demonstrate that F2 reduced calcium current (ICa) in a concentration-dependent manner with an IC50 of 1.19 micromol/L, upshifted the current-voltage curve of ICa, shifted the inactivation kinetics of ICa leftward, and slowed down the recovery of ICa from inactivation. F2 decreased the fluorescent intensity of [Ca2+]i elevation induced by KCl with an IC50 of 1.61 micromol/L, and had no effects on the intracellular calcium release induced by caffeine and inositol-1,4,5-trisphosphate. These findings indicate that F2 may act as a calcium antagonist, which could account for its cardiovascular benefits.     

细胞外Ba2+对大鼠心肌细胞L型钙通道的影响

目的：观察细胞外Ba²⁺对记录大鼠心肌细胞L型钙通道的影响。方法：采用急性酶解分离法获得大鼠的单个心肌细胞,使用全细胞膜片钳技术记录L型钙通道电流。采用Ba²⁺替换台式液中的Ca²⁺和直接向台式液中加入Ba²⁺（0~8 mmol/L）,观察峰值电流15 min内的变化,数据采用5个以上细胞进行重复。结果：（1）台式液中的Ca²⁺被Ba²⁺替换后,L型钙通道电流的失活速率明显减慢（P<0.01）;在台式液中加入少量Ba²⁺（0.2,0.4 mmol/L）时L型钙通道电流的失活速率无明显改变（P>0.05）,加入0.8 mmol/L Ba²⁺时失活速率明显减慢（P<0.05）。（2）与正常台式液比较,在细胞外液中加入Ba²⁺（0.2,0.4 mmol/L）峰值电流衰减减弱,其中10 min和15 min两个时间点衰减差异明显（P<0.01）。（3）在细胞外液中加入Ba²⁺可下移电流电压曲线,改变翻转电位,减弱丹酚酸A对钙电流的抑制强度,使量效关系曲线右移。结论：在细胞外液中加入一定浓度的Ba²⁺,能够减弱全细胞膜片钳技术记录大鼠心室肌细胞L型钙通道时出现的峰值电流衰减,改变通道的电压依赖特性,影响药物量效关系。     

Effect of tetramethyl pyrazine on L-type calcium channel in rat ventricular myocytes

To elucidate possible ionic mechanisms of antimyocardial ischemia and antiarrythmia of tetramethyl pyrazine (TP), we studied L-type Ca2+ currents (I(Ca.L)) in adult rat ventricular myocytes using the whole-cell patch-clamp technique. The results showed: (i) under physiological conditions, 0.25 mmol/L TP decreased amplitude of I(Ca.L) to 60.6% and this inhibition was increased with increasing concentration of TP. ID50 was 0.20 mmol/L. (ii) The Ca2+-antagonistic effect of TP was voltage-dependent. A marked negative shift of the steady-state inactivation curve was observed with long (10 s) conditioning prepulses, but not with short (350 ms) ones. (iii) The time course of inhibition during TP treatment was increased with an increase in drug concentration, and recovery from TP-induced inactivation of I(Ca.L) was slower than in control cases. (iv) Tonic block and use-dependent block with TP treatment, which was induced by increasing the frequency of stimulation, occurred. We suggest that TP inhibits the I(Ca.L) mainly by binding to inactivated Ca2+ channels. The high affinity of TP for the inactivated state of I(Ca.L) may play an important role in developing therapies for pathological conditions.     

白藜芦醇降低大鼠心室肌细胞内游离钙浓度

实验旨在研究白藜芦醇(resveratrol)对大鼠心室肌细胞内钙浓度(intracellular calcium concentratoin,[Ca2+]i)的影响.应用激光共聚焦显微镜技术记录心室肌细胞内的钙荧光强度.结果表明在正常台氏液和无钙台氏液中,白藜芦醇(15～60μmol/L)呈浓度依赖性地降低[Ca2+]i.蛋白酪氨酸磷酸酶抑制剂正钒酸钠(sodium orthovanadate,1.0 mmol/L)和L型Ca2+通道激动剂Bay K8644(10 μmol/L)可部分抑制正常台氏液中白藜芦醇的效应.但NO合酶阻断剂L-NAME(1.0 mmol/L)对白藜芦醇的作用无影响.白藜芦醇也能明显抑制无钙台氏液中由低浓度ryanodine(1.0 nmol/L)引起的[Ca2+]i增加.当细胞外液钙浓度由1 mmol/L增加到10 mmol/L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,白藜芦醇(60 μmol/L)可降低钙波的传播速度和持续时间,最终阻断钙波.结果提示,白藜芦醇能够降低心室肌细胞内游离钙浓度,此作用可能与其抑制电压依赖性Ca2+通道、酩氨酸激酶和肌浆网内钙释放有关.     

Modulation of L-type Ca2+ currents and intracellular calcium by agmatine in rat cardiomyocytes

It is shown that agmatine inhibits L-type Ca²⁺ currents in isolated cardiomyocytes of rats in a dose-dependent manner. The inhibitory analysis indicates that imidazoline receptors of type I (I₁Rs) rather than α₂-adrenoceptors (α₂-ARs) are implicated in mediating the effects of agmatine. Agmatine affects the dynamics of intracellular Ca²⁺ concentration changes in spontaneously active cardiomyocytes. The averaged intracellular Ca²⁺ concentration ([Ca²⁺]_in) varied biphasically, depending on the agmatine dose: at 1–500 μM, agmatine decreased [Ca²⁺]_in; at 500 μM-2 mM, [Ca²⁺]_in remained unchanged, and at concentrations above 2 mM agmatine caused an increase of [Ca²⁺]_in. The effects of low agmatine concentrations were inhibited by 7NI, an inhibitor of NO synthases (NOS), as well as by the inhibitors of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) thapsigargin and cyclopiazonic acid. In contrast, ODQ, a blocker of NO-sensitive guanylate cyclase, and the antagonist of I₁Rs efaroxan were ineffective. At low concentrations agmatine did not affect the increase in [Ca²⁺]_in induced by stimulating doses of ryanodine (40 nM). In addition, agmatine at low doses was found to markedly stimulate NO production. When efaroxan (10 μM) or ryanodine (200 μM) were added to the bath to inhibit I₁Rs and ryanodine receptors (RyRs), respectively, [Ca²⁺]_in became much less sensitive to millimolar agmatine. In contrast to low concentrations (100 μM), high agmatine doses (10–15 mM) did not stimulate the NO synthesis but were effective as NOS inducer in cells pretreated with efaroxan. The selective I₁R agonist rilmenidine increased [Ca²⁺]_in in a dose-dependent manner. The effect of rilmenidine was similar to that of agmatine at high doses and was abolished by RyRs inhibition. Our findings indicate that in spontaneously active cardiomyocytes agmatine at low concentrations decreases [Ca²⁺]_in, does not stimulate I₁Rs but most likely enhances NO synthase followed by an increase in SERCA activity due to the direct nitrosylation of SERCA and/or phospholamban. The effects of high agmatine doses are apparently mediated by I₁Rs and involve RyRs.     

Inhibition of L-type Ca<Superscript>2+</Superscript> current in guinea pig ventricular myocytes by cisapride

The effect of cisapride on L-type Ca²⁺ current (I _Ca,L) was studied in guinea pig ventricular myocytes using a whole-cell voltage-clamp technique and a conventional action potential recording method. Myocytes were held at –40 mV, and internally dialyzed and externally perfused with Na⁺- and K⁺-free solutions; cisapride elicited a concentration-dependent block of peakI _Ca,L, with a half-maximum inhibition concentration (IC₅₀) of 46.9 µM. There was no shift in the reversal potential, nor any change in the shape of the current-voltage relationship ofI _Ca,L in the presence of cisapride. Inhibition of cisapride was not associated with its binding to serotonin or to -adrenergic receptors because ketanserin, SB203186, and prazosin had no effect on the inhibitory action of cisapride onI _Ca,L. Cisapride elicited a tonic block and a use-dependent block ofI _Ca,L. These blocking effects were voltage dependent as the degree of inhibition at –40 mV was greater than that at –70 mV. Cisapride shifted the steady-state inactivation curve ofI _Ca,L in the negative direction, but had no effect on the steady-state activation curve. Cisapride also delayed the kinetics of recovery ofI _Ca,L from inactivation. At a slow stimulation frequency (0.1 Hz), the action potential duration in guinea pig papillary muscles showed biphasic effects; it was prolonged by lower concentrations of cisapride, but shortened by higher concentrations. These findings suggest that cisapride preferentially binds to the inactivated state of L-type Ca²⁺ channels. The inhibitory effect of cisapride onI _Ca,L might play an important role in its cardiotoxicity under pathophysiological conditions, such as myocardial ischemia.     

Decreasing expression of alpha1C calcium L-type channel subunit mRNA in rat ventricular myocytes upon manganese exposure

Manganese is an essential trace element found in many enzymes. As it is the case of many essential trace elements, excessive level of manganese is toxic. It has been proven that excessive manganese could cause heart problems. In order to understand the mechanism of manganese toxicity in the heart, the effects of manganese on isolated rat ventricular myocytes were studied. The L-type calcium channel current was measured by whole-cell patch clamp recording mode. In the electrophysiology experiments, both 50 microM Mn2+ and 100 microM Mn2+ could effectively decrease the channel current amplitude density by 35.7% and 68.2%, respectively. Moreover, Mn2+ shifted the steady-state activation curve toward more positive potential and the steady-state inactivation curve toward more negative potential. Investigation by RT-PCR showed that the mRNA expression of alpha1C/Cav1.2 treated with manganese was decreased depending on its concentration, while the mRNA expression of alpha1D/Cav1.3 was almost unchanged. Fluo-3/AM was utilized for real-time free calcium scanning with laser scanning confocal microscopy (LSCM), and the results showed that Mn2+ could elicit a slow and continuous increase of [Ca2+]i in a concentration-dependent manner. These results have suggested that manganese could interfere with the function of the L-type calcium channel, downregulate the mRNA expression of alpha1C/Cav1.2, and thus causing long-lasting molecular changes of L-type calcium channel which have probably been triggered by overloading of calcium in myocytes.     

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.     

Inwardly rectifying single-channel and whole cell K+ currents in rat ventricular myocytes

Summary The voltage-dependent properties of inwardly rectifying potassium channels were studied in adult and neonatal rat ventricular myocytes using patch voltage-clamp techniques. Inward rectification was pronounced in the single-channel currentvoltage relation and outward currents were not detected at potentials positive to the calculated reversal potential for potassium (E _k). Single-channel currents having at least three different conductances were observed and the middle one was predominant. Its single-channel conductance was nonlinear ranging from 20 to 40 pS. Its open-time distribution was fit by a single exponential and the time constants decreased markedly with hyperpolarization fromE _k. The distribution of the closed times required at least two exponentials for fitting, and their taus were related to the bursting behavior displayed at negative potentials. The steady-state probability of being open (P _o) for this channel was determined from the single-channel records; in symmetrical isotonic K solutionsP _o was 0.73 at –60 mV, but fell to 0.18 at –100 mV. The smaller conductance was about one-half the usual value and the open times were greatly prolonged. The large conductance was about 50 percent greater than the usual value and the open times were very brief. TheP _o(V) relation, the kinetics and the conductance of the predominant channel account for most of the whole cell inwardly rectifying current. The kinetics suggest that an intrinsic K⁺-dependent mechanism may control the gating, and the conductance of this channel. In the steady state, the opening and closing probabilities for the two smaller channels were not independent of each other, suggesting the possibility of a sub-conductance state or cooperativity between different channels.     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Developmental changes in time course of recovery from inactivation in L-type calcium currents of rabbit ventricular myocytes

The mechanisms of recovery from inactivation of the L-type calcium current (I(Ca)) are not well established, and recovery is affected by many experimental conditions. Little is known about developmental changes of recovery from inactivation of I(Ca). We studied developmental changes of recovery from inactivation in I(Ca) using isolated adult and newborn (1-4 days) rabbit ventricular myocytes. We used broken-patch and perforated-patch techniques with physiological extracellular ionic concentrations of calcium and sodium and interpulse conditioning potentials of -80 or -50 mV. We also maximized I(Ca) with forskolin. We found that recovery from inactivation did not differ between adult and newborn cells when either EGTA or BAPTA was used to buffer intracellular calcium. Maximizing I(Ca) with forskolin slowed recovery from inactivation in newborn but not in adult cells. In contrast, when the intracellular buffering of the cell was left nearly intact (perforated patch), recovery from inactivation (half-time of recovery) in the newborn cells was significantly slower than for the adult cells when either a conditioning potential of -80 mV (140 +/- 9 vs. 58 +/- 4 ms, newborn vs. adult; P < 0.05) or -50 mV (641 +/- 106 vs. 168 +/- 15 ms, newborn vs. adult; P < 0.05) was used. Forskolin significantly increased half-time of recovery for both adult and newborn cells. Dialysis with no calcium buffer showed a slower recovery from inactivation in newborn cells. Intracellular dialysis with a calcium buffer masked differences in recovery from inactivation of I(Ca) between newborn and adult rabbit ventricular cells.     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.