Inhibition by nickel of the L-type Ca channel in guinea pig ventricular myocytes and effect of internal cAMP

The characteristics of nickel (Ni) block of L-type Ca current (I(Ca, L)) were studied in whole cell patch-clamped guinea pig cardiac myocytes at 37 degrees C in the absence and presence of 100 microM cAMP in the pipette solution. Ni block of peak I(Ca,L) had a dissociation constant (K(d)) of 0.33 +/- 0.03 mM in the absence of cAMP, whereas in the presence of cAMP, the K(d) was 0.53 +/- 0.05 mM (P = 0.006). Ni blocked Ca entry via Ca channels (measured as I(Ca, L) integral over 50 ms) with similar kinetics (K(d) of 0.35 +/- 0.03 mM in cAMP-free solution and 0.30 +/- 0.02 mM in solution with cAMP, P = not significant). Under both conditions, 5 mM Ni produced a maximal block that was complete for the first pulse after application. Ni block of I(Ca,L) was largely use independent. Ni (0. 5 mM) induced a positive shift (4 to 6 mV) in the activation curve of I(Ca,L). The block of I(Ca,L) by 0.5 mM Ni was independent of prepulse membrane potential (over the range of -120 to -40 mV). Ni (0.5 mM) also induced a significant shift in I(Ca,L) inactivation: by 6 mV negative in cAMP-free solution and by 4 mV positive in cells dialyzed with 100 microM cAMP. These data suggest that, in addition to blocking channel conductance by binding to a site in the channel pore, Ni may bind to a second site that influences the voltage-dependent gating of the L-type Ca channel. They also suggest that Ca channel phosphorylation causes a conformational change that alters some effects of Ni. The results may be relevant to excitation-contraction coupling studies, which have employed internal cAMP dialysis, and where Ni has been used to block I(Ca,L) and Ca entry into cardiac cells.     

Calcium flux in turtle ventricular myocytes

The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca(2+)-sensitive dye Fura-2 elicited Ca(2+) transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca(2+) currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca(2+) currents originating from L-type Ca(2+) channels (I(Ca)). The density of I(Ca) was 3.2 +/- 0.5 pA/pF, which led to an overall total Ca(2+) influx of 64.1 +/- 9.3 microM/l. NCX activity was measured as the Ni(+)-sensitive current at two concentrations of intracellular Na(+) (7 and 14 mM). Total Ca(2+) influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 +/- 7.7 micromol/l and 26.7 +/- 3.2 micromol/l at 14 and 7 mM intracellular Na(+), respectively. In the absence of the SR and L-type Ca(2+) channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca(2+) transport, and most of the Ca(2+) used for contraction originates from the L-type Ca(2+) channel.     

Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution

There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+ current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+ current (I(Ca,L)) in myocytes dialyzed with 10 mM K+ solution and superfused with Tyrode's solution. Although depolarizations from holding potential (Vhp) -40 to 0 mV elicited relatively small inward I(Ca,L) in these myocytes, removal of external K+ or addition of 0.2 mM Ba2+ more than doubled the amplitude of the current. The basis of the enhancement of I(Ca,L) was the suppression of a large transient outward K+ current. Similar enhancement was observed when Vhp was moved to -80 mV and test depolarizations were preceded by short prepulses to -40 mV. Investigation of the time and voltage properties of the outward K+ transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+ solution, accelerated in myocytes dialyzed with 100 microM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic "time-independent" inwardly rectifying K+ current.     

Normal contractions triggered by I(Ca,L) in ventricular myocytes from rats with postinfarction CHF

Attenuated L-type Ca(2+) current (I(Ca,L)), or current-contraction gain have been proposed to explain impaired cardiac contractility in congestive heart failure (CHF). Six weeks after coronary artery ligation, which induced CHF, left ventricular myocytes from isoflurane-anesthetized rats were current or voltage clamped from -70 mV. In both cases, contraction and contractility were attenuated in CHF cells compared with cells from sham-operated rats when cells were only minimally dialyzed using high-resistance microelectrodes. With patch pipettes, cell dialysis caused attenuation of contractions in sham cells, but not CHF cells. Stepping from -50 mV, the following variables were not different between sham and CHF, respectively: peak I(Ca,L) (4.5 +/- 0.3 vs. 3.8 +/- 0.3 pApF(-1) at 23 degrees C and 9.4 +/- 0.5 vs. 8.4 +/- 0.5 pApF(-1) at 37 degrees C), the bell-shaped voltage-contraction relationship in Cs(+) solutions (fractional shortening, 15.2 +/- 1.0% vs. 14.3 +/- 0.7%, respectively, at 23 degrees C and 7.5 +/- 0.4% vs. 6.7 +/- 0.5% at 37 degrees C) and the sigmoidal voltage-contraction relationship in K(+) solutions. Caffeine-induced Ca(2+) release and sarcoplasmic reticulum Ca(2+)-ATPase-to-phospholamban ratio were not different. Thus CHF contractions triggered by I(Ca,L) were normal, and the contractile deficit was only seen in undialyzed cardiomyocytes stimulated from -70 mV.     

Alterations in outward K(+) currents on removal of external Ca(2+) in human atrial myocytes

External divalent cations are known to play an important role in the function of voltage-gated ion channels. The purpose of this study was to examine the sensitivity of the voltage-gated K(+) currents of human atrial myocytes to external Ca(2+) ions. Myocytes were isolated by collagenase digestion of atrial appendages taken from patients undergoing coronary artery-bypass surgery. Currents were recorded from single isolated myocytes at 37 degrees C using the whole-cell patch-clamp technique. With 0.5 mM external Ca(2+), voltage pulses positive to -20 mV (holding potential = -60 mV) activated outward currents which very rapidly reached a peak (I(peak)) and subsequently inactivated (tau = 7.5 +/- 0.7 msec at +60 mV) to a sustained level, demonstrating the contribution of both rapidly inactivating transient (I(to1)) and non-inactivating sustained (I(so)) outward currents. The I(to1) component of I(peak), but not I(so), showed voltage-dependent inactivation using 100 msec prepulses (V(1/2) = -35.2 +/- 0.5 mV). The K(+) channel blocker, 4-aminopyridine (4-AP, 2 mM), inhibited I(to1) by approximately 76% and reduced I(so) by approximately 33%. Removal of external Ca(2+) had several effects: (i) I(peak) was reduced in a manner consistent with an approximately 13 mV shift to negative voltages in the voltage-dependent inactivation of I(to1). (ii) I(so) was increased over the entire voltage range and this was associated with an increase in a non-inactivating 4-AP-sensitive current. (iii) In 79% cells (11/14), a slowly inactivating component was revealed such that the time-dependent inactivation was described by a double exponential time course (tau(1) = 7.0 +/- 0.7, tau(2) = 90 +/- 21 msec at +60 mV) with no effect on the fast time constant. Removal of external Ca(2+) was associated with an additional component to the voltage-dependent inactivation of I(peak) and I(so) (V(1/2) = -20.5 +/- 1.5 mV). The slowly inactivating component was seen only in the absence of external Ca(2+) ions and was insensitive to 4-AP (2 mM). Experiments with Cs(+)-rich pipette solutions suggested that the Ca(2+)-sensitive currents were carried predominantly by K(+) ions. External Ca(2+) ions are important to voltage-gated K(+) channel function in human atrial myocytes and removal of external Ca(2+) ions affects I(to1) and 4-AP-sensitive I(so) in distinct ways.     

Effect of SIN-1 in rat ventricular myocytes: interference with beta-adrenergic stimulation

We have examined the effects of the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on Ca(2+) transients, L-type Ca(2+) current (I(Ca,L)), and cGMP/cAMP content in electrically-stimulated rat ventricular myocytes in the absence and presence of the beta-adrenergic stimulation with isoproterenol. SIN-1 had no effect at low concentrations, but decreased the amplitude of electrically-induced Ca(2+) transients at higher concentrations. SIN-1 attenuated the increase in Ca(2+) transients induced by isoproterenol in a concentration-dependent manner. SIN-1 Also reduced the amplitude of caffeine-induced Ca(2+) transients, and the increase in I(Ca,L) induced by isoproterenol. These effects of SIN-1 were associated with an increased cGMP and a decreased cAMP content in ventricular myocytes in either the absence or presence of isoproterenol. These data suggest that the inhibitory effect of SIN-1 on basal and beta-adrenergic stimulated Ca2+ signal in ventricular myocytes could be due to the depression in the SR function and I(Ca,L), possibly mediated by a cGMP/cAMP-dependent mechanism. Taken together, the present study supports the idea that NO acts as an inhibitory modulator of the cardiac function during pathological conditions associated with an abnormal production of NO such as septic shock.     

Intracellular Ca(2+) regulates responsiveness of cardiac L-type Ca(2+) current to protein kinase A: role of calmodulin

The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca(2+) current (ICa) is affected during transient increases in intracellular Ca(2+) concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca(2+) external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 microM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca(2+) concentration was reduced to 0.5 mM or replaced with Ba(2+). This Ca(2+)-dependent inhibition was also observed when the cells were treated with 1 microM isoproterenol, 100 microM 3-isobutyl-1-methylxanthine, or 500 microM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca(2+)/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca(2+)-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba(2+). Thus Ca(2+) entering the myocyte through the voltage-gated Ca(2+) channel regulates the PKA responsiveness of ICa.     

Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

In cardiac cells that lack macroscopic transient outward K(+) currents (I(to)), the removal of extracellular Ca(2+) can unmask "I(to)-like" currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca(2+) channels underlies these currents. Removal of extracellular Ca(2+) and extracellular Mg(2+) induced time-independent currents at all potentials and time-dependent currents at potentials greater than -50 mV. Either K(+) or Cs(+) could carry the time-dependent currents, with reversal potential of +8 mV with internal K(+) and +34 mV with Cs(+). Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value (V(1/2)) = -24 mV and slope = -9 mV for activation; V(1/2) = -58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC(50) = 2 microM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I(to)-like currents are due to monovalent cation flow through L-type Ca(2+) channels, which in pig myocytes show low sensitivity to nifedipine.     

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).     

Modulation of Ca2+ channel-gated Ca2+ release by W-7 in cardiac myocytes.

Cardiac muscle excitation-contraction coupling is controlled by the Ca(2+)-induced Ca2+ release mechanism. The present study examines the effects of a calmodulin antagonist W-7 on Ca2+ current (ICa)-induced Ca2+ release in whole cell-clamped rat ventricular myocytes. Exposure of cells to W-7 suppressed ICa, but the intracellular Ca(2+)-transients showed a lesser degree of reduction, suggesting possible enhancement of Ca(2+)-induced Ca2+ release. The effects of W-7 on the efficacy of Ca2+ release were most prominent at negative potentials. At test potentials of -30 mV, 20 microM W-7 almost completely blocked ICa, but significant Ca(2+)-transients remained, thus causing a four to six-fold increase in the efficacy of Ca(2+)-induced Ca2+ release. The depolarization-dependent Ca(2+)-transients were eliminated in absence of extracellular Ca2+, blocked by Cd2+, and were absent when the sarcoplasmic reticulum was depleted of Ca2+, implicating dependency on Ca(2+)-signaling between the L-type channel and the ryanodine receptor. W-7 mediated increase in the efficacy of Ca(2+)-induced Ca2+ release was eliminated when myocytes were dialyzed with the internal solution containing gluathione (5 mM), suggesting the possible role of cellular redox state in the regulation of Ca2+ release by the calmodulin antagonist.     

Cyclic AMP-mediated increase in L-type calcium current (ICa,L) by nitroglycerin in guinea-pig ventricular myocytes.

The effects of nitroglycerin (NG) on the L-type Ca2+ current (ICa,L) were investigated in guinea-pig ventricular myocytes. NG did not affect the basal ICa,L significantly. The ICa,L was increased by NG when the ICa,L was augmented by the pre-treatment with isoproterenol (Iso), and this increase was abolished by the pretreatment with methylene blue (MB), a guanylate cyclase inhibitor. NG also increased ICa,L in the myocytes undergoing the pretreatment with isobutylmethylxanthine (IBMX). The NG-induced increase in ICa,L was also observed in the myocytes intracellularly dialyzed with cyclic adenosine monophosphate (cAMP). The order of the Iso-induced increase in ICa,L was Iso, IBMX and cAMP. Relatively a lower concentration of the extracellularly applied 8bromo-cyclic guanosine monophosphate (8Br-cGMP) increased ICa,L in the myocytes dialyzed with cAMP, whereas the higher concentration of 8Br-cGMP decreased ICa,L. NG produced a marked increase in cGMP levels, and a slight increase in cAMP levels in the ventricular tissues. These results suggest that the stimulatory effect of NG on the ICa,L is due to an inhibition of cGMP-inhibitable cAMP-phosphodiesterase (PDE) and a possible activation of cGMP-dependent protein kinase via the NG-induced increase in cGMP levels in guinea-pig ventricular myocytes.     

Effects of permeant ion concentrations on the gating of L-type Ca2+ channels in hair cells

We determined the gating and permeation properties of single L-type Ca(2+) channels, using hair cells and varying concentrations (5-70 mM) of the charge carriers Ba(2+) and Ca(2+). The channels showed distinct gating modes with high- and low-open probability. The half-activation voltage (V(1/2)) shifted in the hyperpolarizing direction from high to low permeant ion concentrations consistent with charge screening effects. However, the differences in the slope of the voltage shifts (in VM(-1)) between Ca(2+) (0.23) and Ba(2+) (0.13), suggest that channel-ion interaction may also contribute to the gating of the channel. We examined the effect of mixtures of Ba(2+) and Ca(2+) on the activation curve. In 5 mM Ca(2+), the V(1/2) was, -26.4 +/- 2.0 mV compared to Ba(2+), -34.7 +/- 2.9 mV, as the charge carrier. However, addition of 1 mM Ba(2+) in 4 mM Ca(2+), a molar ratio, which yielded an anomalous-mole fraction effect, was sufficient to shift the V(1/2) to -34.7 +/- 1.5 mV. Although Ca(2+)-dependent inactivation of the L-type channels in hair cells can yield the present findings, we provide evidence that the anomalous gating of the channel may stem from the closed interaction between ion permeation and gating.     

Inhibition of L-type Ca2+ channel current and negative inotropy induced by arachidonic acid in adult rat ventricular myocytes

We have previously shown an increase in arachidonic acid (AA) release in response to proinflammatory cytokines in adult rat ventricular myocytes (ARVM). AA is known to alter channel activities; however, its effects on cardiac L-type Ca(2+) channel current (I(Ca,L)) and excitation-contraction coupling remain unclear. The present study examined effects of AA on I(Ca,L), using the whole cell patch-clamp technique, and on cell shortening (CS) and the Ca(2+) transient of ARVM. I(Ca,L) was monitored in myocytes held at -70 mV and internally equilibrated and externally perfused with Na(+)- and K(+)-free solutions. Exposure to AA caused a voltage-dependent block of I(Ca,L) concentration dependently (IC(50) 8.5 microM). The AA-induced inhibition of I(Ca,L) is consistent with its hyperpolarizing shift in the voltage-dependent properties and reduction in maximum slope conductance. In the presence of AA, BSA completely blocked the AA-induced suppression of I(Ca,L) and CS. Intracellular load with AA had no effect on the current density but caused a small depolarizing shift in the I(Ca,L) activation curve, suggesting a site-specific action of AA. Moreover, intracellular AA had no effect on the extracellular AA-induced decrease in I(Ca,L). Pretreatment with indomethacin, an inhibitor of cyclooxygenase, or addition of nordihydroguaiaretic acid, an inhibitor of lipoxygenase, had no effect on AA-induced changes in I(Ca,L). Furthermore, AA suppressed CS and Ca(2+) transients of intact ARVM with no significant effect on SR function and myofilament Ca(2+) sensitivity. Therefore, these results suggest that AA inhibits contractile function of ARVM, primarily due to its direct inhibition of I(Ca,L) at an extracellular site.     

Na(+)/Ca(2+)-exchange activity regulates contraction and SR Ca(2+) content in rainbow trout atrial myocytes

We have used the whole cell configuration of the patch-clamp technique to measure sarcolemmal Ca(2+) transport by the Na(+)/Ca(2+) exchanger (NCX) and its contribution to the activation and relaxation of contraction in trout atrial myocytes. In contrast to mammals, cell shortening continued, increasing at membrane potentials above 0 mV in trout atrial myocytes. Furthermore, 5 microM nifedipine abolished L-type Ca(2+) current (I(Ca)) but only reduced cell shortening and the Ca(2+) carried by the tail current to 66 +/- 5 and 67 +/- 6% of the control value. Lowering of the pipette Na(+) concentration from 16 to 10 or 0 mM reduced Ca(2+) extrusion from the cell from 2.5 +/- 0.2 to 1.0 +/- 0.2 and 0.5 +/- 0.06 amol/pF. With 20 microM exchanger inhibitory peptide (XIP) in the patch pipette Ca(2+) extrusion 20 min after patch break was 39 +/- 8% of its initial value. With 16, 10, and 0 mM Na(+) in the pipette, the sarcoplasmic reticulum (SR) Ca(2+) content was 47 +/- 4, 29 +/- 6, and 10 +/- 3 amol/pF, respectively. Removal of Na(+) from or inclusion of 20 microM XIP in the pipette gradually eliminated the SR Ca(2+) content. Whereas I(Ca) was the same at -10 or +10 mV, Ca(2+) extrusion from the cell and the SR Ca(2+) content at -10 mV were 65 +/- 7 and 80 +/- 4% of that at +10 mV. The relative amount of Ca(2+) extruded by the NCX (about 55%) and taken up by the SR (about 45%) was, however, similar with depolarizations to -10 and +10 mV. We conclude that modulation of the NCX activity critically determines Ca(2+) entry and cell shortening in trout atrial myocytes. This is due to both an alteration of the transsarcolemmal Ca(2+) transport and a modulation of the SR Ca(2+) content.     

Modulation of L-type Ca2+ current by fast and slow Ca2+ buffering in guinea pig ventricular cardiomyocytes.

Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.     

L, P-/Q- and T-type Ca2+ channels in smooth muscle cells from human umbilical artery.

The electrophysiological and pharmacological properties of Ca(2+) current (I(Ca)) were determined by the whole-cell configuration of the patch-clamp technique in smooth muscle cells from human umbilical artery. Using 5 mM extracellular Ca(2+), depolarizing step pulses from -60 to 50 mV from a holding membrane potential of -80 mV evoked an I(Ca) which activated at membrane potentials more positive than -50 mV and exhibited a maximum current density in a range of 10-20 mV. Steady-state inactivation protocols using a V(test) of 10 mV gave a voltage at one-half inactivation and a slope factor of -35.6 mV and 9.5 mV, respectively. Nifedipine (1 microM), an L-type Ca(2+) channels antagonist, completely inhibited I(Ca), while the L-type Ca(2+) channels agonist Bay-K 8644 (1 microM) significantly increased I(Ca) amplitude. Moreover, the selective blocker of P-/Q-type Ca(2+) channels omega-agatoxin IVA partially blocked I(Ca) (about 40 % inhibition at +20 mV by 20 nM). These pharmacological results suggest that L- and P-/Q-type Ca(2+) channels, both nifedipine-sensitive, underlie the I(Ca) registered using low extracellular Ca(2+). The presence of the P-/Q-type Ca(2+) channels was confirmed by immunoblot analysis. When I(Ca) was recorded in a high concentration (30 mM) of extracellular Ca(2+) or Ba(2+) as current carrier, it was evident the presence of a nifedipine-insensitive component which completely inactivated during the course of the voltage-step (75 ms) at all potentials tested, and was blocked by the T-type Ca(2+) channels blocker mibefradil (10 microM). Summarizing, this work shows for the first time the electrophysiological and pharmacological properties of voltage-activated Ca(2+) currents in human umbilical artery smooth muscle cells.     

三羟异黄酮对豚鼠心室肌细胞L-型钙通道电流的影响

本实验用全细胞膜片钳技术观察三羟异黄酮(genistein,GST)对豚鼠心室肌细胞L-钙通道电流(ICa、L)的影响。结果如下:(1)GST(10、50、100 μmol/L)可浓度依赖性地降低ICa,L(n=6,P<0.01)。GST的非活性结构类似物daidzein(100μmol/L),在同一浓度范围对ICa,L没有影响(n=5,P>0.05)。(2)GST使I-V曲线上移,但对ICa,L的电压依赖特征和最大激活电压无明显影响。(3)GST对ICa,L的激活动力学特性也无影响,但可使钙电流稳态失活曲线左移。V0.5从对照的-28.6±0.6 mV变为-32.8±1.1mV,κ值从对照的5.8±0.5 mV升至6.5±0.9 mV(n=6,P<0.05)。(4)GST明显使复活曲线右移,从而使ICa,L从失活状态下恢复明显减慢(n=7,P<0.01)。(5)酪氨酸磷酸酶抑制剂正钒酸钠(1 mmol/L)显著对抗GST引起的ICa,L抑制效应(n=6,P<0.01)。根据以上结果得出的结论是:GST抑制ICa,L加速钙通道失活和钙通道在失活状态下恢复减慢;GST对ICa,L的这种抑制作用与蛋白酪氨酸激酶(PTK)抑制有关。     

Altered spatial calcium regulation enhances electrical heterogeneity in the failing canine left ventricle: implications for electrical instability

Myocytes across the left ventricular (LV) wall of the mammalian heart are known to exhibit heterogeneity of electrophysiological properties; however, the transmural variation of cellular electrophysiology and Ca(2+) homeostasis in the failing LV is incompletely understood. We studied action potentials (APs), the L-type calcium (Ca(2+)) current (I(Ca,L)), and intracellular Ca(2+) transients ([Ca(2+)](i)) of subendocardial (Endo), midmyocardial (Mid), and subepicardial (Epi) tissue layers in the canine normal and tachycardia pacing-induced failing left ventricles. Heart failure (HF) was associated with significant prolongation of the AP duration in Mid myocytes. There were no differences in I(Ca,L) density in normal Endo, Mid, and Epi myocytes, whereas in the failing heart, I(Ca,L) density was downregulated by 45% and 26% (at +10 mV) in Endo and Mid myocytes, respectively. The rates of sarcoplasmic reticulum (SR) Ca(2+) release and decay of the [Ca(2+)](i) were slowed, and the amplitude of the [Ca(2+)](i) was depressed in Endo and Epi myocytes isolated from failing, compared with normal, hearts. Experiments in sodium (Na(+))-free solutions showed that Epi and Mid myocytes of the failing ventricle exhibit a greater reliance on the Na(+)-Ca(2+) exchanger to remove cytosolic Ca(2+) than myocytes isolated from normal hearts. Simulation studies in Endo, Mid, and Epi canine myocytes demonstrate the importance of L-type current density and SR Ca(2+) uptake in modulating the potentially arrhythmogenic repolarization in HF. In conclusion, these results demonstrate that spatially heterogeneous decreases in I(Ca,L) and defective cytosolic Ca(2+) removal contribute to the altered [Ca(2+)](i) and AP profiles across the canine failing LV. These distinct electrophysiological features in myocytes from a failing heart contribute to a characteristic electrogram arising from increased dispersion of refractoriness across the LV, which may result in significant arrhythmogenic sequellae.     

Changes in membrane cholesterol of pituitary tumor (GH3) cells regulate the activity of large-conductance Ca2+-activated K+ channels

The effects of changes in membrane cholesterol on ion currents were investigated in pituitary GH3 cells. Depletion of membrane cholesterol by exposing cells to methyl-beta-cyclodextrin (MbetaCD), an oligosaccharide, resulted in an increase in the density of Ca2+-activated K+ current (IK(Ca)). However, no significant change in IK(Ca) density was demonstrated in GH3 cells treated with a mixture of MbetaCD and cholesterol. Cholesterol depletion with MbetaCD (1.5 mg/ml) slightly suppressed the density of voltage-dependent L-type Ca2+ current. In inside-out patches recorded from MbetaCD-treated cells, the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels was enhanced with no change in single-channel conductance. In MbetaCD-treated cells, voltage-sensitivity of BK(Ca) channels was increased; however, no change in Ca2+-sensitivity could be demonstrated. A negative correlation between adjacent closed and open times in BK(Ca) channels was observed in MbetaCD-treated cells. In inside-out patches from MbetaCD-treated cells, dexamethasone (30 microM) applied to the intracellular surface did not increase BK(Ca)-channel activity, although caffeic acid phenethyl ester and cilostazol still opened its probability effectively. However, no modification in the activity of ATP-sensitive K+ channels could be seen in MbetaCD-treated cells. Current-clamp recordings demonstrated that the cholesterol depletion maneuver with MbetaCD reduced the firing of action potentials. Therefore, the increase in BK(Ca)-channel activity induced by membrane depletion may influence the functional activities of neurons or neuroendocrine cells if similar results occur in vivo.     

Angiotensin II stimulates cardiac L-type Ca(2+) current by a Ca(2+)- and protein kinase C-dependent mechanism

Angiotensin II (ANG II) evokes positive inotropic responses in various species. However, the effects of this peptide on L-type Ca(2+) currents (I(Ca)) are still controversial. We report in this study that the effects of ANG II on I(Ca) differ depending on the mode of patch-clamp technique used, standard whole cell (WC) or perforated patch (PP). No significant effects of ANG II (0.5 microM) were observed when WC in cells dialyzed with high EGTA was used. However, when the intracellular milieu was preserved using PP, ANG II induced a significant 77 +/- 6% increase in I(Ca) (-2.2 +/- 0.3 in control and -3.9 +/- 0.6 pA/pF in ANG II, n = 8, P < 0.05). When WC was used in cells dialyzed with low Ca(2+) buffer capacity (EGTA 0.1 mM), ANG II was able to induce an increase in I(Ca) (-3.5 +/- 0.3 in control vs. -4.8 +/- 0.4 pA/pF in ANG II, n = 13, P < 0.05). This increase was prevented when the cells were also dialyzed with the protein kinase C (PKC) inhibitor chelerythrine (50 microM) or calphostin C (1 microM). The above results allow us to conclude that strong intracellular Ca(2+) buffering prevents the physiological actions of ANG II on cardiac I(Ca), which are also dependent on activation of PKC.