Rikuo Ochi

Elementary currents through Ca2+ channels in Guinea pig myocytes

Elementary Ca2+ and Ba2+ currents were recorded from cell-attached membrane patches of ventricular myocytes from adult guinea pig hearts using the improved patch-clamp technique (Hamill et al. 1981). High concentrations of Ba2+ or Ca2+ (50 or 90 mM) were used in the pipettes to increase the signal-to-noise ratio. All data were derived from elementary current analyses in patches containing only one channel.1)In response to voltage steps, channel openings occurred singly or in bursts of closely spaced unitary current pulses separated by wider shut intervals. During depolarizations of small amplitude from the resting potential, channel openings occurred almost randomly, whereas during larger depolarizations the events were grouped preferentially at the beginning.2)Channel openings became more probable with increased depolarization; simultaneously, unitary current amplitudes declined in an ohmic manner. Elementary current amplitudes were slightly larger, when 50 mM Ba2+ replaced 50 mM Ca2+ in the pipettes (slope conductances 9 and 10 pS, respectively), but more than doubled, when Ba2+ was increased to 90 mM (slope conductance 18 pS). Clear outward currents through Ca2+ channels were not observed under these conditions.3)Peak amplitudes of reconstructed mean currents doubled when 50 mM Ba2+ replaced 50 mM Ca2+ and were larger still when 90 mM Ba2+ was used in the pipettes. The current-volrage relations of the reconstructed mean currents showed a positive shift along the voltage axis as Ba2+ was increased or substituted equimolarly by Ca2+. Correspondingly, the open state probability-voltage relations (activation curves) showed a parallel shift as Ba2+ was increased, which was less pronounced when Ba2+ was replaced equimolarly by Ca2+.4)Determination of Ca2+ channel inactivation using 90 mM Ba2+ in the pipettes indicated an overlap with channel activation in a limited voltage range, resulting in a steadystate “window” current. Inactivation can occur without divalent cation influx.5)Formation of an inside-out patch resulted in a fast rundown of elementary Ca2+ channel currents.6)Channel openings were often grouped in bursts. The lifetimes of the open state, the bursts, and the closed states were estimated for Ba2+ and Ca2+ as permeating ions. At least two exponentials were needed to fit the histogram of the lifetimes of all closed states. The lifetimes of the individual openings and bursts were mono-exponentially distributed. The kinetics of the Ca+ channel depended on the voltage and the permeating ion. During +30 mV depolarizations, no significant effect of the permeating ion on channel gating could be detected. The significant increase in burst length (tb) during +50 mV depolarizations, however, seemed to be only due to an increase in the lifetime of the open state (to) for Ba2+, whereas for Ca2+,to was only moderately prolonged but simultaneously, the number of openings per burst increased.7)A three-state sequential scheme is peoposed to model the activation pathway of Ca2+ channels. The latency-to-first-event histogram is also consistent with a process in which multiple closed states precede the open state.

Maitotoxin-activated single calcium channels in guinea-pig cardiac cells

1 In order to clarify the mechanism of Ca‐dependent excitatory action of maitotoxin (MTX), the most potent marine toxin known, patch‐clamp techniques were used to analyse electrophysiological effects of MTX on guinea‐pig isolated cardiac myocytes 2 The whole‐cell recordings showed that MTX (0.3 ng ml−1) produced a sustained inward current that was enhanced by adrenaline (2 μm) and abolished by Cd2+ (1 mm). 3 This current was predominantly carried by Ca2+ or Ba2+ and has an almost linear current‐voltage relationship. 4 In cell‐attached patches, MTX added to the pipette solution activated Ca channels with novel properties. The opening events of these channels occurred as long bursts, and the channel gating showed little voltage‐dependence. 5 The unitary conductance was 12 pS in the presence of 50 mm Ba2+. Within a burst, the distribution of opening times was a single exponential with a mean open time of 10.4 ms. 6 The channel described here represents either a new class of voltage‐independent Ca channel or an entirely modified form of voltage‐gated Ca channel. This channel may account for the mechanism of enhanced Ca2+ influx through the cell membrane induced by MTX, and presumably regulates some ionic movements in myocardial cells.

Properties and expression of Ca2+-activated K+ channels in H9c2 cells derived from rat ventricle

H9c2 is a clonal myogenic cell line derived from embryonic rat ventricle that can serve as a surrogate for cardiac or skeletal muscle in vitro. Using whole cell clamp with H9c2 myotubes, we observed that depolarizing pulses activated slow outward K+ currents and then slow tail currents. The K+ currents were abolished in a Ca2+-free external solution, indicating that they were Ca2+-activated K+ currents. They were blocked by apamin, a small-conductance Ca2+-activated K+ (SK) channel antagonist (IC50 = 6.2 nM), and by d-tubocurarine (IC50 = 49.4 μM). Activation of SK channels exhibited a bell-shaped voltage dependence that paralleled the current-voltage relation for L-type Ca2+ currents ( I Ca,L). I Ca,L exhibited a slow time course similar to skeletal I Ca,L, were unaffected by apamin, and were only slightly depressed by d-tubocurarine. RT-PCR analysis of the mRNAs revealed that rSK3, but not rSK1 or rSK2, was expressed in H9c2 myotubes but not in myoblasts. These results suggest that rSK3 channels are expressed in H9c2 myotubes and are primarily activated by I Ca,L directly or indirectly via Ca2+-induced Ca2+ release from sarcoplasmic reticulum.

Decrease in Ca2+ sensitivity as a mechanism of hydrogen peroxide-induced relaxation of rabbit aorta

OBJECTIVE In vascular strips, hydrogen peroxide (H2O2) relaxes alpha 1-adrenergic agonist-induced but not high-K(+)-induced contractions. The aim of this study was to explore H2O2-induced changes in [Ca2+]i of vascular smooth muscle and to elucidate the mechanisms of action of H2O2. METHODS Isolated rabbit aortic strips were isometrically contracted with high-K+ (64.7 mM) or phenylephrine (PE, 0.3 microM). The effects of 300 microM H2O2 on [Ca2+]i of endothelium-denuded vascular smooth muscle and tension were determined simultaneously by the fura-2 method. Changes in [Ca2+]i were expressed as percentages of high-K(+)-induced values measured at the beginning of the experiments. In another series of experiments, the relaxant effect of 300 microM H2O2 was examined in high-K+ (20 mM)-induced contraction in the presence of the protein kinase C activator, phorbol 12,13-dibutyrate (PDBu). RESULTS Hydrogen peroxide caused a reversible rise in [Ca2+]i of vascular smooth muscle under both resting conditions and in the precontracted state. During high-K(+)-induced contraction, H2O2 further increased [Ca2+]i by 26.6(s.e.m. 1.7)% accompanied by a small increase in tension of 6.5(1.9)% of high-K(+)-induced tension. By contrast, during PE-induced contraction, although H2O2 caused a comparable additional increase in [Ca2+]i (26.4(4.7)%), muscle tension fell by 28.9(2.2)% of the steady-state PE-induced tension. Hydrogen peroxide had a relaxant effect on augmented high-K(+)-induced contraction in which Ca2+ sensitivity of the contractile apparatus was elevated by PDBu. CONCLUSIONS In spite of its effect of increasing [Ca2+]i of vascular smooth muscle, hydrogen peroxide causes relaxation of endothelium-denuded, PE-precontracted rabbit aorta. The mechanism is probably through suppression of agonist-induced augmentation of Ca2+ sensitivity of the contractile apparatus.

L-type calcium channel activity in human atrial myocytes as influenced by 5-HT

Summary5-Hydroxytryptamine (10 μmol/l; 5-HT) exerted a positive inotropic effect associated with an increase in the Ca2+ current (ICa) in the human right atrium. For detailed analysis, L-type Ca2+ channel currents were recorded from cell-attached patches using 100 mmol/l Ba2+ as charge carrier. Ca2+ channel activity was identified, first, by burstlike inwardly directed currents and, second, by the appearance of long channel openings promoted by Bay K 8644 (1 μmol/l) upon repetitive depolarizations from − 80 to 0 mV The unitary conductance of the Ca2+ channel amounted to 25.8 pS. During superfusion with 5-HT, ensemble averaged (mean) current was enhanced by about 60%. The increase in mean current was brought about by an increase in the channel availability, defined as the ratio of sweeps containing Ca2+ channel activity to the total number of depolarizations. The open probability of a single Ca2+ channel within a sweep with channel activity, unitary conductance, mean open and mean shut times of the channel, however, remained unaffected during superfusion with 5-HT (n = 10). The 5-HT-induced increase in macroscopic ICa in the human atrium can therefore be explained by an enhanced availability of Ca2+ channels to open upon depolarization. The observed changes in gating properties of the human Ca2+ channel by 5-HT are very similar to those which are known from isoprenaline-induced CAMP-dependent phosphorylation of the Ca2+ channel protein in other tissues.

Inhibition of the slow inward current and the time-dependent outward current of mammalian ventricular muscle by gentamicin

The aminoglycoside antibiotic, gentamicin (GM), depressed the plateau phase and shortened the duration of the action potential in guinea pig papillary muscle. Its effect on the membrane currents was studied by a single sucrose gap voltage clamp method. The slow inward current (is) was remarkably diminished by GM with little change in its time course, in the voltage-dependency of the steady-state inactivation and activation or in its reversal potential. The maximal amplitude of is, obtained by subtracting the Co2+-resistant current, was reduced to 57% by 0.1 mmol/l GM and almost reduced to zero by 1 mmol/l GM. The efficacy of GM in inhibiting is was reduced by increasing the external Ca2+ concentration from 1.8 to 5.4 or 10.8 mmol/l, but not by the application of adrenaline. The time-dependent outward current (iK) was also decreased by GM but only at higher concentrations. It is proposed that the depressant action of GM on is was due to a blockade of slow channels, whereby GM may have dislocated Ca from the binding sites at slow channels on the external surface of the membrane.

Glucose-6-phosphate dehydrogenase is a regulator of vascular smooth muscle contraction.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 μM), or U46619 (100 nM) significantly (p < 0.05) increased both G6PD activity and glucose flux through the pentose phosphate pathway. The effect was Ca(2+) independent, and there was a corresponding increase in protein kinase C (PKC) activity. Activation of G6PD by KCl was blocked by the PKCδ inhibitor rottlerin (10 μM) or by knocking down PKCδ expression using siRNA. Phorbol 12, 13-dibutyrate (10 μM), a PKC activator, significantly increased G6PD phosphorylation and activity, whereas single (S210A, T266A) and double (S210A/T266A) mutations at sites flanking the G6PD active site significantly inhibited phosphorylation, shifted the isoelectric point, and reduced enzyme activity. Knocking down G6PD decreased NADPH and reactive oxygen species generation, and reduced KCl-evoked increases in [Ca(2+)](i) and myosin light chain phosphorylation, thereby reducing CA contractility. Similarly, aortas from G6PD-deficient mice developed less KCl/phorbol 12, 13-dibutyrate-evoked force than those from their wild-type littermates. Conversely, overexpression of G6PD augmented KCl-evoked increases in [Ca(2+)](i), thereby augmenting CA contraction. Our findings demonstrate that G6PD activity and NADPH is increased in activated CA in a PKCδ-dependent manner and that G6PD modulates Ca(2+) entry and CA contractions evoked by membrane depolarization.

Hyperpolarization and lysophosphatidylcholine induce inward currents and ethidium fluorescence in rabbit ventricular myocytes

Strong electric pulses produce reversible or irreversible membrane breakdown (electroporation). We analysed the permeation properties of minute pores caused by hyperpolarization or lysophosphatidylcholine (LPC) by comparing the amount of charge carried by irregular inward currents (Ihi) with changes in ethidium bromide (EB) fluorescence in isolated rabbit ventricular myocytes. Forty‐second negative pulses from a holding potential of −20 mV induced Ihi whose conductance increased with hyperpolarization; the mean conductance (Ghi) was 63.6 ± 9.9 pS pF−1 (mean ±s.e.m., n= 9) at −160 mV. EB fluorescence increased during voltage pulses in parallel with the time integral of Ihi (Qhi), with the magnitude of the increases in nuclear EB fluorescence being 5.3 times greater than in the cytoplasm at −160 mV. Similar hyperpolarization‐induced parallel increases in Ihi and EB fluorescence were also obtained in Na+‐free, N‐methyl‐d‐glucamine (NMDG) solution. LPC (10 μm) induced large (101.2 ± 21.2 pS pF−1, n= 16), rapid (rise times, 1‐10 ms) Ihi with slow relaxation rates at −80 mV that reflected increases in Ghi to 94.3 ± 24.8 pS pF−1 (n= 8) at 6 min. Plots of EB fluorescence vs. Qhi were well fitted by a common Hills equation with a Hill coefficient of 0.97. Taken together, our findings indicate that hyperpolarization and LPC produced pores having the same filter properties for the permeation of small ions, including ethidium+, and that Ihi (carried in part by Ca2+) generated by membrane breakdown are capable of supplying sufficient ions to evoke abnormal excitation and contraction in cardiac myocytes.

ACTIVATED STELLATE (ITO) CELLS POSSESS VOLTAGE-ACTIVATED CALCIUM CURRENT

We previously reported stellate (Ito) cells possess voltage-activated Ca2+ current. The activation of stellate cells has been indicated to contribute to liver fibrosis and the regulation of hepatic hemodynamics. The aim of this study was to investigate the relationship between voltage-activated Ca2+ current and activation of stellate cells. Voltage-activated Ca2+ current in stellate cells isolated from rats were studied using whole-cell patch clamp technique. L-type voltage-activated Ca2+ current was hardly detected in stellate cells cultured for less than 9 days. Ca2+ current was detected 12.5 and 69% of cells at the 10th and 14th day of culture, respectively. BrdU incorporation indicated cell proliferation was recognized over 50% of cells at the 3rd and 5th day of culture, respectively, then decreased significantly in a time-dependent manner. On the other hand, the expression of alpha-smooth muscle actin indicated cell activation increased from 7th day of culture and collagen type I mRNA appeared remarkably in cells cultured for more than 10 days. In this study, we concluded L-type voltage-activated Ca2+ current was recognized in activated stellate (myofibroblast-like) cells.

Low K+‐induced hyperpolarizations trigger transient depolarizations and action potentials in rabbit ventricular myocytes

1 The effects of large reductions of [K+]o on membrane potential were studied in isolated rabbit ventricular myocytes using the whole‐cell patch clamp technique. 2 Decreasing [K+]o from the normal level of 5.4 mm to 0.1 mm increased resting membrane potential (Vrest) from −75.6 ± 0.3 to −140.3 ± 1.9 mV (means ± s.e.m; n= 127), induced irregular, transient depolarizations with mean maximal amplitudes of 19.5 ± 1.5 mV and elicited action potentials in 56.7 % of trials. The action potentials exhibited overshoots of 37.9 ± 1.5 mV (n= 72) and sustained plateaux. 3 Addition of 0.1 mm La3+ in the presence of 0.1 mm[K+]o significantly increased Vrest but decreased the amplitude of transient depolarizations and suppressed the firing of action potentials. 4 Replacement of external Na+ or Cl− with N‐methyl‐D‐glucamine or aspartate, respectively, or internal dialysis with 10 mm EGTA or BAPTA had little effect on low [K+]o‐induced membrane potential changes. 5 Hyperpolarizing voltage clamp pulses to potentials between −110 and −200 mV activated irregular inward currents that increased in amplitude and frequency with increasing hyperpolarization and were depressed by 0.1 mm La3+. 6 The generation of transient depolarizations by low [K+]o can be explained as being a consequence of decreasing the inward rectifier K+ current (IK1) and the appearance of inward currents reflecting electroporation resulting from strong electric fields across the membrane.