Tohru Sawanobori

Nitric oxide-dependent modulation of the delayed rectifier K+ current and the L-type Ca2+ current by ginsenoside Re, an ingredient of Panax ginseng, in guinea-pig cardiomyocytes.

Ginsenoside Re, a major ingredient of Panax ginseng, protects the heart against ischemia–reperfusion injury by shortening action potential duration (APD) and thereby prohibiting influx of excessive Ca2+. Ginsenoside Re enhances the slowly activating component of the delayed rectifier K+ current (IKs) and suppresses the L‐type Ca2+ current (ICa,L), which may account for APD shortening. We used perforated configuration of patch‐clamp technique to define the mechanism of enhancement of IKs and suppression of ICa,L by ginsenoside Re in guinea‐pig ventricular myocytes. S‐Methylisothiourea (SMT, 1 μM), an inhibitor of nitric oxide (NO) synthase (NOS), and N‐acetyl‐L‐cystein (LNAC, 1 mM), an NO scavenger, inhibited IKs enhancement. Application of an NO donor, sodium nitroprusside (SNP, 1 mM), enhanced IKs with a magnitude similar to that by a maximum dose (20 μM) of ginseonside Re, and subsequent application of ginsenoside Re failed to enhance IKs. Conversely, after IKs had been enhanced by ginsenoside Re (20 μM), subsequently applied SNP failed to further enhance IKs. An inhibitor of guanylate cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μM), barely suppressed IKs enhancement, while a thiol‐alkylating reagent, N‐ethylmaleimide (NEM, 0.5 mM), clearly suppressed it. A reducing reagent, di‐thiothreitol (DTT, 5 mM), reversed both ginsenoside Re‐ and SNP‐induced IKs enhancement. ICa,L suppression by ginsenoside Re (3 μM) was abolished by SMT (1 μM) or LNAC (1 mM). NEM (0.5 mM) did not suppress ICa,L inhibition and DTT (5 mM) did not reverse ICa,L inhibition, whereas in the presence of ODQ (10 μM), ginsenoside Re (3 μM) failed to suppress ICa,L. These results indicate that ginsenoside Re‐induced IKs enhancement and ICa,L suppression involve NO actions. Direct S‐nitrosylation of channel protein appears to be the main mechanism for IKs enhancement, while a cGMP‐dependent pathway is responsible for ICa,L inhibition.

Subcellular mechanism for Ca(2+)-dependent enhancement of delayed rectifier K+ current in isolated membrane patches of guinea pig ventricular myocytes.

Intracellular Ca2+ augments delayed rectifier K+ current (IK) in cardiac myocytes, which may play a major modulatory role in repolarization of action potentials. We investigated subcellular mechanisms for Ca(2+)-induced enhancement of IK in large-pipette inside-out membrane patches excised from isolated guinea pig ventricular myocytes. When [Ca2+]i was raised from 10(-8) to 10(-6) mol/L, the amplitude of IK measured at +80 mV was increased from 12.0 +/- 2.2 to 19.5 +/- 3.3 pA (P < .01). The enhancement of IK by Ca2+ was dose dependent, with an EC50 of 3.8 x 10(-8) mol/L. A calmodulin antagonist, W7 (50 mumol/L), calmidazolium (100 mumol/L), or HT-74 (20 mumol/L), added to the intracellular solution abolished enhancement of IK by Ca2+, whereas the inactive form of the W7 analogue, W5, had no effect on IK. In the presence of a protein kinase inhibitor with a relatively high specificity for protein kinase C (H7), for protein kinase A (H8 or peptide-type inhibitor PKI), or for calmodulin kinase II (KN-62) or a nonspecific inhibitor of serine/threonine protein kinases (staurosporine), increases in [Ca2+]i still enhanced IK. Ca(2+)-induced enhancement of IK was also observed when Mg2+ and ATP were omitted from the intracellular solution to delete exogenous phosphate donors and when adenylylimidodiphosphate was added to preclude trapped cytoplasmic substrates. Thus, cardiac IK was enhanced by increases in [Ca2+]i at a physiological range via a calmodulin-dependent pathway, which did not involve a phosphorylation process.

Mechanism Initiating Ventricular Fibrillation Demonstrated in Cultured Ventricular Muscle Tissue

Ventricular muscle strips of the rat embryo heart were used for tissue culture preparations without adding trypsin. Fibrillation-like arrhythmia was induced by adding aconitine or strophanthin, and the potentials were recorded with one or two microelectrodes. The tracing by one microelectrode was no different than tracings from fibrillating adult mammalian hearts. The size and shape of the action potential varied beat by beat, and its time of appearance was quite irregular and rapid. But, when tracings obtained simultaneously by two microelectrodes were compared, most of the action potentials were roughly synchronous and thus unlike adult cardiac fibrillation. This ruled out the possibility of multiple reentry or multifocal origin in this preparation. Because of the small size of the cultured tissue, reentry of the excitation wave probably could not occur, and the conclusion that fibrillation originated from a single focus is thus supported. Three varieties of the onset of fibrillation, i.e., gradual increase of tachycardia with progressively steeper slow diastolic depolarization, a prominent positive afterpotential followed by a negative afterpotential, and abortive action potentials superimposed on the repolarization of the preceding action potentials or on the negative afterpotential, supported the unifocal onset.

Electrophysiological effects of ginseng and ginsenoside Re in guinea pig ventricular myocytes.

Panax ginseng is a folk medicine with various cardiovascular actions; however, its underlying mechanisms of action are not well known. In the present study, we examined the effects of ginseng and its main component, ginsenoside Re, on action potentials and membrane currents recorded from isolated guinea pig ventricular myocytes with the whole-cell patch clamp technique. Ginseng (1 mg/ml) shortened the action potential duration in a rate-dependent manner. Ginseng depressed the L-type Ca2+ current (I(Ca-L)) in a mode of both tonic block and use-dependent block, and enhanced the slowly activating component of the delayed rectifier K+ current (I(Ks)). Ginsenoside Re 3 microM exhibited similar electrophysiological effects to those of 1 mg/ml ginseng, but of slightly smaller magnitude. Inhibition of I(Ca,L) and enhancement of I(Ks) by ginsenoside Re appear to be one of the main electrophysiological actions of ginseng in the heart, although contributions from other ingredients should be considered.

Multiple modulations of action potential duration by different calcium channel blocking agents in guinea pig ventricular myocytes.

Effects of extracellular applications of different types of Ca2+ channel blocking agents (Mn2+, verapamil, and nisoldipine) on action-potential duration and membrane currents were studied by the whole-cell patch-clamp technique in guinea pig ventricular myocytes. Low concentrations of Mn2+ (1 mM) and verapamil (1 microM) prolonged action-potential duration at 90% repolarization (APD90) with a suppressed plateau phase. Increases in Mn2+ (5 mM) and verapamil (5 microM) shortened APD90 with a further depression of the plateau. Nisoldipine (0.2-1 microM) shortened APD90 without lengthening it. Applications of Mn2+ and verapamil suppressed amplitudes of the L-type Ca2+ current (ICa), the delayed outward K+ current (IK), and the inward rectifier K+ current (IK1). Furthermore, the ratios of ICa:IK inhibition were similar by low and high concentrations of Mn2+ and verapamil. Nisoldipine selectively suppressed ICa without effect on IK and IK1. A low concentration (1 mM) of Mn2+ not only decreased the peak amplitude of ICa but also delayed its decay time course, which caused an increase in late ICa amplitude at the end of a 200-ms depolarizing pulse. Both verapamil and nisoldipine suppressed peak ICa without affecting its decay. Whereas Mn2+ suppressed IBa without changing its decay time course, verapamil and nisoldipine speeded up the IBa decay with suppressed amplitude of IBa. We conclude that different types of Ca2+ channel blocking agents (Mn2+, verapamil, and nisoldipine) diversely modulate APD because of their multiple modes of actions on ICa and IK.

Action of nicorandil on ATP‐sensitive K+ channel in guinea‐pig ventricular myocytes

1 Patch‐clamp techniques were used to study the effects of nicorandil (2‐nicotinamiodethyl nitrate) on the adenosine 5′‐triphosphate (ATP)‐sensitive K+ channel current (IK.ATP) in guinea‐pig ventricular myocytes. 2 Nicorandil activated the time‐independent outward current. This effect was dependent on intracellular ATP concentration ([ATP]i) showing a larger effect at 2 mm than at 10 mm [ATP]i The nicorandil‐induced outward current was inhibited by application of 0.3 μm glibenclamide. 3 In the inside‐out patch configuration, 0.3–1.0 mm nicorandil increased the open‐state probability of IK.ATP without a change in its conductance value (about 90pS). This effect was inhibited by glibenclamide. Analysis of the open and closed time distributions showed that nicorandil had no effect on open and closed distributions shorter than 5 ms. On the other hand, nicorandil increased the life time of bursts and decreased the interburst intervals. 4 The inward rectifier K+ channel current was not influenced by internal application of nicorandil. 5 Therefore, we conclude that IK.ATP is the only K+ current activated by nicorandil, and the main effect of nicorandil is on the kinetics of the IK.ATP bursting behaviour. These actions are similar to that of pinacidil on this preparation.

Stilbene disulfonates block ATP-sensitive K+ channels in guinea pig ventricular myocytes

Effects of stilbene disulfonates on single KATP channel currents were investigated in inside-out and outside-out membrane patches from guinea pig ventricular myocytes. All drugs tested, 4,4′-diisothiocyanatostilbene, 2,2′-disulfonic acid (DIDS), 4-acetamido0-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), and 4,4′-diaminostilbene-2,2′-disulfonic acid (DADS), inhibited the KATP channel when they were applied to the intracellular, but not extracellular side of the membrane patch. Inhibitory actions of DIDS and SITS were irreversible, whereas those induced by DNDS and DADS were reversible. KATP channel inhibition was concentration dependent with an order of potency of DIDS>SITS ≈ DNDS > DADS; the Hill coefficient was close to unity for each drug. No change in channel conductance was observed during exposure to DIDS or DNDS; however, channel kinetics was altered. Distribution of the open time within bursts and that between bursts could be described by a single exponential relation in the absence and presence of DIDS or DNDS. The time constant of the open time within bursts was not altered, but that between bursts was decreased by DIDS (from 40.0±8.1 to 29.8±6.7 msec, P< 0.05) and by DNDS (from 43.1±9.3 to 31.9±7.1 msec, P<0.05). Distributions of closed time within bursts were also fitted to a single exponential function both in the absence and presence of drugs, while those of the closed time between bursts were fitted to a single exponential function in the absence of drugs, but a double exponential function was required in the presence of drugs. The rates of onset and development of channel inhibition by DIDS and DNDS appeared to be concentration dependent; a longer time was required to reach a new steady-state of channel activity as drug concentration was decreased. Inhibition by DIDS or DNDS was regulated by intracellular pH; inhibition was greater during acidic conditions. For DIDS (0.1 mm), the open probability (Po) expressed as a fraction of the value before drug application was 42.9±8.3% at pH 7.4 and 8.2±6.6% at pH 6.5 (P<0.01); corresponding values for DNDS (1 mm) were 39.6±17.6 and 8.9 ±5.8%, respectively (P<0.01). From these data, we conclude that stilbene disulfonates block the KATP channel by binding to their target site with one-to-one stoichiometry. Similar to glibenclamide, the binding of stilbene disulfonates may reflect interpolation in an “intermediate lipid compartment” between the cytosolic drug and the site of drug action.

Abnormal Automaticity in Canine Purkinje Fibers Focally Subjected to Low External Concentrations of Calcium

The false tendon was isolated from the dog heart and mounted under a partition plate in the center of a chamber which was filled with Tyrodes solution. Two microelectrodes were positioned closely on either side of the plate and inserted into the false tendon. The partition did not alter repolarization of the action potential in normal Tyrodes solution. When EDTA was added to one side of the preparation, a low-plateau potential appeared after repolarization on the other side, which contained normal Tyrodes solution. This change occurred concurrently with a prolongation of the action potential in the calcium-free Tyrodes solution. The low-plateau potential was a reflection of the electrotonic potential from the nearby cells whose action potentials were prolonged. Repetitive firing of the action potential was frequently observed when abrupt differences in repolarization were recorded at close interelectrode distances. These results suggest that differences in repolarization of closely apposed cells can initiate arrhythmias.

Use-dependent block of Na+ currents by mexiletine at the single channel level in guinea-pig ventricular myocytes.

1 The mechanism of use‐dependent block of Na+ current by mexiletine was studied at the single channel level in guinea‐pig ventricular myocytes by the patch‐clamp techniques. All experiments were performed using stimulation protocols to enable us to analyze the strict dependence of changes in channel properties on channel use. 2 In cell‐attached patches, bath or pipette application of mexiletine (40 μm) produced a use‐dependent reduction of the peak average current without changes in single channel conductance. Null sweeps were increased and the number of openings per sweep decreased with successive pulses, whereas no significant change in the mean open time was detected during the train. 3 Block by mexiletine became greater when pulse duration was extended beyond the period in which channels were open, suggesting that block progressed without channel opening. 4 At near threshold potentials, mexiletine decreased the later occurrence of first openings. Additionally, late openings were reduced in a use‐dependent way. 5 We conclude that mexiltine binds to the inactivated closed states of the Na+ channel and then causes a failure of late openings as well as early, which results in null sweeps on subsequent depolarization.

The catalytic subunit of cyclic AMP-dependent protein kinase directly inhibits sodium channel activities in guinea-pig ventricular myocytes

We investigated the effects of the purified catalytic subunit (C subunit) of the cAMP-dependent protein kinase (A-kinase) on the cardiac Na+ channel currents. Single Na+ channel currents in guinea-pig ventricular myocytes were recorded using the patch clamp technique of the inside-out configuration. Application of C subunit decreased the peak average current and slowed the current decay, effects which were caused by decrease in the open probability of Na+ channels and increase in the first latency, whereas the unitary current amplitude and mean open times were not affected. We conclude that the cardiac Na+ channel is directly modulated by phosphorylation process through A-kinase.