Yoshizumi Habuchi

Ethanol inhibition of Ca2+ and Na+ currents in the guinea-pig heart

The effects of ethanol on L-type Ca2+ and fast Na+ currents (ICa and INa, respectively) were examined using the whole-cell patch-clamp experiments on guinea-pig ventricular cells. At a clinically relevant concentration of 24 mM, ethanol slightly but significantly shortened the action potential duration, and reduced the ICa by 7 +/- 4% (mean +/- S.D.). This concentration of ethanol did not affect INa, but a lethal concentration of ethanol (80 mM) significantly inhibited INa by 13 +/- 5%. The voltage dependence of INa activation was not affected by ethanol, whereas the inhibitions of ICa by 80 mM ethanol and INa by 240 mM were both accompanied by a several mV shift in the channel availability curve toward more negative potentials, suggesting that the channels in the inactivated state are more susceptible to ethanol. The ICa inhibition by ethanol at clinically relevant concentrations could contribute to a negative inotropic effect, action potential shortening and development of arrhythmias, while the pathophysiological significance of ethanol inhibition of INa seems less important.

Tyrosine kinase-dependent modulation by interferon-α of the ATP-sensitive K+ current in rabbit ventricular myocytes

We examined the effects of interferon‐α on the ATP‐sensitive K+ current (I K,ATP) in rabbit ventricular cells using the patch‐clamp technique. I K,ATP was induced by NaCN. Whole‐cell experiments indicated that interferon‐α (5×102–2.4×104 U/ml) inhibited I K,ATP in a concentration‐dependent manner (60.7±7.5% with 2.4×104 U/ml). In cell‐attached configuration, interferon‐α (2.4×104 U/ml) applied to the external solution also inhibited the activity of the single ATP‐sensitive K+ (KATP) channel by 56.0±5.8% without affecting the single channel conductance. The inhibitory effect of I K,ATP by interferon‐α was blocked by genistein and herbimycin A, tyrosine kinase inhibitors, but was not affected by N‐(2‐metylpiperazyl)‐5‐isoquinolinesulfoamide (H‐7), an inhibitor of protein kinase C and cAMP‐dependent protein kinase. These findings suggest that interferon‐α inhibits the cardiac KATP channel through the activation of tyrosine kinase. The tyrosine kinase‐mediated inhibition of I K,ATP by cytokines may aggravate cell damage during myocardial ischemia.

Electrophysiological effects of ibutilide on the delayed rectifier K+ current in rabbit sinoatrial and atrioventricular node cells

Biophysical and pharmacological characteristics of the delayed rectifier K(+) current (I(K)) of rabbit sinoatrial (SA) node and atrioventricular (AV) node cells have been studied using the whole-cell patch clamp technique together with a recently developed antiarrhythmic agent, ibutilide. Ibutilide is a potent blocker of the rapid delayed rectifier K(+) current, I(Kr). Superfusion with ibutilide (10(-7) M) caused a decrease in the spontaneous firing frequency, depolarization of the maximal diastolic potential and prolongation of the action potential duration in both SA and AV node cells. In whole cell voltage clamp experiments done on myocytes from SA node, ibutilide (10(-7) M) blocked I(K) strongly (40%) and had smaller effects on Ca(2+) current (10%) and hyperpolarization-activated inward current, I(f) (11%). In AV node cells, the corresponding reductions were I(K) (68%), I(Ca) (13%) and I(f) (10%), respectively. A 10-fold increase in the concentration of ibutilide further decreased I(K) in SA node cells (67+/-8%), and blocked I(K) almost completely in AV node cells. These results are consistent with the hypothesis that the delayed rectifier K(+) current in SA node cell is generated by both I(Kr) and I(Ks), whereas I(Kr) predominates in AV node cells. Knowledge of the differences in the distribution of I(Kr), as well as the different sensitivity to blockers of I(Kr) in nodal cells, is important for understanding modifications of the automaticity, conduction velocity, and refractoriness by class III antiarrhythmic agents.

Dopamine stimulation of cardiac β‐adrenoceptors: the involvement of sympathetic amine transporters and the effect of SKF38393

1 Mechanisms underlying β‐adrenoceptor stimulation by dopamine were examined on guinea‐pig Langendorff‐perfused hearts and isolated cells from the right atrium, by using the chronotropic effects and the enhancement of L‐type Ca2+ current (ICa,L) in the presence of prazosin as indicators of β‐adrenoceptor stimulation. Dopamine‐induced overflow of noradrenaline (NA) concentrations was measured by high‐performance liquid chromatography. 2 Dopamine caused positive chronotropic effects with an EC50 of 2.5 μm and induced NA overflow with a similar EC50 (1.3 μm). The chronotropic effect of dopamine was abolished by bisoprolol (1 μm). 3 The effects of dopamine were maintained during prolonged application, whereas the effects of tyramine faded with time. Dopamine (3 μm) restored the chronotropic effects and the NA release suppressed by pretreatment with tyramine, suggesting a de novo synthesis of NA during the exposure to dopamine. 4 Dopamine (3 μm)‐induced NA release was not affected by tetrodotoxin, ω‐conotoxin, rauwolscine, ICI118551 or sulpiride, but was inhibited by desipramine, a NA uptake inhibitor (IC50 ∼1 μm). It was also not affected by GBR12909 and bupropion, dopamine uptake inhibitors in the central nervous system. 5 SKF38393, a D1 receptor partial agonist, potently inhibited the 3 μm dopamine‐induced release of NA (IC50 ∼0.1 μm). D1 receptors are not involved in the DA‐induced release of NA, since SCH23390 (3 μm), a potent D1 antagonist, inhibited the NA release only slightly, and dihydrexidine (1 μm) and chloro‐APB (1 μm), full D1 agonists, caused no significant NA release. 6 SKF38393 inhibited tyramine‐induced overflow of NA, and potentiated the field stimulation‐induced NA release. SKF38393 and desipramine retarded the decay of the stimulation‐induced tachycardia in a similar manner. These results indicate that SKF38393 is a potent monoamine transport inhibitor and a useful tool for the functional evaluation of indirectly‐acting sympathomimetic agonists in the heart. In the presence of SKF38393 (10 μm), dopamine at 1 μm showed no chronotropic effect. 7 Voltage clamp experiments with isolated atrial cells revealed that dopamine is a weak partial agonist. The EC50 for ICa,L stimulation by dopamine was high (13 μm). As a result, dopamine at 1 μm did not affect ICa,L. Bisoprolol abolished the stimulation of ICa,L by dopamine (30 μm), and dihydrexidine (1 μm) did not affect ICa,L. 8 It was concluded that the cardiac effects of dopamine at clinically relevant concentrations (<1 μm) result almost exclusively from the indirect effect of β adrenoceptor stimulation, involving the release of NA from sympathetic nerve terminals. The roles of the direct stimulation of β adrenoceptors by dopamine at these concentrations and the stimulation of postjunctional D1 receptors seem negligible. The desipramine‐ and SKF38393‐sensitive monoamine transporter mediates the release of NA.

Negative chronotropic actions of endothelin-1 on rabbit sinoatrial node pacemaker cells

1 The effects of endothelin‐1 (ET‐1) on sinoatrial (SA) node preparations of the rabbit heart were studied by means of whole‐cell clamp techniques. 2 ET‐1 at 1 nM slowed the spontaneous beating activity and rendered half of the cells quiescent. At a higher concentration of 10 nM, the slowing and cessation of spontaneous activity were accompanied by hyperpolarization. 3 In voltage‐clamp experiments, ET‐1 decreased the basal L‐type Ca2+ current (ICa(L)) dose‐dependently with a half‐maximal inhibitory concentration (EC50) of 0.42 nM and maximal inhibitory response (Emax) of 49.5%. The delayed rectifying K+ current (IK) was also reduced by 33.2±11.1% at 1 nM. In addition, an inwardly rectifying K+ current was activated by ET‐1 at higher concentrations (EC50=4.8 nM). These ET‐1‐induced changes in membrane currents were abolished by BQ485 (0.3 μM), a highly selective ETA receptor antagonist. 4 When ICa(L) was inhibited by ET‐1 (1 nM), subsequent application of 10 μM ACh showed no additional decrease in ICa(L), suggesting the involvement of cyclic AMP in the effects of ET‐1 on ICa(L). In contrast, 1 nM ET‐1 further decreased ICa(L) in the presence of 10 μM ACh, suggesting that ET‐1 activates some additional mechanism(s) which inhibit ICa(L). The ET‐1‐induced ICa(L) inhibition was abolished by protein kinase A inhibitory peptide (PKI, 20 μM) or H‐89 (5 μM). However, the ICa(L) inhibition was not affected by methylene blue (10 μM), suggesting a minor role for cyclic GMP in the effect of ET‐1 under basal conditions. 5 ET‐1 failed to inhibit ICa(L) when the pipette contained GDPβS (200 μM). However, incubation of the cells with pertussis toxin (PTX, 5 μg ml−1, >6 h) only reduced the ET‐1‐induced inhibition to 21.5±9.5%, whereas it abolished the inhibitory effect of ACh on ICa(L). 6 Intracellular perfusion of 8‐bromo cyclicAMP (8‐Br cyclicAMP, 500 μM) attenuated, but did not abolish the inhibitory effect of ET‐1 on ICa(L). This 8‐Br cyclicAMP‐resistant component (17.5±14.4%, n=20) was not affected by combined application of 8‐Br cyclicAMP with 8‐bromo cyclicGMP (500 μM), ryanodine (1 μM) or phorbol‐12‐myristate‐13‐acetate (TPA; 50 nM). 7 In summary, ET‐1 exerts negative chronotropic effects on the SA node via ETA‐receptors. ET‐1 inhibits both ICa(L) and IK, and increases background K+ current. The inhibition of ICa(L) by ET‐1 is mainly due to reduction of the cyclicAMP levels via PTX‐sensitive G protein, but some other mechanism(s) also seems to be operative.

Blockade of Na+ current by promethazine in guinea‐pig ventricular myocytes

1 To elucidate the antiarrhythmic mechanism of promethazine, its effects on the fast Na+ current (INa) were examined in single guinea‐pig ventricular myocytes by whole‐cell voltage clamp methods. 2 Promethazine blocked INa with a KD of 42.6 μm and Hills coefficient of 1.1 at a holding potential of — 140 mV. 3 The INa blockade was enhanced at a less negative holding potential of − 80 mV with a change of KD to 4.4 μm. Although 10 μm promethazine did not change the inactivation time constants of INa, it shifted the steady‐state inactivation curve (h∞ curve) toward more negative potentials by 19.5 mV with the slope factor unaffected. 4 Double pulse experiments revealed that the development of blockade followed two‐exponential functions having time constants of 7 and 220 ms at − 20 mV. 5 Promethazine slowed the repriming of INa. This was associated with the development of slow phase having a time constant of 1160 ± 59 ms. 6 Promethazine produced a profound use‐dependent block when the cell was repeatedly stimulated with interpulse intervals shorter than 1 s. However, short pulses of 2 ms duration hardly produced such a use‐dependent block. Hence, open channel blockade is considered to play a minor role in the promethazine action on INa. 7 These results suggest that promethazine blocks cardiac INa in a manner similar to class I antiarrhythmic drugs and that this effect may account for its antiarrhythmic action.

Effects of intracerebroventricular and intravenous injections of endothelin-1 on blood pressure and sympathetic activity in urethane-anesthetized rats.

Because endothelin-1 (ET-1) is believed to be an endogenous calcium-channel agonist, it was thought that it may affect not only vascular smooth muscle cells but also nerve activity. Intravenous injections of ET-1 (0.01 nmol) did not affect cardiovascular parameters or renal sympathetic activity. ET-1 (1 nmol) initially decreased blood pressure for a few minutes, and then caused an increase for 5-10 min. Blood pressure then returned to baseline but later there was a rise in pressure for more than 60 min. Sympathetic activity was markedly suppressed during the initial hypertensive phase but increased during the later phase. Intracerebroventricular injections of ET-1 increased blood pressure even with the small dose (0.01 nmol). The increase was maintained for about 10 min, and returned to baseline. It then decreased abruptly with marked bradycardia, and finally returned to the baseline 60-90 min later. These results indicate that ET-1 affects not only the vascular smooth muscle but also the autonomic nervous system to influence cardiovascular functions.

Modulation by chloramine-T of 4-aminopyridine-sensitive transient outward current in rabbit atrial cells

The effects of an oxidizing agent, chloramine-T, on the 4-aminopyridine-sensitive transient outward current (ITO) were investigated in rabbit atrial myocytes by using patch-clamp techniques. Extracellular application of chloramine-T at 20 microM irreversibly slowed the time course of inactivation of the whole-cell ITO, and increased the peak by 19.3% (n = 19) at +40 mV. At 100 microM, chloramine-T decreased the peak by 22.5% (n = 9) of the control, and subsequently induced a glibenclamide-sensitive time-independent outward K+ current. Under superfusion with dithiothreitol (3 mM), chloramine-T (100 microM) produced no change in ITO. The chloramine-T-induced slowing of ITO inactivation was partially reversed by subsequent application of 3 mM dithiothreitol. In single-channel recordings with the cell-attached patch configuration, chloramine-T (20 microM) increased the open probability of the ITO channel from 0.15 to 0.46 at a potential 100 mV positive to the resting potential, and the mean open lifetime from 5.1 ms to 7.0 ms (n = 5). The unitary current amplitude was not affected. As a result, chloramine-T increased the ensemble current in amplitude and slowed its decay. These results indicated that: (1) inactivation of the native A-type channels of rabbit heart is susceptible to oxidation; and (2) oxidation of ITO channels may contribute to the genesis of arrhythmias.

Effects Of Hydrogen Peroxide On The Transient Outward Current In Rabbit Atrial Myocytes

1. In the present study, we investigated the effects of hydrogen peroxide (H2O2) on the 4‐aminopyridine‐sensitive transient outward current (ITO) in rabbit atrial myocytes using the amphotericin B‐perforated patch voltage‐clamp method.

The mechanisms underlying heart stimulation by dopamine, with special reference to direct and indirect β adrenoceptor stimulation

1. The positive chronotropic and norepinephrine-releasing effects of dopamine were examined in the isolated guinea pig heart, using the Langendorff model. 2. The released norepinephrine was estimated from the norepinephrine concentration measured in the post-perfusion solution using HPLC. 3. The dose-response curve for dopamine to stimulate the heart rate (HR) closely resembled that for the norepinephrine release. A selective beta 1 antagonist bisoprolol completely abolished the positive chronotropic effect, but did not affect the norepinephrine release. 4. The HR increase in response to 3 mumol/L dopamine was 54 +/- 15% (n = 14) of the control in normal hearts. The response was decreased to 15 +/- 7% (n = 6) by pretreatment with reserpine. 5. A D1 antagonist, SKF83742, (3 mumol/L) shifted the dose-response curve for the dopamine-induced norepinephrine release toward the right, indicating the involvement of D1-like dopamine receptors. 6. Voltage clamp experiments using single cells isolated from the right atrium revealed that dopamine is a weak partial agonist for beta adrenoceptors. Dopamine stimulated the L-type Ca2+ current with a threshold concentration of 3 mumol/L. 7. These findings indicate the important role of the norepinephrine release in the stimulation of beta adrenoceptors by dopamine at clinically relevant concentrations.