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The mechanisms underlying the activation of large conductance Ca2+-activated K+ (BK) channel by nordihydroguaiaretic acid (NDGA) were examined in human embryonic kidney (HEK293) cells, where BK channel alpha (BKalpha) or a plus beta1 subunit (BKalphabeta1) was heterologously expressed, and also in freshly isolated porcine coronary arterial smooth muscle cells (PCASMCs). The activity of both BKalpha and BKalphabeta1 channels was increased by 10 microM NDGA in similar manners, indicating the selective action on the a subunit to increase Ca2+ sensitivity. The application of NDGA to PCASMCs induced outward current and hyperpolarization under voltage and current clamp, respectively, in a concentration-dependent manner (> or = 3 microM). These effects were blocked by 100 nM iberiotoxin. Electrical events induced by NDGA (> or = 10 microM) were, unexpectedly, associated with the increase in [Ca2+]i. After the treatment with caffeine and ryanodine, the [Ca2+]i increase by NDGA was markedly reduced and the hyperpolarization by NDGA was attenuated. The Ca2+ release by 10 microM NDGA was preceded by membrane depolarization of mitochondria. These results indicate that BK channel opening by NDGA in PCASMCs is due to the direct action on a subunit and also to Ca2+ release from sarcoplasmic reticulum, presumably via, at least in part, the inhibition of mitochondria respiration.

The usefulness of bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials (MPs) was evaluated in HEK293 cells, where alpha or alpha plus beta1 subunits of large conductance Ca2+-activated K+ (BK) channels were expressed (HEKBK alpha and HEKBK alphabeta). The fluorescent intensity of DiBAC4(3) was measured at various potentials under voltage-clamp for calibration to estimate the absolute MP semi-quantitatively. The resting MPs measured with DiBAC4(3) were roughly comparable to those recorded with a microelectrode; the MP in HEKBK alphabeta was 10-20 mV more negative than that in native HEK. In HEKBK alpha, the membrane hyperpolarization induced by 10 microM Evans blue, a BK channel opener, was detected with DiBAC4(3). NS-1619, another BK channel opener, induced gradual but substantial change in F/F(K) even in native HEK, while the BK channel opening effect was detected. Oscillatory membrane hyperpolarization was induced in HEKBK alphabeta by application of 10 microM acetylcholine via increase in intracellular Ca2+ concentration. The oscillatory hyperpolarization was, however, detected only as a slow hyperpolarization with DiBAC4(3). It can be concluded that relatively slow effects of BK channel modulators can be semi-quantitatively measured by use of DiBAC4(3) in HEKBK, while the limited temporal resolution and possible artifacts should be taken into account.

Simultaneous recording of Ca2+-images in one confocal plane from vascular smooth muscle cells (SMCs) and endothelial cells (ECs) of an intact rat femoral artery segment was performed using indo-1 and a confocal microscope. During application of 10 microM acetylcholine (ACh), elevation and oscillation of intracellular Ca2+ concentration ([Ca2+]i) were observed in ECs but not in SMCs. Sequential conduction of Ca2+ oscillation from an EC to the neighboring ECs in one longitudinal direction was often observed in the presence of ACh. On the other hand, the activation of voltage-dependent Ca2+ channels by external 30 mM K+ resulted in the elevation of [Ca2+]i only in SMCs. When 10 microM ACh was added in the presence of 30 mM K+, it was observed in one confocal plane that [Ca2+]i in ECs and SMCs was almost simultaneously increased and decreased, respectively. The simultaneous recording method in this intact preparation will provide a line of valuable information about the interactions between SMCs and ECs, based on spatio-temporal analyses of absolute values of [Ca2+]i in individual cells.

The single channel current of intermediate conductance Ca2 +-activated K+ channel (IK channel) was measured in mouse urinary bladder myocytes (MBM), and the molecular basis of the channel was suggested to be the SK4 subtype by RT-PCR. Among Ca2+-activated K+ channel subtypes (SK2, SK3, SK4 and BK), the mRNAs of SK4 and BK were predominantly expressed in MBM. IK channel currents recorded from MBM showed: 38.7 pS slope conductance under symmetrical 140 mM K+ conditions, Ca2+-dependent activation, and blockade by charybdotoxin and econazole. These results strongly suggest that SK4 encodes IK channels in MBM.

Induction of electrical abnormalities (EAs) under simulated ischemic conditions and after reperfusion was measured from single cardiac myocytes isolated from guinea pig ventricle using whole-cell voltage or current clamp with perforated patch variation. Conditions of simulated ischemia were produced by the exchange of medium from the standard one oxygenated with 95% O2-5% CO2 gas (pH 7.4) to the modified one, which contained no glucose, 8 mM K+ and 30 mM sodium-D,L-lactate and was gassed with 90% argon-10% CO2 (pH 6.6). Under the simulated ischemia for 20 min, EAs such as delayed afterdepolarization, early afterdepolarization, automatic activity or transient inward current were observed in about 37% of myocytes driven electrically at 1 Hz. Irreversible hypercontracture occurred in myocytes of 10% or less. Upon reperfusion with the standard solution, EAs and hypercontracture were observed in about 43% and 22% of cells, respectively. Glibenclamide-sensitive current was detected during ischemia, but tended to be enhanced during reperfusion. Amplitude of Ca2+ current and ATP-sensitive K+ current after reperfusion varied widely with time and from cell to cell. When myocytes were pretreated for 10 min with 10 nM benidipine, a 1,4-dihydropyridine derivative Ca2+ blocker, the incidence of EAs and hypercontracture was markedly reduced, suggesting the protective effect of benidipine against cardiac cell injury during ischemia and reperfusion.

Effects of K+ channel modulators, cromakalim and E4031 [1-[2-(6-methyl-2-pyridyl)-ethyl]-4-(4-methylsulfonylaminobenzoyl) piperidine], on the relationship between the action potential duration (APD) and Ca2+ transients were examined in single myocytes isolated from guinea pig cardiac left ventricle. Application of cromakalim decreased APD at 90% repolarization (APD90) and Ca2+ transient elicited at 0.5 Hz (IC50s=0.6 and 3 microM, respectively). Application of 0.3 microM E4031 increased these parameters by 110% and 45%, respectively. Under voltage-clamp, the relation between the duration of depolarization to 0 mV and Ca2+ transients could be described by the sum of two exponential components; the time constants were approximately 5 and 280 msec, respectively. The first component was abolished by 10 microM ryanodine, suggesting the involvement of Ca2+-induced Ca2+ release (CICR). Neither cromakalim nor E4031 directly affected Ca2+ current and Ca2+ transients under voltage clamp. When APD was changed by K+ channel modulators, the relation between APD90 and Ca2+-transients was almost similar to that obtained by changing the depolarization duration under voltage-clamp. CICR was changed significantly only when APD90 was markedly shortened by cromakalim. The extensively prolonged AP and Ca2+ transient in the presence of E4031 were reduced by an addition of cromakalim. It is concluded that these two K+ channel modulators can significantly alter the AP-induced Ca2+ transient mainly by changing APD, which regulates both Ca2+ influx and extrusion.

Ca2+ spark is a local and transient Ca2+ release from sarcoplasmic reticulum (SR) through the ryanodine receptor Ca2+-releasing channel (RyR). In cardiac myocytes, Ca2+ spark is an elementary unit of Ca2+-induced Ca2+ release (CICR) by opening of RyR(s) in junctional SR (jSR), which is triggered by Ca2+-influx through L-type Ca2+ channels to the narrow space between a transverse tubule and jSR. Ca2+ spark has, therefore, been described as the evidence for "the local control of excitation-contraction coupling". In contrast, Ca2+ sparks in smooth muscle have been reported in relation to Ca2+-dependent K+ (K(Ca)) channel activation and muscle relaxation. A spontaneous Ca2+ spark in a superficial area activates 10-100 K(Ca) channels nearby and induces membrane hyperpolarization, which reduces Ca2+ channel activity. In several types of smooth muscle cells, which have relatively high membrane excitability, an action potential (AP) elicits 5-20 Ca2+ hot spots (evoked sparks with long life) in the early stage via CICR in discrete superficial SR elements and activates K(Ca)-channel current highly responsible for AP repolarization and afterhyperpolarization. CICR available for contraction may occur more slowly by the propagation of CICR from superficial SR to deeper ones. The regulatory mechanism of ion channel activity on plasma membrane by superficial SR via Ca2+ spark generation in smooth muscle cells may be analogously common in several types of cells including neurons.

Effects of sematilide, a novel class III antiarrhythmic agent, on the delayed rectifier K+ current (Ik) were examined in guinea pig atrial myocytes using a voltage clamp technique. Sematilide inhibited both time-dependent outward current upon depolarization and tail currents (Ik-tail) at -40 mV. The concentration of sematilide required for a 50% decrease in Ik-tail was approximately 50 microM. The sematilide-sensitive current obtained using a triangular voltage command exhibited marked inward rectification and had the maximum amplitude at -30 mV. These results suggest that sematilide inhibits rapidly activating Ik in guinea pig atrial myocytes, resulting in the prolongation of action potential duration and refractoriness.

Electrophysiological effects of sematilide, a novel class III antiarrhythmic agent, were examined and compared with those of (+/-)sotalol in guinea pig left atrium by a conventional microelectrode technique. Application of 0.1-1000 microM sematilide or 1-1000 microM (+/-)sotalol concentration-dependently prolonged the duration of action potentials (APD) that were elicited by electrical stimulation at 1 Hz. Other parameters of action potentials such as the maximum upstroke velocity of phase 0 depolarization, action potential amplitude and resting membrane potential were not affected significantly by these drugs in the concentration ranges employed. The prolongation of APD by sematilide or (+/-)sotalol was accompanied by a corresponding increase in the effective refractory period (ERP). Approximately a 30% increase in ERP was obtained by the treatment with 5 microM sematilide or 100 microM (+/-)sotalol, suggesting that sematilide as a class III antiarrhythmic agent is approximately 20 times more potent than (+/-)sotalol on a molecular basis. When the stimulation rate was increased stepwise from 0.2 to 2 Hz, the relative increase in APD at 90% repolarization by the treatment with sematilide and (+/-)sotalol was slightly larger at 2 Hz than at 0.2 Hz, indicating that "reverse rate-dependence" was not observed under these conditions. These results may suggest a possibility that sematilide effectively blocks atrial arrhythmia.