Cardiac capillary cells release biologically active nitric oxide at an early stage of in vitro development.

OBJECTIVE: Coronary microvascular endothelial cells (EC) may regulate the myocardial contractile function by releasing cardioactive agents such as nitric oxide (NO). However, understanding of these regulatory mechanisms is complicated by the fact that EC exhibit marked phenotypic changes, such as the loss of endothelial NO synthase (eNOS), when they are placed into culture. Recently, it has been shown that eNOS gene expression is regulated by specific cell-cell interactions with mural cells depending on vascular beds. Since EC and pericytes (PL) are closely associated in capillaries, we have enzymatically isolated these cells from rat hearts to develop a primary culture of capillary cells favoring the re-establishment of cell interactions in vitro. METHODS: Expression of transcripts for both eNOS and the inducible isoform (iNOS), was evaluated by using reverse transcription, polymerase chain reaction and Southern blot analysis. Expression of NOS proteins was detected with specific rhodamine-labeled antibodies. Production of NO was assessed (i) from nitrite measurements in culture supernatants by the Griess reaction, and (ii) from its antiproliferative action on cardiac fibroblasts (FIB) in non-contacted cocultures (reporter-cell bioassay) compared to that of sodium nitroprusside in homotypic FIB cultures. Fura-2 fluorescence was used to measure agonist-induced changes in cytosolic free calcium levels. RESULTS: In our heterotypic cultures, EC firstly proliferated to form spots of monolayers (i.e. first phase) before to be covered by PL on the following days (i.e. second phase). The data from RT-PCR analysis demonstrate the presence of mRNAs of both eNOS and iNOS at all developmental stages of the culture. However, eNOS protein was only detected and restricted to EC. During the first phase of cell growth (5-8 days), cells released nitrite and a labile factor, clearly identified as NO, that inhibited the FIB proliferation in reporter-cell bioassay. These effects, not observed during the second phase of cell growth (15-20 days), were prevented by hemoglobin (50 microM) and by N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM). At the two periods of culture, EC increased rapidly their cytosolic Ca(2+) concentration in response to bradykinin (10 nM). However, this calcium response was associated with an increase in nitrite production only in older cultures. CONCLUSIONS: Our data indicate that heterotypic cultures of native capillary cells preserve the eNOS expression by EC. This enzyme is basally active at an early stage of in vitro development, and then becomes activatable by a Ca(2+)-mobilizing agonist. NO released by growing EC downregulates the proliferation of cardiac FIB, an effect which could be important in the cardiovascular plasticity.

Dilation and action potential lengthening in cardiomyopathic Syrian hamster heart.

The aim of our study was to determine the main ionic mechanisms responsible for the electrophysiological alterations of ventricular action potentials associated with cardiac dilation in a strain of cardiomyopathic Syrian hamsters which does not develop hypertrophy during the first five months of life. Right and left ventricular action potentials (APs) were recorded in Langendorff perfused isolated hearts from dilated cardiomyopathic (MS 200) and normal hamsters at 60, 120, and 180 days of age. AP characteristics differed in the two ventricles and in different regions (base, apex) of the left ventricle in both strains. When recorded in a given region (apex), the plateau was always of higher amplitude and longer duration, i.e., of larger area, in diseased as compared to normal hearts. The participation of the calcium-independent 4-aminopyridine (4-AP) sensitive transient outward current, Ito1, in the left ventricular AP plateau repolarization was smaller in dilated than in control hearts at any age and AP area was the same in both strains at 60 days of age in the presence of 4-AP. The participation of the cadmium (Cd) sensitive L-type Ca current was investigated in the development of AP plateau at 120 days of age and was smaller in dilated than in control hearts. The participation of the Na-Ca exchange inward current, INa-Ca, in the development of the AP plateau was similar in both strains at 60 days of age; later on, it strongly decreased in control hearts, whereas it remained high in diseased hearts. The tetrodotoxin sensitive slowly inactivating inward current was not increased in dilated hearts compared to control hearts. Our results show that the AP lengthening observed, in dilated non-hypertrophic hamster hearts, results essentially from a reduced participation of Ito1 at 60 days of age, whereas it results from both a reduced participation of Ito1 and an increased participation of INa-Ca at 120 and 180 days of age.

Contrasting effects of intracellular redox couples on the regulation of maxi-K channels in isolated myocytes from rabbit pulmonary artery.

1. The effects of intracellular redox couples were investigated on the activation by voltage, Ca2+ and NS 1619 of maxi-K channels in enzymatically isolated smooth muscle cells from large pulmonary arteries of rabbits. 2. In inside-out membrane patches, maxi-K channels were characterized by a single-channel conductance of 266 pS in symmetrical 140 mM KCl solutions. The relationship between the open-state probability (Po) and the membrane potential could be fitted to the Boltzmann equation. The activating action of intracellular Ca2+ was reversible, concentration dependent, and was manifested as the reduction in the voltage necessary to half-activate the channel (V1/2) with no change in the slope factor. NS 1619 also predisposed the maxi-K channel to open at more hyperpolarized membrane potentials. 3. The oxidizing agent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB, 1 mM) activated maxi-K channels by inducing a negative shift of the activity-voltage curve, while the reducing agent 2-hydroxy-1-ethanethiol (beta-mercaptoethanol) (BME, 1 mM) had no effect. DTNB increased the efficacy of Ca2+ in activating maxi-K channels. The action of DTNB was not reversible upon wash-out, but could be counteracted by BME. 4. Maxi-K channel activity was unaffected by other oxidizing agents, such as NAD (2 mM) and glutathione disulphide (GSSG, 5 mM), or by their reduced forms (NADH and GSH). Mg-ATP (0.1 and 1 mM) increased the channel activity in a dose-dependent manner, while guanine nucleotides (100 microM GTP gamma S, 500 microM GDP and 200 microM GDP beta S) had no effect. 5. Our data suggest that a change in the intracellular redox state, which would be expected during acute hypoxia, does not alter the activity of maxi-K channels of large pulmonary artery smooth muscle cells. The sulfhydryl-specific redox reagents (DTNB and BME) must act through another regulatory mechanism.

Effects of halothane and isoflurane on bradykinin-evoked Ca2+ influx inbovine aortic endothelial cells.

BACKGROUND: Volatile anesthetics, such as halothane and isoflurane, have been reported to affect the endothelium mediated relaxation of vascular smooth muscle cells. Because the activity of the constitutive nitric oxide synthase in endothelial cells depends on the availability of intracellular Ca2+, there is a definite possibility that the observed inhibitory effect of volatile anesthetics involves an action on the agonist-evoked internal Ca2+ mobilization and/or Ca2+ influx in these cells. Therefore, a study was undertaken to determine how halothane and isoflurane affect the Ca2+ signalling process in vascular endothelial cells. METHODS: The effect of halothane and isoflurane on the Ca2+ response to bradykinin of bovine aortic endothelial (BAE) cells was investigated using the fluorescent Ca2+ indicator fura-2. Halothane or isoflurane was applied either to resting cells or after bradykinin stimulation. The agonist-evoked Ca2+ influx in BAE cells was estimated by measuring either the rate of fura-2 quenching induced by Mn2+ or the increase in cytosolic Ca2+ concentration initiated after readmission of external Ca2+ after a brief exposure of the cells to a Ca(2+)-free external medium. The effects of halothane on cell potential and intracellular Ca2+ concentration were measured in cell-attached patch-clamp experiments in which a calcium-activated K+ channel and an inward rectifying Ca(2+)-independent K+ channel were used as probes to simultaneously monitor the intracellular Ca2+ concentration and the cell transmembrane potential. In addition, combined fura-2 and patch-clamp cell-attached recordings were carried out, to correlate the variations in internal Ca2+ caused by halothane and the activity of the Ca(2+)-dependent K+ channels, which are known in BAE cells to regulate intracellular potential. Finally, a direct action of halothane and isoflurane on the gating properties of the Ca(2+)-activated K+ channel present in these cells was investigated in patch-excised inside-out experiments. RESULTS: The results of the current study indicate that the initial Ca2+ increase in response to bradykinin stimulation is not affected by halothane, but that pulse applications of halothane (0.4-2 mM) or isoflurane (0.5-1 mM) reversibly reduce the sustained cytosolic Ca2+ increase initiated either by bradykinin or by the Ca2+ pump inhibitor thapsigargin. In addition, halothane appeared to dose-dependently inhibit the Ca2+ influx evoked by bradykinin, and to cause, concomitant to a decrease in cytosolic Ca2+ concentration, a depolarization of the cell potential. Halothane failed, however, to affect internal Ca2+ concentration in thapsigargin-treated endothelial cells, which were depolarized using a high K+ external solution. Finally, halothane and isoflurane decreased the open probability of the Ca(2+)-dependent K+ channel present in these cells. CONCLUSIONS: These observations suggest that the effects of halothane and isoflurane on Ca2+ homeostasis in BAE cells reflect, for the most part, a reduction of the thapsigargin- or bradykinin-evoked Ca2+ influx, which would be consequent to a cellular depolarization caused by an inhibition of the Ca(2+)-dependent K+ channel activity initiated after cell stimulation.

Ionic basis of the action potential prolongation in ventricular myocytes from Syrian hamsters with dilated cardiomyopathy.

OBJECTIVE: The aim of our study was to determine the main electrophysiological alterations associated with cardiac dilation in MS200 strain Syrian hamsters, a model of genetically determined cardiomyopathy. METHODS: Ventricular action potentials (APs) were recorded with standard microelectrodes in isolated hearts from 120-day-old cardiomyopathic (strain MS200) and age-matched control (strain CHF148) Syrian hamsters. Ionic currents were recorded from single ventricular myocytes using the whole-cell patch-clamp technique. RESULTS: In MS200, AP was prolonged and the plateau phase was markedly increased as compared to CHF148. Differences in both AP duration and 4-aminopyridine-induced AP lengthening between epicardial and endocardial tissues were less marked in MS200 than in CHF148 ventricles. Cell size and membrane capacitance were not higher in MS200 than in CHF148 myocytes, indicating the absence of cell hypertrophy in myopathic ventricles. The L-type calcium current (ICa,L) density was significantly reduced in MS200 and the voltage-dependence of both steady-state activation and inactivation was altered. The voltage-dependent outward current was composed of both transient (Ito1) and sustained (Iss) components, respectively sensitive and insensitive to 4-aminopyridine. Ito1 density was strongly depressed in MS200 compared to CHF148, whereas Iss density was only slightly reduced. The conductance-voltage and steady-state inactivation relationships for Ito1 were shifted to more positive potentials in MS200. The Ito1 recovery process was markedly slower in MS200 than in CHF148. The steady-state current-voltage relationships, in the physiological voltage range, were superimposable in MS200 and CHF148. CONCLUSIONS: In ventricular myocytes from dilated heart of MS200 Syrian hamsters, Ito1 is more drastically depressed than ICa,L. Such an observation might partially explain dilation-induced AP lengthening.

Depressed transient outward current density in ventricular myocytes from cardiomyopathic Syrian hamsters of different ages.

We determined whether the dilated cardiomyopathy which develops between 30 and 140 days of age in the Syrian hamster strain MS200, before the onset of cardiac hypertrophy and failure, is associated with alterations in both the action potential (AP) and the Ca(2+)-independent transient outward current, Ito1. AP was recorded in perfused hearts using microelectrodes and Ito1 was recorded in single ventricular myocytes using the whole-cell patch-clamp. The MS200 strain was compared to the control CHF148 strain at different periods of age (60, 90, 120 and 180 days). APs were markedly lengthened in MS200 compared to CHF148 hearts at all ages studied. Cell membrane capacitance increased with age in the two strains, but was not significantly higher in MS200 than in CHF148 of a given age, indicating the absence of cell hypertrophy. At 60 days of age, Ito1 density was the same in the two strains. Later on, Ito1 density increased markedly at 90-120 days then decreased at 180 days in CHF148, whereas this increase was delayed and of reduced amplitude in MS200. The sustained component of outward current, Isus, was not sizeably different in the two strains. The conductance-voltage and steady-state inactivation relationships were shifted with age towards positive potentials by 15 mV in the two strains, but earlier in MS200 (90 days) than in CHF148 (180 days). Similarly, the recovery of Ito1 from inactivation exhibited a slow component which increased with age in the two strains but was larger in MS200 than in CHF148. In conclusion, alterations of Ito1 may contribute to changes in shape of AP, but cannot entirely explain dilation-induced AP lengthening.

Time-dependent fading of the activation of KATP channels, induced by aprikalim and nucleotides, in excised membrane patches from cardiac myocytes.

1. The effects of the potassium channel opener (KCO) aprikalim (RP 52891) on the nucleotide-induced modulation of ATP-sensitive K+ (KATP) channels in freshly dissociated ventricular myocytes of guinea-pig heart, were studied by use of the inside-out patch-clamp technique. The internal surface of the excised membrane patch was initially bathed with a standard solution (Mg(2+)-free with EDTA), then sequentially superfused with solutions containing nucleoside diphosphates (NDPs: 200 microM ADP and 50 microM GDP) and NDPs plus 1 mM MgCl2 (with EGTA; referred to as Mg-NDP solution). 2. The normalized concentration-response (channel closing) relationship to ATP was shifted to the right when the standard solution was replaced by the Mg-NDP solution. Hence, the internal concentration of ATP ([ATP]i) inhibiting the channel activity by half (Ki) increased from 56 microM to 180 microM, with an apparently constant slope factor (s = 2.37). NDPs in the absence of Mg2+ did not decrease the sensitivity of the channels to ATP. 3. In standard solution, aprikalim (100 microM) activated KATP channels in the presence of a maximally inhibitory [ATP]i (500 microM). This effect was strongly enhanced when aprikalim was applied to patches exposed to Mg-NDP solution, as demonstrated by the 9 fold increase in Ki for [ATP]i (from 180 microM to 1.5 mM and s = 2.37). 4. The ability of aprikalim to overcome the channel closing effects of ATP in Mg-NDP solution waned rapidly. Similarly, the NDP-induced activation of ATP-blocked channels was also time-dependent. Both activation processes disappeared before the channel run-down phenomenon appeared in ATP-free conditions. 5. In conclusion, aprikalim is much more potent in opening KATP channels in membrane patches bathed in Mg-NDP solution than in standard solution. However, under the former experimental conditions, the effect of aprikalim waned rapidly. It is proposed that the waning phenomenon results from changes in the intrinsic enzymatic activity of the KATP channel protein (possibly linked to the experimental conditions) which lead to the channel closure.

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. I. Effects of caffeine on intracellular Ca2+ stores.

The effects of agents known to interfere with Ca2+ release processes of endoplasmic reticulum were investigated in bradykinin (BK)-stimulated bovine aortic endothelial cells (BAE cells), via the activation of Ca(2+)-activated potassium channels [K(Ca2+) channels]. In cell-attached patch experiments, the external application of caffeine (1 mM) caused a brief activation of K(Ca2+) channels in Ca(2+)-free and Ca(2+)-containing external solutions. The application of BK (10 nM) during cell stimulation by caffeine (1-20 mM) invariably led to a drastic channel activation which was maintained during a recording period longer than that observed in caffeine-free conditions. In addition, the cell exposure to caffeine (20 mM) during the BK stimulation enhanced systematically the channel activation process. Since a rapid inhibition of BK-evoked channel activity was also produced by removing caffeine from the bath medium, it is proposed that the sustained single-channel response recorded in the concomitant presence of both agents was due to their synergic action on internal stores and/or the external Ca2+ entry pathway resulting in an increased [Ca2+]i. In addition, the local anesthetic, procaine, depressed the initial BK-induced K(Ca2+) channel activity and completely blocked the secondary phase of the channel activation process related to the external Ca2+ influx into stimulated cells. In contrast, this blocking effect of procaine was not observed on the initial caffeine-elicited channel activity and could not suppress the external Ca(2+)-dependent phase of this channel activation process. Our results confirm the existence of at least two pharmacologically distinct types of Ca(2+)-release from internal stores in BAE cells: an inositol 1,4,5-triphosphate (InsP3)-dependent and a caffeine-induced Ca(2+)-release process.

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. II. Effects of thapsigargin on the cellular Ca2+ homeostasis.

Evidence was provided, in the preceding paper (Thuringer & Sauvé, 1992), that the external Ca(2+)-dependent phase of the Ca2+ signals evoked by bradykinin (BK) or caffeine in bovine aortic endothelial cells (BAE), differ in their respective sensitivity to procaine. To examine whether the emptying of the InsP3-sensitive Ca2+ store is the signal for activating the agonist-evoked Ca2+ entry, we have investigated the effects of thapsigargin (TSG), a known inhibitor of the microsomal Ca(2+)-ATPase activity in a variety of cell types, via the activity of calcium-activated potassium channels [K(Ca2+) channels]. In cell-attached experiments, the external application of TSG caused a sustained or oscillatory activation of K(Ca2+) channels depending on both the cells and doses tested. The TSG-evoked channel activity could be reversibly blocked by removing extracellular Ca2+, and strongly decreased by adding 10 mM procaine to the bath medium. In Ca(2+)-free external conditions, TSG did not promote an apparent Ca2+ discharge from internal stores but prevented in a dose- and time-dependent manner the subsequent agonist-evoked channel activity related to the release of internally sequestered Ca2+. These results confirm that TSG and BK release Ca2+ from the same internal stores but with different kinetics. Because the channel response to caffeine was found to be poorly sensitive to procaine, in contrast to that evoked by BK and TSG, it may be concluded that both BK and TSG activate the same Ca2+ entry pathway. Therefore, the emptying of the InsP3-sensitive Ca2+ store is likely to be the main signal for activating the agonist-evoked Ca2+ entry in BAE cells.

A hyperpolarization-activated inward current in human myocardial cells.

Normally-polarized tissue from the human atrial myocardium usually exhibits a diastolic depolarization phase which can be suppressed reversibly by Cs+ or enhanced by inhibiting the inward rectifier K+ current, iK1, with Ba2+. (Escande et al., 1986). Because the suppression of the diastolic slope by Cs+ leads to a hyperpolarization of the cell membrane at the end of the diastolic phase, it was suggested that Cs+ might inhibit an inward current responsible for diastolic depolarization. Among the ionic mechanisms underlying the diastolic depolarization phase of cardiac tissues, the hyperpolarization-activated inward current, if, fits well to explain the small diastolic slope of human atrial fibres. In other preparations, this inward current carried both by Na+ and K+ ions is rapidly deactivated during the action potential and entirely blocked by millimolar concentrations of Cs+ (DiFrancesco 1981; DiFrancesco, et al., 1986; Kokubun et al., 1982; Callewaert et al., 1984; Denyer and Brown, 1990). Such a current in human myocardial cells has not been characterized so far although its existence in human atrial trabeculae was previously reported in an abstract (Carmeliet, 1984). In the present study, we describe an inward current which activates upon hyperpolarization in patch-clamped single human atrial cells and shares similar characteristics with the if pacemaker current described in unicellular and intact preparations of mammalian cardiac tissues.

Modulation by extracellular pH of bradykinin-evoked activation of Ca(2+)-activated K+ channels in endothelial cells.

We used the patch-clamp technique to investigate, via the activation of Ca(2+)-activated potassium channels [K(Ca2+)channels], the effects of extracellular pH (pHo) on the bradykinin (BK)-stimulated rise in cytosolic Ca2+ concentration in bovine aortic endothelial cells (BAE). In cell-attached experiments, the external application of BK caused a transient activation of the K(Ca2+) channels. Increasing pHo from 7.3 to 9 maintained the channel activity, which was not inhibited by withdrawing the agonist. The channel-activation process could be blocked either by removing external Ca2+ or by depolarizing the cells with a high-K+ external solution. These results indicate that the Ca2+ influx triggered by BK contributes in maintaining the agonist-evoked response in high pHo. Changes in pHo produced a slight increase in the intracellular pH (pHi) measured fluorimetrically with the H+ indicator dye 2',7-bis(carboxyethyl)5(6')-carboxyfluorescein. However, increasing pHi by the external application of NH4Cl at physiological pHo caused a rapid decline and not an increase in the K(Ca2+) channel activity triggered by BK. In fura-2-loaded cells, alkaline pHo had no effect on the time course of the Ca2+ response to BK in external Ca(2+)-free conditions, suggesting that the Ca2+ extrusion process is not affected by pHo. Our results suggest that the BK-evoked Ca2+ influx, which is required to reload internal Ca2+ stores, is controlled by a mechanism depending on extracellular H+.

Potassium accumulation in the globally ischemic mammalian heart. A role for the ATP-sensitive potassium channel.

We investigated the contribution of opening of the ATP-sensitive K+ channel to extracellular accumulation of K+ during ischemia with the use of glibenclamide, a specific blocker of this K+ channel. To characterize the electrophysiological effects of glibenclamide during metabolic inhibition (by either application of dinitrophenol or hypoxia) we performed patch-clamp studies in isolated membrane patches of guinea pig myocytes and in intact guinea pig myocytes and studied action potential parameters in isolated superfused guinea pig papillary muscle. We studied the effect of glibenclamide on extracellular accumulation of K+ and H+ in isolated retrogradely perfused globally ischemic hearts of rat, guinea pig, and rabbit. Experimental evidence is presented that supports the conclusions that glibenclamide 1) effectively blocks open K+ATP channels, 2) reverses the dinitrophenol-induced increase of the outward current and prevents the hypoxia-induced shortening of the action potential, 3) decreases the rate of K+ accumulation during the first minutes of ischemia in stimulated hearts, an effect which was entirely absent in quiescent hearts, and 4) does not influence the rate and extent of ischemia-induced extracellular acidification.

Effect of 2,3-butanedione 2-monoxime on slow inward and transient outward currents in rat ventricular myocytes.

The effect of 2,3-butanedione 2-monoxime (BDM), a substance possessing phosphatase-like activity, was studied on action potentials of isolated rat heart and on the slow inward calcium current and outward current (including the 4-aminopyridine (4-AP)-sensitive transient outward component), in rat ventricular myocytes. In contrast to what was observed by other authors in different species and cardiac tissues, BDM increased markedly the amplitude and duration of the rat ventricular action potential plateau. On the other hand, in the presence of 4-AP and ryanodine BDM shortened action potential duration. BDM decreased in a dose dependent manner the amplitude of both the slow inward calcium current and the transient outward current, accelerated their inactivation and shifted their steady-state inactivation-voltage relationships towards negative potentials. BDM also depressed other components of outward current. It is suggested that the lengthening effect of BDM on action potential duration results mainly from the simultaneous reduction of both the slow inward calcium current and the transient outward current, two antagonistic currents with unequal influences on action potential plateau development. The similarity of effect of BDM on these two currents also suggests that ionic channels generating them might require similar phosphorylation for their functioning.

Apparent competition between ATP and the potassium channel opener RP 49356 on ATP-sensitive K+ channels of cardiac myocytes.

The mechanism whereby RP 49356, a novel potassium channel opener, activates ATP-sensitive K+ channels (K+-ATP channels) in isolated cardiac cells was investigated with the patch-clamp technique. When directly applied onto the inner face of an inside-out membrane patch, RP 49356 (300 microM) had no effect on K+ channels opened in an ATP-free solution. In contrast, the same concentration of the drug reactivated K+-ATP channels that had experienced spontaneous "run-down" of their activity following long recording periods. In cell-attached experiments, externally applied RP 49356 (300 microM) opened K+-ATP channels at 35 degrees in spite of the high intracellular ATP concentration, which was sufficient to prevent channel openings in the absence of the drug. In control conditions, the dose-response relation for ATP closing the channels had a Hill coefficient of 2.37 and a half-inhibition concentration of 56 microM. With 30 microM RP 49356 present in the intracellular medium, the slope factor of this relation was unchanged but the curve was shifted to the right, with a half-inhibition concentration of 515 microM. Conversely, the dose-response relation of RP 49356 activating K+-ATP channels was shifted to the right in a parallel manner under the influence of increasing concentrations of ATP. It is concluded that RP 49356 acts on cardiac K+-ATP channels by decreasing their sensitivity to ATP. Our results are consistent with an apparent competition between ATP and RP 49356.

Potassium channel openers act through an activation of ATP-sensitive K+ channels in guinea-pig cardiac myocytes.

In a previous article (Escande et al. 1988a), we have shown that cromakalim (BRL 34915), a potassium channel opener (PCO), is a potent activator of ATP-sensitive K+ channels in cardiac cells. In the present article, the influence on K+ channels of two other potassium channel openers chemically unrelated to cromakalim, RP 49356 and pinacidil, has been investigated in patch-clamped isolated cardiac myocytes. In the whole-cell configuration, K+ currents were recorded in the presence of 50 microM TTX and 3 microM nitrendipine or 3 mM cobalt. Like cromakalim, RP 49356 or pinacidil activated a time-independent outward current at 33-35 degrees C but not at 19-21 degrees C, which showed little voltage-dependency in the potential range -60 to +60 mV. Its amplitude was a function of the agonist concentration, e.g. it was 2.1 +/- 0.4 nA at +60 mV with 30 microM RP 49356 and 4.3 +/- 0.8 nA with 300 microM. In control conditions, glibenclamide, a blocker of K+-ATP channels in pancreatic and heart cells, affected neither the inward rectifier, iK1, nor the delayed K+ current, iK. At 3 microM, glibenclamide fully prevented the effects of 300 microM RP 49356 or pinacidil. At lower concentrations, glibenclamide partially counteracted the activation by PCOs of a K+ current. In the cell-attached configuration, externally applied RP 49356 or pinacidil caused opening of large channels which reversed around O mV in a high K+ external medium. In inside-out patches, both RP 49356 or pinacidil activated K+-ATP channels by increasing the time period for which the channels remained in the open state. It is concluded that, like cromakalim, RP 49356 and pinacidil are potent activators of K+-ATP channels in cardiac myocytes.

The potassium channel opener cromakalim (BRL 34915) activates ATP-dependent K+ channels in isolated cardiac myocytes.

In cardiac myocytes, cromakalim (BRL 34915), a potassium channel opener, activates a time-independent K+ current exhibiting poor voltage-sensitivity. This effect of cromakalim is antagonized by low concentrations of glibenclamide, a specific blocker of ATP-dependent K+ channels in cardiac cells. Direct recording of the activity of K+ channels in inside-out membrane patches, confirmed that cromakalim is a potent activator of ATP-dependent K+ channels in cardiac myocytes.