Comparison of Na(+)/Ca(2+) exchanger current and of its response to isoproterenol between acutely isolated and short-term cultured adult ventricular myocytes.

The Na(+)/Ca(2+) exchanger protein is present in the cell membrane of many tissue types and plays key roles in Ca(2+) homeostasis, excitation-contraction coupling, and generation of electrical activity in the heart. The use of adult ventricular myocyte cell culture is important to molecular biological approaches to study the roles and modulation of the cardiac Na(+)/Ca(2+) exchanger. Therefore, we characterised the functional expression of the exchanger in adult guinea-pig ventricular myocytes maintained in short-term culture (for 4 days) and compared the response of ionic current (I(NaCa)) carried by the exchanger from acutely isolated and Day 4 cells to beta-adrenoceptor activation with isoproterenol (ISO). Functional activity of the exchanger was assessed by measuring I(NaCa) using whole cell patch clamp, under selective recording conditions. I(NaCa) amplitude measured at both +60 and -100mV declined significantly by Day 1 of cell culture, showing a further small decline by Day 4. However, cell surface area (assessed by measuring membrane capacitance) also declined over this time-frame. I(NaCa) normalised to membrane capacitance (I(NaCa) density) did not differ significantly between acutely isolated and cells cultured for 4 days. However, although ISO (1 microM) increased I(NaCa) in acutely isolated myocytes, it exerted no significant effect on I(NaCa) from Day 4 cells. This was not due to an inherent inability of these cells to respond to ISO, as L-type calcium current amplitude from Day 4 cells was increased by ISO to a similar extent as that from acutely isolated cells. Our data suggest that the functional expression of the Na/Ca exchanger is well maintained during short-term culture of adult ventricular myocytes. The lack of response to ISO of I(NaCa) from Day 4 cells suggests: (a) that, despite a well-maintained I(NaCa) density, cultured adult myocytes may not necessarily be suitable for studies of exchanger modulation by some agonists and (b) that there may exist subtle differences between beta-adrenergic regulation of the exchanger protein and of L-type Ca channels.

External nickel blocks human Kv1.5 channels stably expressed in CHO cells.

We have investigated the actions of Nickel (Ni(2+)) on a human cardiac potassium channel (hKv1.5), the main component of human atrial ultra-rapid delayed rectifier current, stably expressed in Chinese hamster ovary cell line using the whole-cell voltage-clamp technique. External Ni(2+) reversibly decreased the amplitude of the current in a concentration-dependent manner. The concentration for half-maximum inhibition of the current at +50 mV was 568 microm. The activation, deactivation, reactivation kinetics of the current were not affected by Ni(2+). Block was not voltage-dependent but frequency-dependent block was apparent. The extent of channel block during the first pulse increased when the duration of exposure to Ni(2+), prior to channel activation, was prolonged indicating that Ni(2+) interacted with hKv1.5 in the closed state. The percentage of current remaining in presence of Ni(2+) decreased steeply over the range of steady-state channel inactivation, consistent with an enhanced block with increased inactivation. This suggests that Ni(2+) preferentially blocks nonconducting hKv1.5 channels, either in the resting or inactivated state in a concentration-dependent manner. The data indicate that the mechanisms of hKv1.5 channel inhibition by Ni(2+) are distinct from those of other K(+) channels.

Effects of anorexinogen agents on cloned voltage-gated K(+) channel hKv1.5.

Appetite suppressants have been associated with primary pulmonary hypertension (PPH), inhibition of voltage-gated potassium channels, membrane depolarization, and calcium entry in pulmonary artery smooth muscle cells. In cells taken from pulmonary arteries of primary pulmonary hypertensive patients, voltage-gated potassium channels appear to be dysfunctional and in particular, reduced hKv1.5 gene transcription and hKv1.5 mRNA instability have been shown. We have compared the effects of anorexinogen agents on hKv1.5 channels stably expressed in mammalian cell line. We found that aminorex, phentermine, dexfenfluramine, sibutramine, and fluoxetine cause a dose-dependent inhibition of hKv1.5 current. Aminorex, phentermine, and dexfenfluramine had a K(D) of inhibition greater than to 300 microM and are not potent inhibitors of hKv1.5. Sibutramine and fluoxetine inhibited hKv1.5 current with lower K(D) values of 41 and 21 microM, respectively. Block by both drugs increased rapidly between -20 and +10 mV, coincident with channel opening and suggested an open channel block mechanism. This was confirmed by a slower deactivation time course resulting in a "crossover" phenomenon when tail currents recorded under control conditions and in the presence of either drug were superimposed. Single channel experiments demonstrated that open probability and open duration of hKv1.5 were decreased by fluoxetine and sibutramine. These results indicate that among the anorexinogen agents tested, sibutramine and fluoxetine are the most potent toward hKv1.5 channel, which they preferentially block in the open state. Nevertheless, their inhibitory effects do not correlate with their ability to produce PPH neither with their previously reported therapeutic plasma concentrations.

Characterization of mibefradil block of the human heart delayed rectifier hKv1.5.

The goal of this study was to analyze the effects of mibefradil on a human cardiac K(+) channel (hKv1.5) stably expressed in Chinese hamster ovary cells using the whole-cell configuration of the patch-clamp technique. Mibefradil inhibited in a concentration-dependent manner the hKv1.5 current with a K(D) value of 0.78 +/- 0.05 microM and a Hill coefficient of 0.97 +/- 0.06. Block induced by mibefradil was voltage dependent, consistent with a value of electrical distance of 0.13. The apparent association (k) and dissociation (l) rate constants measured at +50 mV were found to be 7.3 +/- 0.5 x 10(6) M(-1).s(-1) and 4.3 +/- 0.1 s(-1), respectively. Block increased rapidly between -20 and +10 mV, coincident with channel opening and suggested an open channel block mechanism, which was confirmed by a slower deactivation time course resulting in a "crossover" phenomenon when tail currents recorded under control conditions and in the presence of mibefradil were superimposed. Shifts toward negative potentials of the maximum conductance and the activation curve were observed, confirming the voltage dependence of block. Mibefradil induced a significant use-dependent block when trains of depolarization at frequencies between 0.02 and 2 Hz were applied. In the presence of mibefradil, recovery of inactivation was faster than under control conditions, suggesting that mibefradil might compete with the inactivation gate of hKv1.5. These results indicate that mibefradil blocks hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutic range.

Pharmacological properties of Ca(V)3.2, a low voltage-activated Ca2+ channel cloned from human heart.

Three genes encoding T-type Ca2+ channels have been described but their correspondence to the various native T-type Ca2+ currents remains uncertain. In particular, Ca(V)3.2 (or alpha1H) was cloned from a human heart library, its message was found abundantly in cardiac tissue, and expressed Ca(V)3.2 was shown to conduct low voltage-activated currents, which inactivate rapidly and are sensitive to Ni2+ and mibefradil. These observations suggested that Ca(V)3.2 might encode native cardiac T-type Ca2+ channels but more information on the pharmacology of Ca(V)3.2 was needed to confirm this hypothesis. In the present study, we compare the pharmacology of Ca(V)3.2 expressed in HEK293 cells and of native T-type Ca2+ channels in guinea pig atrial myocytes ("native-T"). (1) Ca(V)3.2 and native-T are insensitive to TTX and to toxins selective for N-, P-, or Q-type Ca2+ channels (omega-CTx-GVIA, omega-Aga-IVA, omega-CTx-MVIIC). (2) The half-maximal blocking concentration (IC50) of mibefradil on Ca(V)3.2 is near that on native-T and the block is similarly voltage-dependent. (3) Ca(V)3.2 is five- to sixfold less sensitive than native-T to the 1,4-dihydropyridine (DHP) amlodipine, suggesting a difference in the DHP binding site. (4) Both channels display similar (but not identical) sensitivities to the inorganic blockers Ni2+ and Cd2+ and the IC50s are in the range of values found for T-type Ca2+ currents in other cell types. (5) Ni2+ shifts the voltage dependence of Ca(V)3.2 activation but not that of native-T. The many similarities between the two channels support the contention that Ca(V)3.2 encodes cardiac T-type Ca2+ channels. The slight differences may be due to species variations and/or to the choice of splice variant.

Stimulation of Na/Ca exchange by the beta-adrenergic/protein kinase A pathway in guinea-pig ventricular myocytes at 37 degrees C.

We investigated the effect of beta-adrenergic stimulation on Na/Ca exchange in whole-cell patch-clamped guinea-pig ventricular myocytes at 37 degrees C. With ion channel and Na/K pump currents blocked, the Na/Ca exchange current (I(Na-Ca) was measured selectively as membrane current inhibited by 10 mM nickel (Ni) during a voltage ramp applied between +80 and -120 mV. Isoprenaline (1 microM) caused an increase in both inward and outward current generated by the Na/Ca exchange, which was prevented by the beta-adrenoceptor blocker propranolol. These data suggest that isoprenaline caused a receptor-mediated up-regulation of Na/Ca exchange activity. Mimicking beta-adrenoceptor activation, either by stimulation of adenylate cyclase with forskolin or by internal dialysis of cells with cyclic AMP (3':5'-cyclic adenosine monophosphate), also increased I(Na-Ca). Using fluorescence Ca measurement, an increase of internal cAMP was shown to increase the rate of transmembrane Ca transport via the Na/Ca exchange. A selective inhibitor of protein kinase A prevented stimulation of Na/Ca exchange by isoprenaline. These data suggest that the underlying mechanism of stimulation was phosphorylation of the Na/Ca exchange protein by protein kinase A. Isoprenaline did not stimulate I(Na-Ca) when experiments were carried out at 20 degrees C, in contrast to the findings at 37 degrees C. Modulation of Na/Ca exchange by the beta-adrenergic pathway may have important physiological consequences for intracellular Ca regulation and electrical activity during hormonal stimulation, or during sympathetic nerve stimulation.

Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 degrees C, dialysed internally with cAMP-free and cAMP-containing solutions.

In many mammalian tissue types an integral membrane protein--the sodium/calcium (Na/Ca) exchanger--plays a key role in intracellular Ca homeostasis, and evidence suggests that Na/Ca exchange function can be modulated by cAMP-dependent phosphorylation. External Nickel (Ni) ions are used widely to inhibit the exchange but little is known about the mode of Ni action. In guinea-pig ventricular myocytes, we investigated inhibition of Na/Ca exchange by external Ni under phosphorylated (cells dialysed with cAMP) and non-phosphorylated conditions. Ventricular myocytes were isolated from adult guinea-pig hearts, recordings were made at 37 degrees C using the whole-cell patch clamp technique. Internal and external solutions were used which allowed Na/Ca exchange current (INaCa) to be measured during a descending voltage ramp protocol (+80 to -120 mV) applied from a holding potential of -40 mV. The application of 10 mM Ni caused a maximal block of INaCa since inhibition was identical to that when a Na- and Ca-free (0Na/0Ca) solution was superfused externally. Kinetics of Ni-block of INaCa were assessed using applications of different external [Ni] to cells dialysed internally with cAMP-free and 100 microM cAMP-containing solutions. At +60 mV, Ni inhibited INaCa in cells dialysed with a cAMP-free solution with a dissociation constant (KD) of 0.29 +/- 0.03 mM and the data were fitted with a Hill coefficient of 0.89 +/- 0.07 (n = 9 cells). In cells dialysed with 100 microM cAMP the exchange was inhibited by Ni with a KD of 0.16 +/- 0.05 mM, the Hill coefficient was 0.82 +/- 0.16 (n = 6-7 cells). The KD and Hill coefficient values obtained in cells dialysed with cAMP-free and cAMP-containing solutions were not significantly different. Inhibition of INaCa by Ni did not appear to be voltage-dependent, was maximal within 3-4 s of application and was rapidly reversible. With cAMP-free internal dialysate, inhibition was 'mixed' showing competition with external Ca and a degree of non-competitive block. With 100 microM cAMP the inhibition appeared to be more non-competitive. We conclude that, under these experimental conditions, a concentration of external Ni of 10 mM is sufficient to produce maximal inhibition of INaCa in guinea-pig cardiac cells.

Electrophysiological approach of the role of Na+/H+ exchange in low-flow global ischemia and in ischemic preconditioning.

We investigated, at first in low-flow global ischemia and then with ischemic preconditioning, the effects of a compound, (4-isopropyl-3-methylsulphonylbenzoyl)guanidine hydrochloride (HOE 642), known to inhibit the Na+/H+ exchange in rat cardiomyocytes. In rat isolated hearts, perfused on a Langendorff apparatus with Krebs-Henseleit carbonate buffer, the action potentials and the contractile function were measured during a 25-min period of global low-flow ischemia (coronary flow, 0.3 mL.min-1) followed by a 30-min reperfusion. In hearts previously preconditioned, two intermittent periods of total ischemia for 5 min each, separated by 5 min reflow, were performed before low-flow ischemia. Treated hearts received HOE 642 (3.0 x 10(-8) mol.min-1) exclusively during low-flow ischemia. Treatment with HOE 642 during low-flow ischemia improves cardiac performance and lowers the rise in diastolic tension during reperfusion. Concomitantly HOE 642 shortens the action potential, and has striking effects on ventricular arrhythmias during reperfusion as well. These results support the concept that Na+/H+ exchange activation is a contributing factor to low-flow ischemia-reperfusion injuries. HOE 642 exhibited minor effects when combined with the preconditioning protocol, but a lengthening in action potential was observed and ventricular arrhythmias were mostly affected. Preconditioned hearts demonstrated marked glycogen depletion compared with controls. These results support the hypothesis that preconditioning could decrease glycogenolysis and therefore subsequently limit acidification during low-flow ischemia.

Mechanical and electrophysiological effects of preconditioning in isolated ischemic/reperfused rat hearts.

Changes in action potential duration (APD) were studied during ischemic/reperfusion injury preceded or not by preconditioning in isolated rat hearts. Hearts were perfused on a Langendorff apparatus with Krebs-Henseleit carbonate buffer and submitted to 25-min global low-flow ischemia (coronary flow, 0.3 mol x min-1) followed by 30-min reperfusion. In hearts that had been preconditioned, two intermittent periods of total ischemia for 5 min each, separated by 5-min reflow, were performed before low-flow ischemia. At the end of the ischemic period, APs were significantly prolonged in nonpreconditioned hearts; this prolongation was abolished by preconditioning. Moreover, preconditioning increased the recovery of the contractile function. Therefore, ischemia can widen APD. The results also showed that in rats, preconditioning can be produced in a manner qualitatively similar to preconditioning in other species. Verapamil (3 x 10(-9) mol x min(-1)) or 4-aminopyridine (4-AP, 3 x 10(-6) mol x min(-1)) applied exclusively during low-flow ischemia significantly improved postischemic contractile function in nonpreconditioned hearts (25.9 +/- 4.4. and 37.9 +/- 2.4 vs. 12.9 +/- 5.3%, respectively) as well as in preconditioned hearts (61.8 +/- 4.2 and 55.5 +/- 4.7 vs. 36.0 +/- 1.4%, respectively). With verapamil, this protection was associated with a decrease in APD at 90% of repolarization in the nonpreconditioned hearts (APD90 32.2 +/- 0.1 vs. 71.1 +/- 6.7 ms at the end of ischemia). With 4-AP, this same protection was associated with an increase in APD in the preconditioned hearts (APD90 67.7 +/- 0.7 vs. 48.5 +/- 2.6 ms at the end of ischemia). Both agents given during a 25-min ischemic challenge improved myocardial recovery in nonpreconditioned and preconditioned hearts, despite discordant effects on the AP. Furthermore, the action of these agents was cumulative with the effect of preconditioning.

The benzodiazepine midazolam preferentially blocks inactivated Na channels in skeletal muscle fibre.

The effects of the benzodiazepine midazolam were studied on frog skeletal muscle fibres held under current- or voltage-clamp conditions. Midazolam induced a concentration-dependent (10(-5) mol/l to 10(-3) mol/l) block of the action potential and of the underlying Na current. Block of the Na current occurred without any changes in its voltage dependence or in its activation and inactivation kinetics. An apparent dissociation constant of 223 mumol/l was determined for midazolam from the rested Na channels of well polarized fibres. The blocking effect of a threshold concentration (10(-5) mol/l) could be greatly enhanced (up to the complete suppression of the current) by predepolarizations, positive holding potentials or high stimulation frequencies. This apparent voltage- and frequency-dependent block (no use dependence, i.e., no activation block) could be ascribed to a blockade of inactivated Na channels. From the apparent shift towards negative potentials of the steady-state inactivation curve, a dissociation constant of 6.0 mumol/l was calculated for midazolam from the inactivated Na channels, according to the modulated-receptor model. These results show that midazolam preferentially blocks inactivated rather than rested Na channels, and suggest that this mechanism of action might contribute to the well-known myorelaxant effect of the benzodiazepines.