Existence of two calcium currents recorded at normal calcium concentrations in single frog atrial cells.

A low threshold calcium current (ICALT) was found in Cs+-loaded frog atrial cells in addition to the classical (high threshold) calcium current (ICaHT), and was investigated at physiological Ca2+ concentrations using the whole-cell patch-clamp technique. The threshold potentials were approximately -60 mV for ICaLT and -40 mV for ICaHT. The amplitude and time course of ICaLT were almost unaffected by exchanging Ca2+ for Ba2+ or Sr2+, while those of ICaLT were modified. ICaLT was inhibited by Ni2+ (40 x 10(-6) M) but was not affected by Cd2+ (20 x 10(-6) M) while ICaHT was inhibited by Cd2+ and only slightly reduced by Ni2+ at the same concentrations. Co2+ (10(-3) M) inhibited both types of calcium currents while La3+ (5 x 10(-6) M) had a greater blocking effect on ICaHT. ICaLT was neither modified by dihydropyridines (nisoldipine, Bay K) nor by adrenergic agonists (adrenaline, noradrenaline, isoprenaline), in contrast with the effects of these agents on ICaHT. Angiotensin II (40 x 10(-9) M) increased and atrial natriuretic factor (0.1 x 10(-6) M) decreased ICaLT while ICaHT, was not modified by these two substances.

In vitro and in vivo reversal of cancer cell multidrug resistance by the semi-synthetic antibiotic tiamulin.

A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.

Opposite effects of halothane on guinea-pig ventricular action potential duration.

Halothane protects the heart against the reperfusion injury observed after an ischemia. In ischemic or anoxic conditions, a large ATP-sensitive K(+) (K(ATP)) conductance is supposed to provide an endogenous protection to the myocardium. In this study, we tested the possibility that halothane acted by modulating this conductance. Isolated guinea-pig cardiomyocytes were successively studied in current clamp and in voltage-clamp conditions. Action potentials regulation by halothane was tested in control conditions and in situations where the K(ATP) channels were activated. In control conditions, halothane decreased action potential duration of myocytes but did not significantly alter the inward rectifying K(+) current. Conversely, halothane lengthened action potential of cells in which the K(ATP) conductance was activated, by inhibiting the K(ATP) current. In ischemic conditions, simultaneous shortening of long action potentials and lengthening of shortened ones would be expected to homogenize the absolute refractory period at the border between normoxic and anoxic zones. This effect, together with a decrease in calcium load, could protect the myocardium against re-entrant arrhythmias.

The effects of sodium removal on the two types of calcium currents in single frog atrial cells.

The effects of sodium removal on the two types of calcium currents were studied in enzymatically dispersed frog (Rana esculenta) atrial myocytes with the single pipette patch-clamp technique. Reduction of calcium currents was recorded when NaCl was replaced either by TEACl, LiCl, TrisCl, cholineCl or by mannitol. An involvement of the Na-Ca exchange mechanism could be ruled out since the decrease was also observed after replacing external Ca with Ba. The slight shift of the apparent reversal potential recorded in our study suggests that the inward flow of Na ions through calcium channels does not contribute significantly to the L-type calcium current. Once again, the slight negative shift of the steady-state inactivation curve of the L-type calcium current cannot explain this decrease while no shift was recorded for the T-type calcium current. Even if a TTX-resistant Na current was recorded from a few cells this current cannot explain the decrease of calcium currents which was always observed upon sodium removal. To date we have no explanation for this effect.

Membrane responses to large hyperpolarizations in trabecles and single cells of frog atrium.

Atrial trabeculae (studied in voltage-clamping conditions and in the presence of 0.5 mmol/l BaCl2 to abolish gK1) responded to 1 s hyperpolarizations to beyond approximately E = -140 mV (from HP of about E = -80 mV) with an inwardly directed current increasing with time. Quite similar results were obtained with enzymatically dissociated frog atrial cells studied in whole cell voltage clamp with a patch-clamp pipette. This behaviour could be accounted for by assuming the presence of an "if" current at this quite negative range of potentials or by the fact that the cell membrane may undergo reversible electropermeabilization when its potential is brought to values negative to about -140 mV (Stämpfli 1958). When a brief (1 ms) and large (150 mV) hyperpolarization was applied 1 s before the test pulse, an inwardly directed current increasing with time was elicited by test pulses to beyond approximately E = -120 mV. This current was neither abolished in the presence of 1 mmol/l CsCl nor greatly reduced in the absence of Na+ ions, unlike "if" (Di Francesco 1981). We conclude that this current having a time course similar to that of "if" is of different nature and we argue that it might be accounted for by electropermeabilization of the membrane (reversible within about 2.5 min) due to the electrical shock represented by a brief and large hyperpolarization.

Possible role of Na+ ions in intracellular Ca2+ release in frog auricular trabeculae.

Membrane potential-current and mechanical tension of frog atrial muscle were studied in a Ca and Mg-free solution containing 1 mmol/l EGTA (Ca-free solution). Exposure to Ca-free solution resulted in a shortening of action potential duration within 1.5 min and a subsequent lengthening which were paralleled by changes in magnitude and duration of the contraction. Similarly, the slow inward current quickly disappeared and progressively reappeared with a quite slower inactivation time-course. Its reversal potential varied with [Na]0 as for a pure Na current. By 12 min in Ca-free solution, the tension-voltage relation could be interpreted as the sum of two components correlated with the slow inward current and the membrane potential respectively. Contractures in response to sustained large depolarizations had similar time courses in Ca-free solution and Ringer's containing Na-Ca exchange blockers (Mn2+ 15 mmol/l or La3+ 3 mmol/l). Intracellular Na loading by voltage-clamp depolarizations (40 mV from the resting potential for 100 ms, at 0.2 Hz) in the presence of Veratrine (7.5 X 10(-6) g/ml) caused a large progressive increase in tonic tension. An intracellular Ca2+ release is invoked, partly related to Na+ entry and partly to membrane potential changes. The potential dependent part could be influenced by intracellular Na+.

A low threshold calcium current recorded at physiological Ca concentrations in single frog atrial cells.

The presence of a low threshold calcium current (ICa,LT) besides the classical (high threshold) calcium current (ICa,HT) is investigated using the whole-cell patch-clamp technique (10) on Cs-loaded frog atrial cells at physiological Ca concentrations. ICa,LT, which inactivates within tens of milliseconds, gives rise to a shoulder in the negative part of the peak current-voltage curve. It is completely abolished by 40 microM Ni while ICa,HT is not modified. Cd (30 microM) decreases ICa,LT by about 50% while ICa,HT is completely abolished.

Role of the Na+-Ca2+ exchange in the calcium paradox in frog auricular trabeculae.

After a Ca2+-free ouabain perfusion of 8-10 min duration, reperfusion of isolated stimulated frog auricular trabeculae with Ca2+ ouabain containing medium resulted in a large and transient contracture. The contracture was weaker in a quiescent preparation or in the absence of ouabain. In sodium-free (Li+ substitute) ouabain containing medium, the amplitude of the contracture was largely decreased while it disappeared completely in Na+-free medium without ouabain. Moreover this contracture was suppressed by 15 mM Mn2+. Although the approach was only indirect, these results suggest that the contracture is due to an entry of Ca2+ ions through the Na+-Ca2+ exchange mechanism and that this could be the unique route of calcium entry during calcium repletion.

[Demonstration of intracellular release of calcium induced by sodium ions in the auricular trabeculae of the frog]. / Physiologie animale. -Mise en évidence d'une libération intracellulaire de calcium induite par les ions sodium au niveau de trabécules auriculaires de Grenouille.

In a Ca-free, Mg-free medium containing EGTA (10-3M) auricular trabecles develop a slow inward current which is a pure sodium current. After 12 min in this medium, it is still possible to obtain a phasic mechanical activity which shows a perfect correlation with the current. This kind of behavior indicates that a mechanism of sodium-induced calcium release is present at the level of some internal sites.

Forskolin but not isoprenaline increases sodium current from frog cardiac myocytes.

The effect of forskolin on sodium current (INa) was studied in enzymatically dispersed frog atrial myocytes. The single pipette patch-clamp technique was used, with a low external sodium concentration. Forskolin (0.5 microM) increased INa at all potentials between -50 and +30 mV. The forskolin-induced increase of INa was mimicked by 1,9-dideoxyforskolin (0.5 microM) but not by isoprenaline (2 microM). These results suggest that forskolin can modulate INa through a mechanism that does not involve the production of cyclic AMP.

Inotropic effects of potassium rich solutions of frog cardiac muscles.

1. Inotropic effects of potassium rich solutions on frog cardiac muscle have been studied bith in current clamp and voltage clamp conditions, using a double sucrose gap apparatus. 2. Potassium rich solutions cause either a positive or a negative inotropic effect, together with an increase or a decrease in the duration of the action potential, according to the preparation. 3. The phasic phase of contraction and the slow inward current are decreased in amplitude; the reversal potential of the slow inward current is shifted towards more negative values. 4. The tonic phase of contraction is first decreased, the increased; the effects are correlated with modifications of the background current, initially in the inward, then in an outward direction. 5. The tension level obtained in contracture experiments is increased or decreased, according to the direction of the changes in the background current. 6. The effects of potassium rich solutions are still observed in he presence of ouabain, suggesting that they are independent of any effect on the sodium-potassium pump. 7. The effects of potassium rich solutions are still observed when external sodium is replaced by sucrose; they disappear (except the effect on the background current) when external sodium is replaced by lithium. 8. The results, which indicate that potassium ions play a role in the regulation of the intracellular concentration of calcium ions, are discussed in relation to a possible K/Ca exchange mechanism, to the Na/Ca exchange and to the role of intracellular calcium stores.

Reduction of I(Ca,L) and I(to1) density in hypertrophied right ventricular cells by simulated high altitude in adult rats.

The present paper describes the effect of a simulated hypobaric condition (at the altitude of 4500 m) on morphological characteristics and on some ionic currents in ventricular cells of adult rats. According to current data, chronic high-altitude exposure led to mild right ventricular hypertrophy. Increase in right ventricular weight appeared to be due wholly or partly to an enlargement of myocytes. The whole-cell patch-clamp technique was used and this confirmed, by cell capacitance measurement, that chronic high-altitude exposure induced an increase in the size of the right ventricular cells. Hypertrophied cells showed prolongation of action potential (AP). Four ionic currents, playing a role along with many others in the precise balance of inward and outward currents that control the duration of cardiac AP, were investigated. We report a significant decrease in the transient outward (I(to1)) and in the L-type calcium current (I(Ca,L)) densities while there was no significant difference in the delayed rectifier current (I(K)) or in the inward rectifier current (I(K1)) densities in hypertrophied right ventricular cells compared to control cells. At a given potential the decrease in I(to 1) density was relatively more important than the decrease in I(Ca,L) density. In both cell types, all the currents displayed the same voltage dependence. The inactivation kinetics of I(to 1) and I(Ca,L) or the steady-state activation and inactivation relationships were not significantly modified by chronic high-altitude exposure. We conclude that chronic high-altitude exposure induced true right ventricular myocyte hypertrophy and that the decrease in I(to 1) density might account for the lengthened action potential, or have a partial effect.

Reversibility of electrophysiological changes induced by chronic high-altitude hypoxia in adult rat heart.

Recent studies indicate that regression of left ventricular hypertrophy normalizes membrane ionic current abnormalities. This work was designed to determine whether regression of right ventricular hypertrophy induced by permanent high-altitude exposure (4,500 m, 20 days) in adult rats also normalizes changes of ventricular myocyte electrophysiology. According to the current data, prolonged action potential, decreased transient outward current density, and increased inward sodium/calcium exchange current density normalized 20 days after the end of altitude exposure, whereas right ventricular hypertrophy evidenced by both the right ventricular weight-to-heart weight ratio and the right ventricular free wall thickness measurement normalized 40 days after the end of altitude exposure. This morphological normalization occurred at both the level of muscular tissue, as shown by the decrease toward control values of some myocyte parameters (perimeter, capacitance, and width), and the level of the interstitial collagenous connective tissue. In the chronic high-altitude hypoxia model, the regression of right ventricular hypertrophy would not be a prerequisite for normalization of ventricular electrophysiological abnormalities.

Increased sodium-calcium exchange current in right ventricular cell hypertrophy induced by simulated high altitude in adult rats.

Ventricular hypertrophy is associated with an increase in action potential (AP) duration which is potentially arrhythmogenic. The implication of the Na-Ca exchange current (I(Na-Ca)) in the lengthening of the AP is controversial. The role of this current in the increased duration of the low plateau of the AP in hypertrophied adult rat ventricular myocytes by simulated chronic high-altitude exposure ( approximately 4500 m) was evaluated. Electrophysiological experiments were carried out on isolated right ventricular myocytes from exposed and control rats with the perforated patch or the conventional whole-cell technique in current or in voltage clamp condition. With the two techniques, a significant increase of the low plateau duration was observed in hypertrophied myocytes as compared to controls. The low plateau in hypertrophied myocytes was depressed when Na was replaced by Li and was no longer recorded when intracellular Ca was buffered with EGTA. Inward tail currents, evoked either on repolarization to -80 mV following a depolarizing pulse to +10 mV or by interrupted AP technique, were greater in hypertrophied than in control myocytes and were abolished when Na was replaced by Li or when intracellular Ca was buffered with EGTA, indicating an increased Na-Ca exchange activity. The Li-sensitive current-voltage curves, obtained by a voltage clamp ramp protocol with an intracellular calcium buffered solution, were not significantly different in both hypertrophied and control myocytes, suggesting no modification in the density of the Na-Ca exchange protein. This was corroborated by the lack of difference in NCX1 mRNA levels between right ventricles from control and exposed rats. We conclude that increased duration of the low plateau of rat ventricular AP in altitude cardiac hypertrophy may be attributed to an increase of the inward I(Na-Ca). This augmented I(Na-Ca)may result from a modification in the intracellular Ca homeostasis.

Effects of aging on the cardiac remodeling induced by chronic high-altitude hypoxia in rat.

Effects of chronic high-altitude hypoxia on the remodeling of right ventricle were examined in three age groups of rats: 2, 6, and 18 mo. The extent of right ventricular (RV) hypertrophy (RVH) showed an age-associated diminution. RV cell size and pericellular fibrosis showed a significant increase in the 2- and 6-mo-old exposed rats but not in the 18-mo-old exposed rats compared with control. A hyperplasic response was underscored in the three exposed age groups but appeared less pronounced in the 18-mo-old rats. A significant decrease in the transient outward potassium current (Ito) density was observed in RV cell only in the 2-mo-old exposed group compared with the control group. In the control group, there was a clear tendency for Ito density to decrease as a function of age. The sustained outward current density was modified neither by the hypoxia condition nor by the age. Neither the cytochrome c oxidase activity nor the heat shock protein 72 content in the RV was altered after hypoxic exposure regardless of age. The norepinephrine content in the RV was significantly decreased in each age group exposed to hypoxia when compared with their age-matched control group. Our findings indicate that the remodeling (at morphological and electrophysiological levels) induced by chronic hypoxia in the RV can be decreased by the natural aging process.