Reincorporated plasma membrane Ca2+-ATPase can mediate B-Type Ca2+ channels observed in native membrane of human red blood cells.

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1-5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in results between RBC and reincorporated PMCA, most probably attributable to the decrease in ATPase activity following the procedure of reincorporation, the present experimental conditions appear to reveal a channel-mode of the PMCA that shares many similarities with the B-type Ca2+ channel.

Are B-type Ca2+ channels of cardiac myocytes akin to the passive ion channel in the plasma membrane Ca2+ pump?

The present study demonstrates that B-type Ca2+ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca2+-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca2+ channels were completely blocked by internal application of PMCA pump inhibitors, namely La3+ (100 microm), eosin (10 microm) and AIF(3) (100 microm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba2+-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba2+ , mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 microm eosin. These results suggest that B-type Ca2+ channels are some form of the PMCA pump.

Slowly inactivating component of sodium current in ventricular myocytes is decreased by diabetes and partially inhibited by known Na(+)-H(+)Exchange blockers.

Recent evidence has suggested a major role for a slowly inactivating component of Na(+)current (I(NaL)) as a contributor to ischemic Na(+)loading. The purposes of this study were to investigate veratrine and lysophosphatidylcholine (LPC)-induced I(NaL)in single ventricular myocytes of normal and diabetic rats and to analyse the effects on this current of three pharmacological agents, known as Na(+)/H(+)exchange inhibitors, whose selectivity has been questioned in several studies. A decrease in Na(+)/H(+)exchange activity has been previously shown to be associated with diabetes, and this has been found to confer some protection to the diabetic heart after an episode of ischemia/reperfusion. Recordings were made using the whole-cell patch-clamp technique. I(NaL)was stimulated either by veratrine (100 mg/ml) or by LPC (10 micromol/l) applied extracellularly. Veratrine as well as LPC-induced I(NaL)was found to be significantly decreased in ventricular myocytes isolated from diabetic rat hearts. Veratrine- and LPC-induced I(NaL)in ventricular myocytes of normal rats was significantly (in the range 10(-7)to 10(-4)mol/l) inhibited by the Na(+)/H(+)exchange blockers HOE 694, EIPA and HOE 642. HOE 694 was the most potent inhibitor, followed by the amiloride derivative EIPA and HOE 642. The sensitivity of veratrine-induced I(NaL)to inhibition by HOE 694 and EIPA was markedly reduced in diabetic ventricular myocytes, with no observed inhibition by HOE 642. These data may have important implications as to the protection that may be afforded against ischemic and reperfusion injury, especially during ischemia and when ischemia occurs in a diabetic situation.

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.

Establishment of a human thymic myoid cell line. Phenotypic and functional characteristics.

The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. alpha-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells.

[Transient outward potassium current and repolarization of cardiac cells]. / Courant potassique transitoire sortant et repolarisation des cellules cardiaques.

The transient 4-aminopyridine-sensitive outward potassium current, Ito, is one of the ionic membrane currents involved in the repolarization of cardiac action potentials. It is present in several species (rat, dog, human) but not in guinea pig ventricle. It induces both a marked lowering of the ventricular action potential plateau level and an early repolarization wave in the ventricular ECG complex of hypothermic rats. In dog ventricle where Ito is much shorter than the action potential plateau it can induce only a transient initial repolarization (notch). The distribution of Ito is heterogeneous across the dog left ventricular free wall, the current being of sizeable amplitude in epicardial and midmyocardial layers but absent in the endocardial layer. As a result, ventricular action potentials exhibit a notch only in epicardial and mid layers. Although the physiological role of Ito remains unclear, we suggest that it can participate in the control of calcium current intensity by influencing the level of the initial part of the plateau. In pathophysiological conditions, Ito may exert unfavourable effects, specially during simulated ischemia when the notch reaches the cellular repolarization threshold, thus inducing premature termination of the action potential, an obvious cause of drastic electrical heterogeneity and resulting severe arrhythmias. The current Ito is reduced in moderate cardiac hypertrophy and dilatation and almost entirely suppressed in severe hypertrophy. Ito is of larger amplitude in human atrial than in ventricular myocytes. The heterogeneous distribution of Ito described in the dog has also been found in human ventricles. Because Ito is markedly prolonged at low temperatures it is suggested that it can be responsible for the early repolarization wave (J wave) observed in the ECG of subjects submitted to hypothermia.

Late sodium current inhibition in human isolated cardiomyocytes by R 56865.

R 56865, a cytoprotective agent, has been shown to prevent myocardial ischemia and reperfusion injury by blockade of the late sodium current (I(Nal)). The effect of R 56865 on I(Nal) in isolated human atrial myocytes was investigated by using the whole-cell patch-clamp technique. I(Nal) recorded at the end of a 350-ms test pulse evoked from -100 to +20 mV was significantly increased by the addition of veratrine (100 microg/ml: quantity of charge corresponding to total I(Nal): 6.1 +/- 1.2 at baseline vs. 86.9 +/- 15; p < 0.001). Tetrodotoxin (TTX; 1 microM) fully prevented veratrine-induced increases in I(Nal). R 56865 (0.1-10 microM, n = 14) significantly and reversibly decreased veratrine-induced I(Nal) (42.01 +/- 8.6%, n = 6; p < 0.001 at 10 microM). Moreover, R 56865 reduced I(Nal) without significantly affecting kinetic parameters of inactivation [tau1 = 1.04 +/- 0.1 ms and tau2 = 119.3 +/- 2.3 ms (baseline) vs. tau1 = 1.57 +/- 0.5 ms and tau2 = 134.4 +/- 14 ms in the presence of 10 microM R 56865; NS]. The data indicate that R 56865 is a potent blocker of the late inducible component of sodium current in human cardiomyocytes.

B-type Ca2+ channels activated by chlorpromazine and free radicals in membrane of human atrial myocytes.

The present study demonstrates that background or B-type calcium channel activity can be recorded in excised inside-out and cell-attached membrane patches from human atrial myocytes. In control conditions, with Ba2+ or Ca2+ as charge carrier, single-channel activity spontaneously appeared in irregular bursts separated by quiescent periods of 2-17 min, in nearly 25% of tested patches. Channel activity was recorded at steady-state applied membrane potentials including the entire range of physiological values, and displayed no "rundown" in excised patches. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. This type of channel activity was triggered or markedly increased when chlorpromazine (CPZ 20 or 50 microM) was applied to internal face of inside-out patches, with a proportion of active patches of approximately 25%. CPZ-activated channels were potential-independent in the physiological range of membrane potential. In 96 mM Ba2+ solution, three conductance levels: 23, 42 and 85 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. As previously observed by Wang et al. (1995) in membrane of rat ventricular myocytes, increasing free-radicals level and metabolic poisoning readily enhanced B-type channel activity in human atrial myocytes. Application of H2O2 (from 0.1-10 mM) in cell-attached mode induced an activation of Ba2+ permeable channel activity in a dose-dependent manner, with an estimated EC50 of 9.7 mM. In the same type of experiments, 10 mM deoxyglucose also induced similar Ba2+ permeable channel activity. When 500 microM CPZ were applied to myocytes studied in the whole-cell configuration and maintained at a holding potential of -80 mV in the presence of 5 mM external Ca2+, a noticeable inward current could be observed. The mean CPZ-activated current density determined from seven myocytes was 0.63 pA/pF.

[Electrogenic sodium-calcium exchange and electrical activity in cardiac cells]. / Echange sodium-calcium électrogène et activité électrique cellulaire cardiaque.

Several studies have shown that the Na-Ca exchange is electrogenic in cardiac tissues and that the current carried by the exchange, iNa-Ca, participates in the development of the cardiac action potential plateau. The aim of the present study was to assess the contribution of iNa-Ca to the development of the plateau in different preparations, i.e. normal and hypertrophied (DOCA-salt) perfused rat hearts, normal and dilated (MS 200 strain) perfused hamster hearts and normal human atrial myocytes and to examine the repercussion of iNa-Ca suppression on the global electrogram of the perfused guinea-pig heart. iNa-Ca was suppressed by briefly substituting lithium for sodium (Li test). In the normal rat heart, the Li test resulted in a depression of the late component of the plateau. In the severely hypertrophied rat heart, the action potential was lengthened, the two components of the plateau barely distinguishable. The part of the plateau suppressed during Li perfusion was not larger in amplitude than in the normal rat heart, but was of much longer duration. In the dilated heart of hamsters older than 60 days of age the shortening effect of the Li test was much larger than in the normal hamster heart indicating a strongly increased contribution of iNa-Ca to the plateau development during dilatation. The contribution of iNa-Ca to the action potential plateau of human atrial myocytes was as large as that found in animal ventricles and varied with the type of cell studied. In the perfused guinea-pig heart the Li test resulted in a sizeable shortening of QT duration and a marked decrease in T wave amplitude suggesting that iNa-Ca may be a major source of current in the building up of the ventricular complex of the electrocardiogram in normal and pathological conditions.

Action potential and plateau ionic currents in moderately and severely DOCA-salt hypertrophied rat hearts.

Ventricular hypertrophy is associated with an increase in action potential (AP) plateau amplitude and duration. In a model of cardiac hypertrophy by DOCA-pellet implantation in uninephrectomized saline-drinking rats, we have previously demonstrated that the influence of hypertrophy was to reduce Ito1 density, the process being fully reversible after elimination of DOCA pellets. In that study the decrease of Ito1 density appeared to vary from moderate reduction to complete suppression which could explain, at least in part, the AP lengthening. In the present study the effect of the degree of hypertrophy (moderate and severe hypertrophy) was investigated on rat ventricular action potential plateau amplitude and duration, high threshold calcium current, Ica-L, Na-Ca exchange current, INa-Ca, transient outward potassium current, Ito1, and sustained outward potassium current Isus. Ventricular action potentials of isolated perfused hearts were recorded by means of standard floating microelectrodes and ionic currents of single ventricular myocytes were measured using the whole-cell recording patch-clamp technique. We show that: (1) AP plateau amplitude and duration increase more markedly in severe than in moderate hypertrophy; (2) the decrease in Ito1 density is much larger in severe than in moderate hypertrophy whereas ICa-L, INa-Ca and i(sus) densities remain unaltered in either state of hypertrophy. After suppression of Ito1 by 3 mM 4-aminopyridine, action potential plateau amplitude and duration remain increased in severely hypertrophied rat hearts compared to sham rats. Therefore, although these results designate Ito1 reduction as the main cause of hypertrophy-induced AP changes, those occurring in severe hypertrophy cannot be uniquely explained by a quasi-complete extinction of Ito1.

Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure.

Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock. Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 M KCl) activated by stretch. The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances. In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch. These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E. coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes. These conductances could be grouped into three subfamilies of poorly selective channels. In both preparations, the higher the conductance, the higher was the negative pressure needed for activation. We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system.

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.

Effects of chloride ion substitutes and chloride channel blockers on the transient outward current in rat ventricular myocytes.

The Cai(2+)-insensitive transient outward current, ilo was studied at 20-24 degrees C in rat ventricular myocytes with the whole cell recording patch-clamp technique. The current was recorded before and after replacement of chloride by methanesulfonate or aspartate or in the absence and the presence of chloride channel blockers, SITS or 9-anthracene carboxylic acid. In control conditions (in the presence of external divalent cations, Ca2+ and Cd2+, Cd2+ being used to suppress Ca2+ current), ilo inactivation was composed of a fast and a slow component. When methanesulfonate was substituted for external Cl-, the peak current decreased to a variable extent, but the inactivation of the remaining current was still composed of a fast and a slow component. In contrast, the inactivation of the difference current was well fitted by a single exponential. The time to peak of the difference current was shorter than that of the current recorded either in the absence or the presence of methanesulfonate. Both activation- and steady-state inactivation-voltage curves were either unchanged (n = 4) or shifted by a few mV (5.5 mV, n = 14) towards positive potentials when methanesulfonate was substituted for Cl-. The current remaining in methanesulfonate reversed at potentials closed to EK. The difference current was composed of a peak and a steady-state component. The peak was suppressed by 4-aminopyridine whereas the steady-state component was not. The peak was also suppressed when pipette solution contained Cs+ instead of K+ but was still present when the Hepes concentration in both external and pipette media was increased 5-fold (50 mM vs. 10 mM). When aspartate was substituted for Cl- or when 2 mM SITS was added to the external solution (in the absence of Ca2+ and Cd2+ because aspartate is known to chelate Ca2+ ions and possibly other divalent cations), ilo was reduced to a similar extent in the two cases and the difference current was composed of a peak (inactivation fitted by a single exponential) and a steady-state component. The SITS-sensitive transient current reversed at a potential close to ECl. When 5 mM 9-anthracene carboxylic acid was added to external solution (in the presence of Ca2+ and Cd2+), the peak of the difference current was similar to that observed when Cl- was substituted by methanesulfonate. The difference current resulting from the substitution of methanesulfonate for chloride was not changed when the pipette solution contained either 50 mM EGTA (instead of 5 mM) or 10 mM EGTA and 10 mM BAPTA. The nature of Cs(+)- and 4-aminopyridine-sensitive transient outward current suppressed by chloride ion substitutes or chloride channel blockers is discussed.

Divalent cation channels activated by phenothiazines in membrane of rat ventricular myocytes.

Phenothiazines (PTZ) such as chlorpromazine (CPZ) or trifluoperazine (TPZ) induced a sustained divalent cation-permeable channel activity when applied on either side of inside-out patches or on external side of cell-attached patches of adult rat ventricular myocytes. The percentage of active patches was approximately 20%. In the case of CPZ, the Kd of the dose-response curve was 160 microM. CPZ-activated channels were potential-independent in the physiological range of membrane potential and were permeable to several divalent ions (Ba2+, Ca2+, Mg2+, Mn2+). At least three levels of currents were usually detected with conductances of 23, 50 and 80 pS in symmetrical 96 mM Ba2+ solution and 17, 36 and 61 pS in symmetrical 96 mM Ca2+ solution. Saturation curves corresponding to the three main conductances determined in Ba2+ symmetrical solutions (tonicity compensated with choline-Cl) gave maximum conductances of 36, 81 and 116 pS (with corresponding half-saturating concentration constants of 31.5, 38 and 34.5 mM). The corresponding conductance values were estimated to 1.7, 3.3 and 5.2 pS in symmetrical 1.8 mM Ba2+ and to 1.1, 2.4 and 3.7 pS in symmetrical 1.8 mM Ca2+ (the value in normal Tyrode solution). Channels were poorly permeable to monovalent cations, such as Na, with a PBa/PNa ratio of 10. A PTZ-induced channel activity similar to that described in cardiac cells was also observed in cultured rat aortic smooth muscle cells but not in cultured neuroblastoma cells. PTZ-activated channels described in cardiac cells appear very similar to the sporadically active divalent ion permeable channels described in a previous paper (Coulombe et al., 1989). Surprisingly, when 100 microM CPZ were applied to myocytes studied in the whole-cell configuration, and maintained at a holding potential of -80 mV in the presence of 24 mM external Ca2+ or Ba2+, no detectable macroscopic inward current could be observed, whereas the L-type Ca2+ current triggered by depolarizing pulses was markedly and reversibly reduced. The possible reasons are discussed.

Reduction of calcium-independent transient outward potassium current density in DOCA salt hypertrophied rat ventricular myocytes.

Saline-drinking, left-nephrectomized rats made hypertensive by deoxycorticosterone acetate (DOCA) pellet implantation at the time of surgery develop a cardiac hypertrophy, which becomes maximal after 6-7 weeks. The hypertrophy results in a marked increase in the amplitude and duration of both the early and the late component of the ventricular action potential plateau recorded in the isolated perfused rat heart. The 4-aminopyridine(4-AP)-sensitive calcium-independent transient outward potassium current was markedly depressed in hypertrophied ventricular myocytes resulting in a highly significant decrease in current density (from 19.9 +/- 3.5 to 6.4 +/- 3.1 pA/pF at +60 mV). Activation/voltage and steady-state inactivation/voltage relationships were moderately although non-significantly shifted towards negative potentials. The steady-state outward current measured at the end of 1-s depolarizing pulses was not significantly changed in hypertrophied myocytes. 4-AP induced a smaller increase in plateau amplitude and duration in hypertrophied rather than in control hearts, a point that is well explained by the depression of the transient outward current resulting from hypertrophy. We also demonstrated that a complete recovery of both cell capacitance and transient outward current amplitude occurs in myocytes from saline-drinking rats studied 13 weeks after DOCA pellet implantation, showing that hypertrophy regresses as a result of pellet elimination. Several mechanisms can be involved in the observed phenomena, including the possibility that the expression of potassium channels responsible for the transient outward current is not enhanced by hypertrophy in contrast with what occurs in the case of calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-dependent cationic channel of Escherichia coli.

A fraction highly enriched with inner membranes of E. coli was fused with liposomes, using the dehydration-rehydration technique, to produce giant liposomes amenable to patch-clamp recordings. Among the several channels present in this type of preparation, one was further characterized. The channel has a conductance of some 200 pS (in 0.1 M KCl) and is weakly selective for cations (PK/PCl = 4). The channel stays open at negative and low positive membrane potentials and shows an increasing probability of closure with increasing voltage. High positive membrane potentials favor transitions to a long-lived inactivated state, following slow kinetics. Voltage-dependent rapid flickerings of the same amplitude, between open state and other short-lived closed states, are superposed on these kinetics. The channel is presumed to be localized in the inner membrane, but its characteristics are also compatible with those of porins of the outer membrane. However, the major porins OmpF and OmpC, purified and reconstituted into giant liposomes, exhibited a marked different behavior.

Large-conductance chloride channels of new-born rat cardiac myocytes are activated by hypotonic media.

Large-conductance chloride (LC-type Cl-) channel activity was studied in rat ventricular myocyte membrane during development. In contrast with results previously obtained in cultured ventricular myocytes of the new-born rat, we failed to record single-channel activity in freshly isolated myocytes whatever the age of the animal (from 2 days old to adult) and the recording patch configuration used. However, spontaneous single-channel activity of LC-type Cl- channels was recorded in bleb membranes of myocytes of rats younger than 12 days, with a higher frequency in excised inside-out membrane patches than in cell-attached membrane patches. In intact neonatal myocytes, application of hypotonic media (150 mOsm) also initiated the channel activity, after variable delays (25-200 s). The channel could not be activated by suction applied through the pipette and was not observable in cells from rats older than 15 days. The LC-type Cl- channels showed properties similar to those reported in other preparations and previously observed in cardiac cultured cells: they had a large single-channel conductance of 400 pS in symmetrical 150 mM NaCl, showed multiple subconductance states, a relatively high selectivity to Cl- ions over Na+ ions (PCl/PNa = 24.6), were blocked by 10 microM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and showed voltage-dependent inactivation. They were not activated by 10 microM colchicine or 3 microM cytochalasin D.(ABSTRACT TRUNCATED AT 250 WORDS)