Inhibitory activity of antibodies against the human atrial 5-HT(4)receptor.

Antibodies directed against the second extracellular loop of G protein-coupled receptors have been shown to exert "agonist-like" activities. In order to test the hypothesis that this is a general phenomenon, antibodies were raised in rabbits against a synthetic peptide corresponding to the second extracellular loop of the newly sequenced human cardiac 5-HT(4)receptor. The antibodies were affinity-purified and shown to recognize the 5-HT(4)receptor in immunoblots of Chinese hamster ovary (CHO) cells expressing the receptor. The antibodies had no intrinsic effect but could depress the activation of L -type calcium channel induced by serotonin in human atrial cells. This antagonist-like effect was exerted both by intact IgG and by Fab fragments. These results are physiologically important since it has been shown that the 5-HT(4)receptor could be a target for autoantibodies in mothers at risk of giving birth to children with neonatal atrio-ventricular block.

Block of gating currents related to K+ channels as a mechanism of action of clofilium and d-sotalol in isolated guinea-pig ventricular heart cells.

1 The possibility that the class III antiarrhythmic drugs clofilium and d-sotalol might affect delayed rectifier potassium channels at the level of their gating currents was assessed with the whole-cell patch-clamp technique in guinea-pig isolated ventricular heart cells. 2 Clofilium (up to 20 microM) and d-sotalol (1 microM) did not decrease the Na current, the L-type Ca current or the background K current iKl, but significantly depressed the time-dependent delayed outward K current iK. 3 Clofilium partially decreased in a dose-dependent manner (1-20 microM) QON of intramembrane charge movements (ICM) elicited by a depolarizing pulse applied from a holding potential of -110 mV or following a 100 ms inactivating prepulse to -50 mV. D-sotalol (1 microM) also decreased QON. Channel density estimated from the clofilium-sensitive ICM closely matched that of the delayed rectifier channels. 4 Clofilium and d-sotalol decreased QOFF seen on repolarization in a dose- and voltage-dependent manner. The kinetics of the decay of the OFF gating currents were not affected, and only the fast phase was depressed. 5 In control conditions, QON availability with voltage was most of the time well described by two inactivating components. In the presence of clofilium and d-sotalol, a complex behaviour of QON availability was observed, unmasking additional components. The reactivation kinetics of QON after a 500 ms inactivating pulse to 0 mV was not affected. 6 We conclude that delayed rectifier K channels significantly contribute to QON and QOFF of ICM in guinea-pig ventricular heart cells, besides Na and Ca channels, and that clofilium and d-sotalol directly interact with these K channels proteins by affecting their gating properties.

Ruthenium red as an effective blocker of calcium and sodium currents in guinea-pig isolated ventricular heart cells.

1. The effect of ruthenium red on calcium and sodium currents was studied in guinea-pig isolated ventricular heart cells with the whole cell patch-clamp technique. 2. Ruthenium red very efficiently blocked the L-type calcium current in a dose-dependent manner. A significant block was observed for concentrations as low as 0.3 microM. Analysis of the dose-response curve with the logistic equation indicated an EC50 of 0.8 microM, a maximum inhibition of 85% reached at 5 microM, and a coefficient of 2.37. 3. There was no shift in the voltage-dependence of the Ca current activation, nor in that of its steady-state inactivation determined with a 1 s prepulse. However, removal of Ca current inactivation at positive voltage was considerably reduced in the presence of concentrations of ruthenium red above 1 microM. A slowing of the time-course of inactivation of the Ca current was also observed. 4. At 10 microM, a concentration generally used to block the sarcoplasmic Ca release channels or the mitochondrial Ca uptake, ruthenium red blocked 26.7+/-4.3% (n=8) of the sodium current, and slowed its inactivation time-course. No effect was observed on the voltage-dependence of the current activation or inactivation. The peak sodium current was also decreased at a 10 times lower concentration by 7.6+/-2.7% (n=3). 5. Thus, at concentrations used to assess intracellular Ca movements, ruthenium red induced in heart cells a significant block of both Ca and Na channels.

In vivo and in vitro inhibition of the L-type calcium current in isolated guinea-pig cardiomyocytes by the immunosuppressive agent cyclosporin A.

Cyclosporin A (CsA), an immunosuppressive agent used to reduce rejection after organ transplantation, induces secondary effects in heart tissue. We have studied the effects in vivo and in vitro of CsA on L-type Ca2+ current (ICa) and the associated gating currents of isolated guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. For in vivo experiments, a group of animals (n=28) was treated for 21 days by subcutaneous injection of CsA (15 mg/kg/day). Blood level of CsA was 1191+/-221 ng/ml (n=9). In cells from these animals (n=65, 19 animals), ICa was reduced to about 75% of that recorded from control cells (n=32, six animals). CsA decreased the availability of Ca2+ channels at potentials more positive than +30 mV. Isoproterenol (100 nM) was still able to increase ICa but only by 30+/-6% (n=9), whereas in control it increased ICa by 290+/-22% (n=5). Gating currents related to L-type Ca2+ channels were not altered in cells from CsA-treated animals. In in vitro experiments, CsA reduced ICa when applied directly to cardiomyocytes. CsA affected the kinetics of ICa inactivation, slowing down the rapid phase and accelerating the slow phase (n=4). The steady-state inactivation curve of ICa was shifted to more negative voltages and the degree of availability at -80 mV decreased by in vitro application of CsA. The half inactivation potential (V1/2) changed from -23+/-0.6 mV in control to -31+/-2 mV, -48+/-0.6 mV and -49+/-0.6 mV, in 1, 50 and 80 microM CsA, respectively. In these cells, the gating currents related to L-type Ca2+ channels were also not altered by CsA. CsA does not modify the Ca2+ channel density, although it induces a decrease in the beta1-adrenergic stimulation of ICa. The results are explained by a direct effect on the calcium channel inactivation of CsA and a non specific indirect effect.

Effect of sulfhydryl oxidation on ionic and gating currents associated with L-type calcium channels in isolated guinea-pig ventricular myocytes.

OBJECTIVE: L-type calcium currents (ICa) and gating currents modification by extracellular application of the selective free sulfhydryl oxidant p-hydroxy-mercuric-phenylsulphonic acid (PHMPS) were studied. METHODS: Both currents were obtained with the whole cell patch clamp technique in guinea-pig ventricular cardiocytes. RESULTS: The main finding was a reduction of ICa clearly differentiable from a "run down" process. This effect was protected, stopped and in some cases partially reversed by dithiothreitol, a protective reagent for -SH groups. We also found a decrease of the gating currents associated with L-type calcium channels. The calcium modulation and cAMP phosphorylation systems of ICa are unaffected by PHMPS. With barium as charge carrier the current-voltage curves of barium currents were shifted by 10 mV to the positive direction by PHMPS. The same effect was obtained with calcium currents using BAPTA as a fast calcium buffer. CONCLUSION: The results indicate that oxidation of -SH groups carried by the channel protein induces dysfunction of the calcium entry to cardiac cells by altering the gating process. A participation of thiol functions on the gating of the calcium channel is proposed.

The effects of increasing cell length on auxotonic contractions; membrane potential and intracellular calcium transients in single guinea-pig ventricular myocytes.

Until recently the investigation of length-dependent effects in cardiac muscle was restricted to multicellular preparations. We describe our experimental set-up which for the first time, in single cardiac myocytes, permits the effects of changes in cell length on auxotonic contractions (measured by carbon fibre transducers) to be simultaneously recorded with the effects on membrane potential and/or changes in intracellular calcium concentration (using indo-1 AM, acetoxylmethyl form). Consistent with previous findings (in experiments at 20-25 degrees C and 0.25 Hz) we report that following a stretch there was an increase in passive tension and contraction. A stretch which increased sarcomere length by approximately 3% had no significant effect on resting membrane potential or action potential amplitude. There was, however, a significant decrease in the action potential duration (P < 0.01, n = 8). No significant change in the amplitude of the intracellular calcium transient was seen following a stretch but a reduction in its duration was observed (P < 0.025, n = 11). Our observations on intracellular calcium transients are consistent with the hypothesis that, in mechanically loaded preparations, their time course is more dependent on changes in tension than changes in length.

Alteration of the L-type calcium current in guinea-pig single ventricular myocytes by heptaminol hydrochloride.

1. The effects of heptaminol on calcium current amplitude and characteristics were studied in single ventricular myocytes of guinea-pig by use of the whole cell configuration of the patch clamp technique. 2. A concentration-dependent decrease in ICa amplitude was observed. At heptaminol concentration as low as 10(-6) M, this effect was observed in only two cells (n = 6). At 10(-5) M the reduction of ICa was of 30 +/- 15% (n = 11). 3. The current recovery from inactivation at -40 mV holding potential (HP) seemed less sensitive to perfusion with heptaminol (greater than 10(-6) M). However, at -80 mV HP the overshoot of the recovery curve was decreased by heptaminol. 4. Both at -40 mV and -80 mV HP, heptaminol (10(-5) M) significantly increased the steady state inactivation of ICa. 5. As previously proposed by others to explain the effects of membrane active substances, the effects of heptaminol may result from alterations in cell membrane properties and possibly from an increase in intracellular free calcium ion concentration.

Rate dependence of action potential duration and calcium current in isolated guinea-pig cardiocytes.

The duration of the action potential at 50% of its amplitude (APD50) and peak calcium currents (ICa) was measured in single cardiac guinea-pig ventricular cells, using the whole-cell patch-clamp technique in current-clamp and voltage-clamp modes respectively. In the absence of intracellular calcium buffer, pacing at 0.28 Hz from a rest period of 1-2 min induced a transient increase (15.5 +/- 3.5%) in APD50, followed by a gradual shortening. Switching from 0.28 to 0.75 Hz again induced a transient increase of APD50 (6.8 +/- 2.9%). In the presence of EGTA or BAPTA on the cytosolic side of the membrane, this transient phase was prolonged and its amplitude slightly increased (10.6% when switching from 0.28 to 0.75 Hz in 5 mM-BAPA). The same increase in rate induced either a negative or a positive staircase of ICa, depending on the holding potential. At a holding potential of -80 mV, ICa peak was enhanced and the inactivation kinetics was slowed down. This facilitation of ICa seems to be dependent on calcium ions entering the cell via the calcium channels and could partly explain the observed transient increase in APD50.

Stretch-induced increase of resting intracellular calcium concentration in single guinea-pig ventricular myocytes.

Single guinea-pig ventricular myocytes were loaded with the fluorescent Ca2+ indicator Indo-1 AM and stretched by carbon fibres. Stretching increased resting tension. Sarcomere lengths were increased by 2-18%. It was observed that a stretch increased resting [Ca2+]i in seven out of eight cells. The change in [Ca2+]i increased with the size of the stretch and returned to pre-stretch levels on return to resting cell length. These observations suggest a means by which changes in resting muscle length can modify the contractile state of cardiac muscle.

Differences in the hypoxic contraction of small isolated pulmonary arteries of cat and rabbit.

In small (less than 300 microns diameter) pulmonary arterial (PA) rings isolated from the cat, hypoxia induced a transient contraction (250 +/- 120 mg, n = 7), whereas in rings of rabbit PA of the same size, hypoxia had no significant effect (n = 19). Precontraction by 40 mmol KCl.1(-1), noradrenaline (NA) 10(-6) mol.1(-1), or histamine (His 10(-5) mol.1(-1)) did not modify this difference between the two species and did not potentiate the hypoxic contraction of small rings of the cat PA. Large rabbit pulmonary arterial segments (300-2000 microns) exhibited no response to hypoxia before precontraction (n = 15). In the presence of procaine (2%) rabbit PA rings (n = 6, small) exhibited no hypoxic contraction. These results in vitro reflect previous in vivo observations.

A new method of attachment of isolated mammalian ventricular myocytes for tension recording: length dependence of passive and active tension.

The study of the Frank-Starling's law in mammalian single cells has been hindered by a lack of an easily performed method of stretching cells. Some authors have succeeded in this but their methods required a great deal of technical expertise and in most cases they have not had much success. We have developed an easy method of stretching mammalian ventricular cells from slack sarcomere length (S.L.) (Lo, 1.77 +/- 0.05 microns) to about 117% of this length. Thin carbon fibers (12 microns in diameter) which can be bound electrochemically to the cell membrane surface have been used. A flexible long fiber of known compliance (80 microns/microN) was attached to one end of the cell and a stiff double fiber (4 microns/microN) to the other end. The cell attachment was relatively easy to perform and successful results were obtained in 80% of the attempts. The displacement of the flexible fiber allows the quantitative measurements of the resting tension in a group of non-stimulated cells and of auxotonic contractions developed upon stimulation in another group of cells. Increasing S.L. from Lo to 105-106% of Lo, an increase in active tension from 0.21 +/- 0.03 mN/mm to 0.26 +/- 0.01 mN/mm (n = 4) could be noticed with a stimulation frequency of 0.5 Hz. An increase in active tension was also observed at 1 Hz. Staircase kinetics were accelerated with stretching; this confirms at the single cell level the hypothesis of an effect of length-dependent activation on the staircase. Eulerian differential stiffness constant was calculated and found to be 13.5 +/- 1.2, a value which is comparable to that described in intact heart. Thus the important stiffness found in the whole heart may be due to intracellular component(s) such as myofilament and/or connectin.

The effects of aldehydes on the inward rectifier of skeletal muscle fibres from the toad Bufo marinus.

A comparative study of the effects of formaldehyde and glutaraldehyde on the inward rectification in skeletal muscle fibre of the toad Bufo marinus was made using two different techniques. In whole sartorius muscle fibres formaldehyde produced a transient increase in conductance, followed by a decrease while glutaraldehyde only produced the second effect, with a rather fast time course. In cut end single fibres both aldehydes produced only a reduction of conductance and eventually the abolition of the inward rectification. The blocking effect of glutaraldehyde was concentration and voltage dependent while formaldehyde only produced a concentration dependent blocking. The elimination of inward rectifying currents by aldehydes allowed an estimation of the time constants of activation by a subtraction method.

Decrease in internal H+ and positive inotropic effect of heptaminol hydrochloride: a 31P n.m.r. spectroscopy study in rat isolated heart.

1. The cardiotonic effect of heptaminol hydrochloride (Hept-a-myl, Delalande) was studied using 31P-nuclear magnetic resonance (n.m.r.) spectroscopy and left ventricular pressure (LVP) measurements in rat isolated hearts. The possibility of this effect being mediated by an intracellular realkalinisation was tested. 2. Isolated hearts were perfused at 10 ml min-1 by the Langendorff method with Krebs-Henseleit solution at 37 degrees C and stimulated at 5 Hz. Mechanical activity was measured as variations of left ventricular pressure (LVP). 31P-n.m.r. spectra were recorded every 2 min. Changes in cardiac adenosine triphosphate (ATP), phosphocreatine (PCr) and inorganic phosphate (Pi) were followed and intracellular pH (pHi) was estimated from the chemical shift of Pi. 3. The effects of heptaminol were tested in different conditions: normoxia, moderate ischaemia, severe ischaemia, and moderate ischaemia in the presence of amiloride or guanidinium chloride as inhibitors of the Na-H exchange. 4. In normoxia, heptaminol induced a cyclic increase of systolic LVP, associated with an increase in Pi. No significant effect on pHi was observed. In changing from normoxia to moderate ischaemia, PCr and systolic LVP decreased; a mild intracellular acidification (pHi 6.96) was obtained. Heptaminol induced a restoration of pHi and increased LVP. In severe ischaemia, the realkalinization effect and the restoration of LVP induced by heptaminol were no longer observed. During moderate ischaemia, Na-H exchange inhibitors decreased pHi and LVP. Heptaminol applied in the presence of these inhibitors was unable to restore pHi and LVP. In severe ischaemia, the realkalinization effect and the restoration of LVP induced by heptaminol were no longer observed. During moderate ischaemia, Na-H exchange inhibitors decreased pHi and LVP. Heptaminol applied in the presence of these inhibitors was unable to restore pHi and LVP. 5. These results suggest that the positive inotropic effect of heptaminol during moderate ischaemia could be related to a restoration of internal pH, possibly mediated by a stimulation of the Na-H exchange.

Contractile responses to hypoxia of isolated rings from the left branch of rabbit pulmonary artery.

Contractile responses of left extrapulmonary artery segments with either intact or damaged endothelium were examined with changes in PO2 at constant pH. Hypoxia consistently reduced noradrenaline-induced contractions. This hypoxia-induced relaxation was sometimes followed by a contraction and a second relaxation. Hypoxia-induced relaxations were also obtained if precontraction was elicited with KCl (40 mM), but no triphasic response was observed. Relaxations faded away with time and only contractions were then observed. Relaxations were more considerable in rings from young animals and, although always present, decreased with the age of the animal. Endothelium damage reduced hypoxia-induced relaxations. Indomethacin, a potent blocker of cyclooxygenase, increased hypoxia-induced contraction and reduced relaxation in segments with intact endothelium. Without endothelium the indomethacin effect was less significant. It is concluded that the response to hypoxia of the pulmonary artery is similar to that of systemic vessels. Endothelium seems to play a modulatory role in the hypoxia-induced response in extrapulmonary artery. Prostaglandin metabolism seems to play a minor role in this modulation.

Inactivation, reactivation and pacing dependence of calcium current in frog cardiocytes: correlation with current density.

1. Ca2+ currents were measured in single cells isolated from frog ventricle using the whole-cell patch clamp technique and a perfused pipette. K+ currents were blocked with intracellular (120 mM) and extracellular (20 mM) Cs+. 2. A single type of Ca2+ current (ICa) was found in these cells. The current activated at voltages positive to -30 mV, exhibited a symmetrical current-voltage relationship with a peak at 0 mV, and was slowly inactivating with Ba2+ as charge carrier. 3. Large variations in ICa amplitude were observed from cell to cell (ICa at 0 mV = 293.1 +/- 283.3 pA; N = 152). These variations were not due simply to differences in cell membrane area, which was estimated by cell membrane capacitance (Cm), because the density of Ca2+ current (dICa = ICa/Cm) also varied significantly from cell to cell (1.3-28 pA/pF at 0 mV; mean +/- S.D. = 4.49 +/- 3.96; N = 152). 4. The inactivation curve of ICa was a complex function of membrane potential. 200 ms pre-pulses to voltages between -60 and +20 mV progressively inactivated ICa elicited by a subsequent test pulse with half-maximal inactivation occurring for pre-pulses to approximately -40 mV. With pre-pulses positive to +20 mV, ICa elicited by the test pulse became progressively larger. The degree of inactivation induced by a 200 ms depolarization to potentials more positive than +20 mV varied significantly from cell to cell, while no such variations were observed in the negative range of membrane potentials. 5. The time course of reactivation (i.e. removal from inactivation) of ICa at -80 mV often exhibited an overshoot. The amplitude of the overshoot varied between 100% (i.e. no overshoot) and approximately 180% in eighty-one cells. 6. The degree of inactivation at positive potentials (+100 mV) and the amplitude of the overshoot were strongly correlated with the Ca2+ current density. The overshoot was more pronounced, the reactivation was faster, and the inactivation at positive potentials was less in cells with lower ICa density. 7. Increasing the stimulation frequency from 0.125 to 2 Hz induced a positive staircase of ICa in cells with ICa density less than 2 pA/pF and a negative staircase in cells with ICa density greater than 3 pA/pF. 8. Perfusing the patch pipette with 5 mM-BAPTA instead of EGTA reduced the amplitude of the overshoot and slightly slowed the inactivation kinetics. Replacing extracellular Ca2+ ions by Ba2+ ions completely suppressed the overshoot.(ABSTRACT TRUNCATED AT 400 WORDS)

A dual effect of cardiac glycosides on Ca current in single cells of frog heart.

The effects of cardiac glycosides (1 microM ouabain, 50 microM dihydrooubain, 1 microM strophanthidin) on Ca current (ICa) were investigated on Cs-loaded single frog ventricular cells using the whole-cell patch-clamp technique (9). Cardiac glycosides exert both inhibitory and stimulatory effects on ICa in 20 Cs Ringer solution, but have only a stimulatory effect in 0 Cs, when the Na,K pump is blocked. The inhibitory response seems related to the inhibition of the Na,K pump by glycosides. The stimulatory effect on ICa may contribute to the positive inotropic effect of cardiac glycosides.