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R Fischmeister

Publications and source records attributed to R Fischmeister.

At least 73 records · Page 4Linked to original sources
PubMed1989

Cyclic GMP regulates the Ca-channel current in guinea pig ventricular myocytes.

The effect of intracellular perfusion with cyclic GMP (cGMP) on Ca current (Ica) was investigated in Cs-loaded isolated cells from guinea pig ventricle using the whole-cell patch-clamp technique and a perfused patch pipette. cGMP (5 microM) strongly reduced Ica which had been elevated by intracellular perfusion with 50 microM of either cyclic AMP (cAMP) or its hydrolysis-resistant analog 8-Bromo-cAMP. In addition, cGMP prevented the stimulation of Ica by IBMX, a phosphodiesterase inhibitor. The membrane permeant cGMP analog 8-Bromo-cGMP (100 microM), when applied outside the cell, also antagonized the stimulatory effect of IBMX on Ica. It is concluded that cGMP inhibits Ica in guinea pig ventricular cells by a mechanism different from the activation of a cGMP-stimulated phosphodiesterase recently found in frog ventricular cells.

Animals↗
PubMed1989

Interactive effects of isoprenaline, forskolin and acetylcholine on Ca2+ current in frog ventricular myocytes.

1. Calcium currents (ICa) were measured in single cells isolated from frog ventricle using the whole-cell patch-clamp technique and a perfused pipette. The dose-dependent stimulatory effects of isoprenaline (Iso, 0.1-100 microM) and forskolin (Fo. 0.1-50 microM) on ICa were determined in the presence and absence of acetylcholine (ACh, 10 microM) and/or threshold concentrations of Fo (0.2 microM) and Iso (0.05 microM), respectively. EC50 (i.e. concentration of Iso or Fo at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of Ica expressed as percentage increase in ICa with respect to control) were measured under each condition. 2. ACh increased EC50 for the stimulatory action of Iso on ICa from 0.84 to 3.72 microM while it reduced Emax from 658 to 185%. Thus, ACh mainly reduced the efficacy of Iso to stimulate ICa. 3. ACh increased EC50 for the stimulatory action of Fo on ICa from 2.06 to 10.26 microM but only slightly reduced Emax from 893 to 778%. Thus, ACh mainly reduced the potency of Fo to stimulate ICa. 4. Intracellular perfusion with 100 microM of hydrolysis-resistant GTP analogues, GTP-gamma-S [guanosine-5'-O-(3-thiotriphosphate)] and Gpp (NH)p (5'-guanylylimido-diphosphate), had no effect on basal ICa but reduced by greater than 50% the stimulatory effect of 2 microM-Iso on ICa. 5. In the presence of Gpp(NH)p or GTP-gamma-S, Fo (3 microM) reversibly increased ICa by 490%, as compared to a 717% increase in control (GTP) intracellular solution. Although ACh could still inhibit Fo-stimulated ICa, the degree of inhibition was significantly smaller than in the presence of GTP. 6. Extracellular perfusion with low concentrations of a combination of Iso (33 nM) and Fo (330 nM) enhanced ICa to a much greater extent than did either agent alone at 3 times higher concentrations. Thus, low concentrations of Iso and Fo appear to increase ICa in a synergistic fashion. 7. ICa stimulated by a combination of Iso and Fo appeared to be more resistant to inhibition by ACh than when stimulated by either alone. It was the efficacy, rather than the potency, of ACh to inhibit ICa that was reduced upon dual stimulation of ICa. 8. In the presence of 0.2 microM-Fo, EC50 and Emax for the effects of Iso on ICa were 0.27 microM and 619%, respectively. By comparison with the effects of Iso alone, Fo reduced EC50 approximately 3 times with no significant change in maximal stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1988

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)

Action Potentials↗
PubMed1988

Atrial natriuretic factor regulates the calcium current in frog isolated cardiac cells.

The effect of external application of synthetic atrial natriuretic factor (ANF) on calcium current (ICa) was studied in single cells isolated from frog ventricle using the whole-cell patch-clamp technique. Rat atriopeptin III (APIII) and 3-28 ANF rat (rANF) had negligible effects on basal ICa at concentrations up to 200 nM. However, when ICa was increased by isoprenaline, both peptides had significant inhibitory effects. rANF (3 nM) decreased isoprenaline-elevated ICa by an average of 33% after 3-5 minutes. APIII was slightly less effective than rANF. The effects of rANF and APIII were dose-dependent in a complex manner: one stimulatory and two different inhibitory effects were observed, one being responsible for an irreversible rundown of ICa. The effects of ANF were not blocked by atropine and desensitization of the cells to isoprenaline did not play a significant role in the response to ANF. When ICa was elevated by intracellular perfusion with cyclic adenosine 3',5'-monophosphate, added to the patch electrode solution or using a perfused pipette, rANF or APIII had less inhibitory effect, and no rundown of ICa was observed. It is proposed that adenylate cyclase may be one of several mechanisms by which ANF regulates ICa.

Animals↗
PubMed1988

A patch-clamp study of the effects of cicletanine on whole-cell calcium current in ventricular myocytes.

The effects of extracellular application of cicletanine on the voltage-sensitive calcium current (ICa) was studied in isolated cells from frog ventricle. Myocytes were isolated by enzymatic dissociation and ICa was measured using the whole-cell configuration of the patch-clamp technique modified to permit intracellular perfusion with various substances. Cicletanine (10 to 100 microM) had no effect on control ICa. However, when ICa was enhanced by superfusion of the cell with saturating doses of beta-adrenergic agonist (isoprenaline, 2 microM) or by intracellular perfusion with maximal doses of cAMP (20 microM), cicletanine exerted a dual effect on ICa. At 10 microM, cicletanine generally induced a transient or sustained stimulation of ICa (5 to 40%), while 100 microM of the drug generally reduced ICa. The effects of cicletanine were reversible and not voltage-dependent. These results suggest that cicletanine affects ICa by acting on a mechanism occurring after cAMP synthesis, by enhancing cAMP concentration (e.g. through an inhibition of cAMP phosphodiesterase) or facilitating cAMP-dependent phosphorylation of the Ca channels.

Animals↗
PubMed1987

Cyclic guanosine 3',5'-monophosphate regulates the calcium current in single cells from frog ventricle.

1. The effect of intracellular perfusion with cyclic AMP and cyclic GMP on Ca2+ current (ICa) was studied in single cells isolated from frog ventricle using the whole-cell patch-clamp technique and a perfused pipette. 2. Intracellular perfusion with cyclic GMP (0.1-20 microM) had no effect on the basal ICa. However, when ICa was increased by isoprenaline or by intracellular perfusion with cyclic AMP, perfusion with cyclic GMP (20 microM) reduced ICa by an average of 67%. The effect of cyclic GMP on ICa elevated by cyclic AMP was reversible. A half-maximal effect of cyclic GMP was observed at 0.6 microM. Cyclic GMP had no significant effect on the shape of the ICa current-voltage relationship. 3. The effect of cyclic GMP was specific to the 3',5' form; 2',3'-cyclic GMP had no effect. 4. The effect of cyclic GMP was apparently not mediated by stimulation of cyclic-GMP-dependent protein kinase because 8-bromo-cyclic GMP, a very potent activator of the protein kinase, was without effect. 5. Cyclic GMP had no effect on ICa elevated by the non-hydrolysable 8-bromo-cyclic AMP. The effect of cyclic GMP on cyclic-AMP-elevated ICa was partially blocked by the phosphodiesterase inhibitor, methylisobutylxanthine. Thus, it was hypothesized that the effect of cyclic GMP was mediated by hydrolysis of cyclic AMP as a result of a stimulation of a cyclic nucleotide phosphodiesterase by cyclic GMP. 6. The dose-response curve for cyclic AMP on ICa was well fitted by the Michaelis equation with a K50 (i.e. concentration of cyclic AMP at which response is 50% of the maximum) of 0.7 microM and a maximal 11-fold stimulation of ICa. Cyclic GMP shifted the curve one log unit to the right and decreased the maximal stimulation to 8.6-fold. Thus, the effect of cyclic GMP appeared uncompetitive. 7. The products of cyclic AMP and cyclic GMP hydrolysis, 5'-AMP and 5'-GMP, had no effect on ICa. Furthermore, strong buffering of intracellular pH did not reduce the effect of cyclic GMP. 8. It is proposed that cyclic-GMP-stimulation of a cyclic nucleotide phosphodiesterase may be one of several mechanisms by which acetylcholine regulates ICa.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Effect of forskolin and acetylcholine on calcium current in single isolated cardiac myocytes.

The effect of extracellular and intracellular application of forskolin on the voltage-sensitive calcium current, ICa, was studied in myocytes isolated from frog ventricle. Myocytes were isolated by enzymatic dissociation, and ICa was measured using the whole-cell configuration of the patch clamp technique modified to permit intracellular perfusion of various substances. Intracellular perfusion with forskolin (0.1 to 10 microM) had a negligible effect on ICa: ICa was increased 15 +/- 13% (mean +/- SE; N = 5). In contrast, superfusion of the cell with forskolin increased ICa significantly. The EC50 for the forskolin effect was 0.4 microM. A maximal 4.5-fold increase in ICa occurred with 3 microM forskolin. This is somewhat less than the maximal response to isoprenaline seen in this same series of experiments. The effects of forskolin, isoprenaline, and intracellular cAMP were not additive. In contrast, the effects of isoprenaline or intracellular cAMP and calcium channel agonists, such as Sandoz (+)202-791, were additive. This supports the hypothesis that the positive inotropic effects of forskolin are at least partly mediated by an increase in intracellular cAMP and a stimulation of ICa. The effects of forskolin were antagonized by acetylcholine (1 microM) or intracellular perfusion with cGMP. Acetylcholine on the average decreased forskolin-stimulated ICa 57 +/- 11% (N = 17). The relevance of these results to the suggestion that acetylcholine acts by mechanisms other than inhibition of adenylate cyclase is discussed.

Acetylcholine↗
PubMed1986

Some limitations of the cell-attached patch clamp technique: a two-electrode analysis.

With two independent patch electrodes sealed to small clusters of electrically coupled chick embryo cardiac cells, we have measured four parameters: true seal and patch resistance, channel conductance, and membrane potential. One electrode was in the cell-attached mode, and recorded current flowing in parallel through the membrane patch and seal. The second electrode, sealed on a different cell in the cluster, was in the whole cell recording configuration, and served to record or control the membrane potential of the cluster. We fit the four measured parameters to a simple electrical model to reveal errors not usually recognized in the patch-clamp technique. Among these are the following: (1) The apparent seal resistance, determined by changing the potential in a patch electrode, may be a poor estimate of true seal resistance, since it includes the parallel combination of seal- and patch-resistance. (2) Patch resistance may be influenced by the electrode filling solution, and is often much lower than is usually assumed. (3) With a small cell preparation that has an input resistance in the gigaohm range, measurements of single-channel conductance using a cell-attached patch electrode may be inaccurate because cell membrane potential does not remain constant as electrode potential is varied.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Mechanism of action of acetylcholine on calcium current in single cells from frog ventricle.

Ca currents (ICa) were measured by whole-cell patch clamp in single cells isolated from frog ventricle in which K currents were blocked with intracellular (120 mM) and extracellular (20 mM) Cs. Inward currents elicited by depolarizing voltage steps from a holding potential of -80 mV were blocked completely by 0.5 mM-Cd. The quality of the voltage clamp was assessed using two patch electrodes on a single cell. One electrode was used in the voltage-clamp mode to measure membrane currents and the other in current-clamp to measure membrane potential. Ca currents as large as 2 nA were well clamped in cells as long as 210 micron. Acetylcholine (ACh) had no effect on ICa in the absence of beta-adrenergic stimulation but reduced to control levels ICa elevated by isoprenaline. Nanomolar concentrations of ACh were able to reduce significantly ICa elevated by 2 microM-isoprenaline. ACh had no effect on the shape of the I-V curve, on the reactivation (recovery from inactivation), or on the inactivation of ICa. Although isoprenaline increased ICa by an average of 6.5-fold, it had no effect on the shape of the I-V curve or on the inactivation at test potentials negative to +40 mV. However, isoprenaline slowed the half-reactivation time from a control value of 120 +/- 10 ms (mean +/- S.D.) to 153 +/- 12 ms at -80 mV. The effect of cyclic AMP on ICa was investigated using two patch electrodes, one filled with cyclic AMP. Maximal effects of cyclic AMP were observed with 5 microM-cyclic AMP in the pipette. Maximal ICa was recorded several minutes after breaking the patch with the second electrode. After removing the cyclic-AMP-containing electrode, ICa declined to control levels after approximately 10 min. 5 microM-cyclic AMP in the patch electrode increased ICa by an average of 6.9-fold, but had no effect on the shape of the I-V curve or on inactivation. Cyclic AMP had a slowing effect on reactivation (half-reactivation time = 155 +/- 24 ms) similar to that of isoprenaline. ACh (1-10 microM) did not reduce ICa elevated with cyclic AMP (0.1-20 microM-cyclic AMP in the pipette). With low concentrations of cyclic AMP in the pipette (0.1 microM), isoprenaline augmented ICa, but with 5 microM-cyclic AMP in the pipette, isoprenaline was incapable of increasing ICa further. These results suggest that the decrease of ICa produced by ACh can be explained solely by decreases in cyclic AMP levels.

Acetylcholine↗
PubMed1984

Channel currents during spontaneous action potentials in embryonic chick heart cells. The action potential patch clamp.

Single-channel currents were recorded with the cell-attached patch-clamp technique from small clusters (2-20 cells) of spontaneously beating 7-d embryo ventricle cells. Because the preparation was rhythmically active, the trans-patch potential varied with the action potential (AP). The total current through the patch membrane was the patch action current (AC). ACs and APs could be recorded simultaneously, with two electrodes, or sequentially with one electrode. Channel activity, which varied depending on the number and type of channels in the patch, was present during normal cell firing. This method can reveal the kinetics and magnitudes of the specific currents that contributed to the AP, under conditions that reflect not only the time and voltage dependence of the channels, but also environmental factors that may influence channel behavior during the AP.

Action Potentials↗
PubMed1984

Calcium-mediated inactivation of the calcium conductance in cesium-loaded frog heart cells.

Ca current inactivation was investigated in frog atrial muscle under voltage-clamp conditions. To inhibit the outward currents, experiments were performed on Cs-loaded fibers and in 20 mM Cs (K-free) Ringer with 4-AP added. Inactivation, produced by a conditioning pulse, was measured by reducing the current during a subsequent test pulse. The extent of inactivation increased initially with prepulse amplitude and then decreased as the prepulse potential became progressively positive. Relative inactivation follows a U-shaped curve. When Sr was substituted for Ca, both the degree and the rate of inactivation decreased. Relative inactivation appeared to be linearly related to the amount of divalent cations (Ca and Sr) carried into the cell during the prepulse. Elevating Ca enhanced peak current and accelerated its decline. Elevating Mg decreased peak current and slowed its decline. An application of Na-free (LiCl) solution resulted in a somewhat smaller but faster inactivating current. Adrenaline increased and D600 decreased the maximal Ca conductance with little alteration in the inactivation rate; Co decreased both peak current and the rate of inactivation. Enhancement of the outward currents, reduced driving force, and intracellular surface charge screening do not adequately account for the above results. Evidence was considered that Ca entry mediates most of Ca current inactivation in frog atrial fibers. Removal from inactivation was also investigated in normal-Ca, Ca-rich, and Sr solutions. Recovery after partial inactivation by high depolarization was biphasic. Recovery was slowed by 10 Ca and accelerated by 1.8 Sr, whereas opposite effects have been shown on activation.

Animals↗
PubMed1984

Changes in external Na induce a membrane current related to the Na-Ca exchange in cesium-loaded frog heart cells.

The effects of transient alterations in Nao were investigated under voltage clamp conditions in frog heart cells previously loaded with Cs. Tetrodotoxin and Cs were used to inhibit Na and K currents. On applying a Na-poor solution (39.2 mM), an outward current was generated during both depolarizations and hyperpolarizations. The current amplitude described a U-shaped function of the membrane potential. On reapplying the standard solution after 15 min equilibration, an inward current was then induced that exhibited a bell-shaped function of the membrane potential. Current amplitude was sensitive to the external Ca concentration. Increasing pHi by 10 mM NH4Cl enhanced this current, while the internal acidification that occurred on switching back to the control solution greatly reduced it. Variations in the amplitude of this current during repetitive stimulations or long pauses are best explained by subsequent alterations in Nai and pHi; no evidence for a time dependence was found. This current was inhibited by La3+, Co2+, and D600, and was sensitive to adriamycin, quinidine, and disopyramide; lidocaine, another local anesthetic, and nifedipine had no effect. These observations extend previous work on intact heart cells and sarcolemmal vesicles. They suggest that the Na-Ca exchange may generate a current that is outward when Ca ions are moving into the cell.

Amiloride↗
PubMed1983

Variation of intracellular Ca2+ following Ca2+ current in heart. A theoretical study of ionic diffusion inside a cylindrical cell.

The variation in the concentration of a diffusing substance inside a cylindrical cell submitted to a time-dependent flux at the sarcolemmal membrane was studied theoretically. An application was derived to estimate the local modifications of intracellular free Ca2+ concentration ([CA2+]i) induced by the slow inward Ca2+ current (ICa) in frog heart. During a O mV voltage clamp depolarization, [Ca2+]i at the inner side of the membrane rises earlier and faster than [Ca2+]i at the center of the cell. The binding of intracellular Ca2+ to specific sites enhances the deviation between the two concentrations and may generate an accumulation-depletion process of Ca2+ near the membrane. However, it also decreases the overall [Ca2+]i. The relatively slow diffusion of sarcoplasmic Ca2+ does not significantly affect the kinetics of ICa through a modification in the Ca2+ gradient across the membrane.

Animals↗
PubMed1983

Caffeine-induced current in embryonic heart cells: time course and voltage dependence.

Abrupt exposure of 90- to 130-micron diameter chick embryonic myocardial cell aggregates to 10 mM caffeine has been shown to induce a transient inward current. In the present study, we recorded a similar current in small cell clusters (less than 10 cells) in which access of caffeine to each of the cells was rapid. The resulting inward current consisted of a single peak, which decayed exponentially (predominant time constant 335 +/- 130 ms at -40 mV) and had a peak amplitude of up to 15.5 microA/cm2. The caffeine-induced current persisted when the slow inward current was abolished by a 30-s pretreatment with 2 microM D 600 and could be observed at potentials where the fast sodium channels were fully inactivated. The current-voltage relation of the caffeine response was linear between -110 and -40 mV, giving an extrapolated voltage intercept of +12 mV. However, the inward current did not diminish or reverse with further depolarization. A substantial inward current occurred at potentials up to +60 mV, which is more positive than the reversal potential of the tetrodotoxin-sensitive inward current. We conclude that the caffeine-induced current is mediated in part by electrogenic Na+-Ca2+ exchange.

Animals↗
PubMed1982

Slow inward Ca current in frog heart: theoretical evidence against a voltage-dependent inactivation.

The validity of a Hodgkin-Huxley type voltage-dependent inactivation of slow inward Ca current (Isi) was tested in frog heart using a computer simulation. The time course of Isi was calculated during the development of a frog atrial action potential (AP). With a time constant of inactivation (tauf) of 55 ms at a membrane potential (Em) of -15 mV, the variation of Isi was biphasic: after a transient increase followed by a decrease to zero, Isi partially "reactivated" (at the beginning of the AP repolarization phase) and then fully deactivated. The "reactivation" phase of Isi developed whether tauf was an increasing, decreasing, U-shaped, or bell-shaped function of Em. The addition of an independent and slower process responsible for the recovery from inactivation only partly suppressed the "reactivation" phase. However, until now there was no experimental evidence supporting such a biphasic variation of Isi during AP repolarization. Thus our results indicate that the Hodgkin-Huxley type model of the voltage-dependence of Isi-inactivation process may not correctly represent the actual behavior of frog cardiac muscle.

Action Potentials↗
PubMed1981

The electrogenic Na-Ca exchange and the cardiac electrical activity. I--Simulation on Purkinje fibre action potential.

1. The effects of the current (iex) generated by the Na-Ca exchange mechanism have been investigated on the electrical activity of a cardiac cell using the model of MC ALLISTER et al. (1975) for the Purkinje fibre action potential (AP). 2. The reversal potential and the steady-state value of iex were described by the same equations as for the squid axon (MULLINS, 1976). The maximal intensity and the time constant of iex were extrapolated from experiments using frog hearts. 3. A compact program written with the Adams method in Fortran IV (PLANT, 1979) allowed a minicomputer to be used. 4. The addition of iex to the previous model induced a prolongation of the AP and a reduction of the duration of the diastolic phase. 5. Increasing the maximal steady-state amplitude of iex may lead to early after depolarizations and oscillatory behaviour. These effects can be prevented by adequate changes in the amplitude of some potassium outward currents. 6. It is concluded that : (i) alterations of the Na-Ca exchange, e.g., by cellular Na loading, should induce variations of both AP repolarization and diastolic phase durations and, consequently, alter the beating rate ; (ii) iex could interfere with the outward currents whose characteristics should be reconsidered.

Action Potentials↗