Force production in voltage-clamped human atrial muscle.

Human atrial muscle preparations obtained during open heart surgery were mounted in a sucrose gap. Force and membrane currents were recorded during voltage clamp. After a 20-s rest, 10 clamps from a holding potential of -40 to 0 mV at 1.0 Hz were given. This was followed by a test clamp (called 1) of a varied duration and amplitude and two more test clamps (called 2 and 3) as during the priming period. Peak force of contraction 1 (F1) was independent of clamp duration from 2s to about 100 ms but declined at shorter durations. Peak force of contraction 2 (F2) and 3 (F3) increased with the duration and became potentiated. Increasing the clamp amplitude raised F1 to an optimum value at about +10 mV and there was a decline at higher voltages. Both F2 and F3 increased at higher amplitudes. A conventional bell-shaped current-voltage relation for the second inward current was obtained during clamp 1 with maximum inward current around -10 mV. In control experiments on isolated human myocytes peak current was recorded at somewhat more positive potentials. The relation between F3 and F2 was linear both when duration and amplitude of clamp 1 was varied. The slope of the line, interpreted as a measure of recirculation of activator calcium, was 0.4. It is concluded that force during voltage clamp in human atrial muscle is similarly related to membrane voltage as previously reported for guinea pig and ferret preparations.     

Potentiation of the contraction following a prolonged depolarization in isolated ferret myocardium

The contractile force was studied in ferret papillary muscles during voltage clamp depolarizations, using the single sucrose gap method. Prolongation of a test depolarization within a train produced potentiation of the following contraction. The effects of varied duration and membrane potential of the test depolarization upon the potentiated force of the following beat were studied. We assumed that force of a beat was an index of calcium entry on the previous depolarization. The relationship between the peak contractile force of the following potentiated beat and the systolic membrane potential of the test depolarization revealed an equilibrium around ?18 mV. This was manifest after l00 ms of no effect. Positive potentials caused potentiation of force of the following beat; negative potentials caused suppression of force of the following beat. Calcium entry, if carried by an electrogenic exchange mechanism, would be revealed as a membrane current developing after l00 ms. Membrane current at these times was always outward. When the duration of the test depolarization was prolonged, outward current prior to repolarisation progressively increased. When the duration of the test depolarization was held constant, outward current was varied by variation in membrane potential. Force of the following beat was proportional to the test clamp membrane potential. The potentiation of the contraction following a prolonged depolarization was abolished by substituting 75% of the sodium in the perfusion medium with lithium. These results are compatible with the hypothesis that potentiation of force following a prolonged depolarization is derived from calcium entry into myocardial cells by reversed sodium-calcium exchange.     

Force production following transient potential changes in voltage-clamped myocardium

Inter-relationships between force, membrane voltage and currents were studied in ferret and guinea-pig papillary muscles using the single sucrose gap technique (37 degrees C). The preparations were held at -90 or -40 mV and depolarized (excited) to 0 mV for 180 ms at 1.0 Hz. At regular intervals the shape of a single clamp pulse (called '1') was varied and its effects were investigated during the same test cycle and in two subsequent test cycles ('2' and '3'). Peak force of contraction 1 (F1) increased with the duration of the test clamp up to 90 ms and was constant thereafter. F1 increased with clamp amplitude (V1) between -30 and 10 mV and decreased at greater amplitudes. This relation was similar to the relation between peak second inward current (I1) and V1. The peak force of contractions 2 and 3 rose with the clamp duration and clamp amplitudes of cycle 1. The relation between F3 and F2 was linear (slope 0.40), except at the lowest and highest F2 values where there was a small deviation. There was an inverse relation between I2 and F2. The results support the idea that increased duration or amplitude of the voltage clamp pulse leads to a greater calcium entry which is manifested in the following potentiated contraction. The relation between F3 and F2 implies that about 40% of calcium recirculates between the contractions. The inverse relationship between F2 and I2 indicates that the second inward current is regulated by release from the sarcoplasmic reticulum via negative feedback.     

Local control of excitation-contraction coupling in rat heart cells.

1. Cytosolic free calcium ion concentration ([Ca2+]i) and whole-cell L-type Ca2+ channel currents were measured during excitation-contraction (E-C) coupling in single voltage-clamped rat cardiac ventricular cells. The measurements were used to compute the total cellular efflux of calcium ions through sarcoplasmic reticulum (SR) Ca2+ release channels (FSR,rel) and the influx of Ca2+ via L-type Ca2+ channels (FICa). 2. FSR,rel was elicited by depolarizing voltage-clamp pulses 200 ms in duration to membrane potentials from -30 to +80 mV. Over this range, peak FSR,rel had a bell-shaped dependence on clamp pulse potential. In all cells, the 'gain' of the system, measured as the ratio, FSR,rel(max)/FICa(max), declined from about 16, at 0 mV, to much lower values as clamp pulse voltage was made progressively more positive. We named this phenomenon of change in gain as a function of membrane potential, 'variable gain'. At clamp pulse potentials in the range -30 to 0 mV, the gain differed from cell to cell, being constant at about 16 in some cells, but decreasing from high values (approximately 65) at -20 mV in others. 3. At clamp pulse potentials at which Ca2+ influx (FICa) was maintained, FSR,rel also had a small maintained component. When macroscopic Ca2+ influx was brief (1-2 ms, during 'tails' of FICa), FSR,rel rose rapidly to a peak after repolarization and then declined with a half-time of about 9 ms (typically). 4. The rising phase of [Ca2+]i transients could be interrupted by stopping Ca2+ influx rapidly (by voltage clamp). We therefore termed this phenomenon 'interrupted SR Ca2+ release'.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acceleration of relaxation by hyperpolarization of the crayfish muscle fibre membrane

Summary The membrane of single crayfish muscle fibres was depolarized to –50 to zero mV for 50 or 500 ms by a voltage clamp through two microelectrodes. Simultaneously, clamp current, isometric force development and its first time derivative were recorded. After the depolarization pulse, the membrane potential was either clamped back to the holding potential of about –70 mV (repolarization), or clamped to a more negative potential of –95 to –125 mV (hyperpolarization). During such hyperpolarizations, relaxations following large contractions were accelerated. If e. g. the contractions were triggered by depolarizations to –29 to zero mV of 500 ms duration, the average increase in the maximum rate of relaxation due to hyperpolarization was 27±3% (s.e.). The acceleration of relaxation by hyperpolarization started about 100 ms after the end of the depolarization pulse. This latent period was observed after depolarization pulses of 50 as well as 500 ms duration. If chloride in the bathing solution was replaced by nitrate, the acceleration of relaxation by hyperpolarization was increased. If chloride was replaced by propionate, relaxation was slowed. The acceleration of relaxation by hyperpolarization was smaller in propionate than in chloride saline. The latent period for acceleration to appear after the end of the depolarization pulse increased to 450 ms in propionate saline.—The results seem to indicate a state of excitation in parts of the transverse tubular system triggered by depolarization, which cannot fully be terminated by a repolarization of the external membrane for several 100 ms and thus slows relaxation.This study was supported by the Deutsche Forschungsgemeinschaft.     

The effects of changes to action potential duration on the calcium content of the sarcoplasmic reticulum in isolated guinea-pig ventricular myocytes

. We have estimated sarcoplasmic reticulum calcium content using rapid application of caffeine on voltage clamped, isolated guinea-pig ventricular myocytes. Caffeine induces the release of calcium from the sarcoplasmic reticulum and this calcium is extruded from the cells by the sarcolemmal Na/Ca exchange. Integrating the inward Na/Ca exchange current thus allows estimations of sarcoplasmic reticulum calcium content. Ventricular myocytes were stimulated to reach new steady-states by action potential voltage clamps of varying duration. Once contractile steady-state had been reached caffeine was rapidly applied in place of the next action potential and sarcoplasmic reticulum calcium content measured. Prolonging the action potential duration increased sarcoplasmic reticulum calcium content and vice-versa. This calcium loading may underlie the positive inotropic effect of increased action potential duration. Received: 11 July 1996 / Received after revision: 15 October 1996 / Accepted: 26 November 1996     

Sarcoplasmic reticulum calcium mobilization in right ventricular pressure-overload hypertrophy in the ferret: relationships to diastolic dysfunction and a negative treppe

In a model of right-ventricular pressure-overload hypertrophy (POH) in the ferret, action potential duration (to 90% repolarization) was found to be significantly longer (228±11 vs 314±12 ms) with no change in amplitude (85±3 vs 85±2 mV) or resting membrane potential (-79 ±1.5 vs -79±1 mV) for control and POH, respectively. Peak sarcoplasmic reticulum Ca²⁺ release (expressed as the logarithm of the fractional luminescence,-4.2±0.1 vs -4.4±0.3) and resting calcium concentrations (-5.5±0.1vs -5.7±0.1) were not different between the two groups (control vs POH respectively). Muscles from control and POH animals demonstrated a positive force/interval relationship in the presence of physiological extracellular [Ca²⁺]. However, unlike muscles from control animals, muscles from animals with POH subjected to increasing frequencies of contraction in the presence of increased extracellular [Ca²⁺] demonstrated further impairment of diastolic relaxation and a negative treppe. Exposure of muscles from POH animals to isoproterenol returned the slowed Ca²⁺ uptake by the sarcoplasmic reticulum as detected with aequorin to control values, although the relaxation phase of the isometric twitch remained prolonged compared to non-hypertrophied muscles. Exposure to milrinone also abbreviated the time course of the intracellular Ca²⁺ transient, but did not return it to that seen in normal myocardium. The exposure of non-hypertrophied isolated muscles to caffeine resulted in similar prolongation of the isometric twitch duration to that seen in hypertrophied myocardium. Results of these experiments suggest that impaired muscle relaxation in POH reflects changes at the level of the myofilaments. Thus, although slowed intracellular calcium mobilization contributes to diastolic relaxation abnormalities, it can not be the sole factor responsible for the slowed relaxation as has been suggested.     

Properties of a facilitating calcium current in pace-maker neurones of the snail, Helix pomatia.

1. Simultaneous measurements of local voltage clamp currents from patches of soma membrane and K activity at the soma surface were used to analyse the time and voltage dependence of the slow inward current in bursting pace-maker neurones of the snail (Helix pomatia). 2. At low levels of depolarization (less than or equal to mV) a net inward current is recorded simultaneously with an efflux of K ions from the cell. 3. With larger depolarizations (20-170 mV from holding potential of -50 mV) the deficit in net outward charge transfer compared with K efflux and the appearance of inward-going tail currents following repolarization, reveal a persistent inward-going current also under these conditions. This inward current is carried primarily by Ca ions, as demonstrated by its voltage dependence (a minimum at about + 115 mV) and its disappearance in Co-Ringer. It is identified with the slow inward Ca current Iin slow (Eckert & Lux, 1976). 4. The inward current predicted from comparisons of current trajectories reaches a maximum at 15-20 msec (for depolarizations from -50 to 0 mV) and gradually declines with sustained depolarization. 5. Partial inactivation is removed by repolarization to -50 mV and the Ca dependent deficit is greater in the sum of repeated voltage clamp pulses than during sustained depolarization. It is largest for pulses of 25-100 msec duration, decreasing as pulse duration increases. 6. Responses to repeated activation with 100 msec pulses with different repolarization intervals reveal a minimum Iin slow at short intervals (e.g. 20 msec) due to failure to remove partial inactivation. At intermediate intervals (e.g. 200-400 msec) Iin slow shows facilitation. This is revealed in calculations of the net charge transfer and current deficits and is also shown in the tail currents following repolarization. The deficit increases progressively with repetitive stimulation. With longer intervals (e.g. 800-1000 msec) defacilitation during repeated stimulation after the first two pulses is revealed in calculations of deficits, current trajectories and in the tail currents. 7. Although facilitation depends on duration of repolarization between pulses, increasing intermediate hyperpolarizations from the holding potential of -50 mV are usually ineffective in increasing Iin slow. Strong preceding hyperpolarization can even decrease the magnitude of Iin slow and prevent its facilitation with repetitive stimulation,whereas preceding depolarizing pulses can increase Iin slow without preventing its facilitation with repetitive stimulation. 8. The properties of Iin slow are contrasted with previously described membrane conductances and compared with properties attributed to Ca fluxes in other systems.     

Mechanical and electrophysiological effects of milrinone on the force-frequency relationship in mammalian myocardium

Isometric force, action potential and current-voltage relation were studied in guinea-pig and ferret papillary muscles. Milrinone (1 μu) increased peak twitch force by 40 + 4%, reduced time to peak tension (TPT) by 12.1 ± 3% (n= 6, P < 0.01) and reduced time to half relaxation by 17.3 ± 4.1 % (n= 6, P < 0.01). The effect of milrinone was potentiated by rolipram, a RI-PDE inhibitor which in itself had no inotropic effect. After the addition of rolipram peak isometric force was increased by 104 + 8% (n= 6, P < 0.001), TPT was further reduced whereas time to half relaxation was slightly increased after the addition of rolipram. Action potential duration at 75% repolarization was decreased by 11 + 5 ms (n= 6, P < 0.05). Milrinone also potentiated the second inward current (I_st) by 21 + 3.2% (n= 6, P < 0.01). Peak twitch force in response to a test stimulus after an interval, i.e. mechanical restitution was increased at all intervals. The onset of restitution was faster and time to full restitution also shortened. Maximum postextrasystolic potentiation was greater in the presence of milrinone, whereas relative potentiation was smaller in presence of milrinone (46 ± 7%) than in control (74 ± 7%). The recirculation fraction of activator calcium was enhanced by milrinone from 0.35 ± 0.04 to 0.48 ± 0.07. The results support the view that the positive inotropic effect of milrinone is due to a greater inflow of calcium during the action potential and a more efficient intracellular calcium handling. In addition, the effect on mechanical restitution would suggest a more direct action on sarcoplasmic reticulum calcium handling.     

Contractions of single crayfish muscle fibers induced by controlled changes of membrane potential

Summary The membrane potential of short crayfish muscle fibers was changed stepwise by means of the voltage clamp method. Simultaneously, the development of force was measured isometrically. The threshold potential for contraction lies at –60 to –50 mV, at stronger depolarization up to –10 mV, the force increases steeply. Further depolarization, however, does not lead to an appreciable increase of force. The membrane current flowing during such depolarizations is not correlated to the developed force. During depolarization to a certain membrane potential, the maximum force depends strongly on the duration of depolarization: the force reaches a steady state only after 5 to 20 s. The amplitude of the steady state force increases with depolarization. Tension reaches a maximum value of 10 kg/cm² during a depolarization to 0 mV. The relaxation does not depend on the course of the preceding depolarization but is dependent on the maximum force previously achieved.Development of force starts immediately after the onset of depolarization, the rate of rise of the force increases steeply for 160 ms, and then decreases slowly. After repolarization of the membrane, the force increases still for 50 ms or stays constant if the steady state has been reached. After short depolarizing pulses (10 ms), the rate of rise of the force increases for about 50 ms.The time course of the active state and its control by the membrane potential are discussed.This work was supported by the Deutsche Forschungsgemeinschaft.     

Slow inactivation of currents in cardiac Purkinje fibres

1. When membrane currents, associated with repolarization to the holding potential, were measured in cardiac Purkinje fibres after depolarizing voltage clamp steps to the plateau level of the action potential (-20 to -10 mV), tails of inward current were observed which turned into outward current. Amplitude and duration of the tails of inward current increased when the preceding depolarization was progressively shortened. This current could only be recorded in Na-containing solution which strongly supports the view that the inactivation of sodium conductance (g(Na)) has a slow and incomplete component.2. Initial outward current (most probably chloride current (I(Cl))) during strong depolarization (beyond -20 mV) could be inactivated either by increasing the frequency of depolarizations or by applying a conditioning depolarizing prepulse.3. In Na-free solution a small, slowly decreasing inward current was recorded. This is most probably carried by calcium ions and is at least partly responsible for the negative conductance of the current-voltage relation in the voltage range -60 to -30 mV.4. If a strong conditioning depolarization (V(1)) to the inside positive potential range preceded repolarizing clamp steps to different membrane potentials (V(2)) inward current (probably calcium current (I(Ca)) could be activated in Na-free and Na-containing solutions. This current depended on the duration of V(1) as well as on the potential level of V(2). After the maximum inward current, increasing outward current occurred when the duration of V(1) was increased. This current could be explained either by an inactivation of calcium conductance (g(Ca)) with time or by a very slow activation of an additional outward current.5. The main conclusions from these experiments are that (i) slow and incomplete inactivation of g(Na) are important features for the plateau of the action potential and (ii) calcium ions carry additional charge during this phase of the action potential. Chloride conductance (g(Cl)) should be largely inactivated at a heart rate of 90/min.     

Contractile repriming in snake twitch muscle fibres

1. Contractile repriming has been studied in voltage-clamped snake twitch muscle fibres. Maintained depolarization causes a contractile response which inactivates after a few seconds. Repolarization of the fibre can restore its ability to contract to a subsequent depolarization. This restoration, or repriming, depends on the magnitude and the duration of the repolarization. At -100 mV the minimal period of repolarization which restores contractile response is 0.38 sec. The time for recovery to half maximal tension is about 0.68 sec, and restoration is complete at about 4 sec.2. Repolarization to smaller levels of membrane potential results in a slower rate of repriming. For example, at -60 mV the mean minimal time for repriming was 2.89 sec, and nearly 17 sec of repolarization was required for full restoration of contractile response.3. The rate of repriming was not influenced by lowering the external sodium concentration.4. Repriming could be produced by repetitive, brief pulses of repolarization.5. The restoration of contractile response and of outward inactivating current showed similar time courses.     

Currents under voltage clamp of burst-forming neurons of the cardiac ganglion of the lobster (Homarus americanus)

Crustacean cardiac ganglion neuronal somata, although incapable of generating action potentials, produce regenerative, slow (greater than 200 ms) depolarizing potentials reaching -20 mV (from -50 mV) in response to depolarizing stimuli. These potentials initiate a burst of action potentials in the axon and are thus termed driver potentials. The somata of the anterior-most neurons (cells 1 or 2) were isolated by ligaturing for study of their membrane currents with a two-electrode voltage clamp. Inward current is attributed to Ca2+ by reason of dependence of driver potential amplitude on [Ca2+]0, independence of [Na+]0, resistance to tetrodotoxin, and inhibition by Cd (0.2 mM) and Mn (4 mM). Ca-mediated current (ICa) is present at -40 mV. It is optimally activated by a holding potential (Vh) of -50 to -60 mV and by clamps (command potential, Vc) to -10 mV. Time to peak (10-30 ms) and amplitude are strongly voltage dependent. Maximum tail-current amplitudes observed at -70 to -85 mV are ca. 100 nA. Inward tail peaks may not be resolved by our clamp (settling time, 2 ms). Tails relax with a time constant (tau) of approximately equal to 12 ms (at -70 to -85 mV). ICa exhibits inactivation in double pulse regimes. Recovery has a tau of approximately equal to 0.7 s. Tail current analyses indicate an exponential decline (tau approximately equal to 23 ms at -20 mV) toward a maintained amplitude of inward current tails. Analysis of outward currents indicates the presence of three conductance mechanisms having voltage dependences, time courses, and pharmacology similar to those of early outward current (IA), delayed outward current (IK), and outward current (IC) of molluscan neurons. Analysis of tail currents indicates a reversal potential for each of these near -75 mV, indicating that they are K currents. Early outward current, IA, shows a peak at 5 ms followed by rapid decline. Response to a second clamp given within 0.4 s is reduced; recovery is exponential, with a tau of approximately equal to 200 ms (at Vh = -50 mV). The amplitude of IA tested at 0 mV shows activation or deactivation by subthreshold shifts of Vh. The extent and rate of these changes shows voltage dependence (tau approximately equal to 100-500 ms for subthreshold prepulses). At the normal cell resting potential of -50 mV the amplitude of IA is 25% of that tested from -80 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regenerative repolarization of the frog ventricular action potential: a time and voltage-dependent phenomenon.

1. The regenerative repolarization process has been examined in frog ventricular myocardium using a single sucrose gap voltage clamp technique. 2. Application of brief (30-150 msec) anodal voltage clamp pulses during the plateau of the action potential revealed a 'threshold' potential region for immediate repolarization. The response to anodal clamp pulses was not all-or-none but was graded. 3. The threshold potential was strongly dependent on the duration of the test voltage clamp pulses and was more negative for shorter clamps. 4. Regenerative repolarization was also observed in the presence of tetrodotoxin. 5. No threshold for immediate repolarization was observed with very short clamps (2-20 msec in duration). Instead the membrane depolarized upon release of each clamp pulse. 6. Theoretical showed that the de- and repolarizations observed after test clamp steps are not due to geometrical properties or inhomogeneous potential distributions. 7. The results suggest that the instantaneous I-V relation of the membrane during the plateau may be linear.     

Mechanisms for hypothermia-induced increase in contractile force studied by mechanical restitution and post-rest contractions in guinea-pig papillary muscle

Lowering myocardial temperature increases contractile force, presumably by increasing intracellular calcium content. To study the mechanisms behind this, we compared the effects of some known inotropic interventions with hypothermia on mechanical restitution and post-rest contractile force in isolated guinea-pig papillary muscles. In four groups (n = 6 per group), the effects of: (1) reducing the ability for Na/Ca exchange to extrude Ca²⁺ (a) by increasing [Na⁺]₁ with ouabain or (b) by increasing [Ca²⁺]_o; and (2) activation of calcium channels with Bay-K 8644, were compared with lowering temperature from 37 to 27 °C. Normally (at 37 °C and 2 mm CaCl₂), mechanical restitution could be described by a rapid recovery phase with a time constant between 180 and 220 ms, followed by a slowly decaying phase with a time constant between 5000 and 8000 ms and post-rest contractions (1–10 min rest) were markedly depressed compared to steady-state contractions. Steady-state developed force was markedly increased at 27 °C, after 1 μm ouabain, 6 mm CaCl₂ or 0.1 μM Bay-K 8644. At 27 °C the rapid recovery phase of restitution was delayed while the slowly decaying phase was not affected. Ouabain and increased [Ca²⁺]_o caused elevation of the slowly decaying phase of restitution and markedly attenuated the post-rest depression of developed force, which may be attributed to a reduced diastolic extrusion of Ca²⁺ via the Na/Ca exchanger. Hypothermia and Bay-K 8644 on the other hand, augmented this post-rest depression. Hence, this study suggests that increased Ca²⁺ influx due to delayed inactivation of calcium channels may account for the increased developed force during hypothermia rather than reduced diastolic extrusion of Ca²⁺ via the Na/Ca exchanger.     

Quantification of calcium release from the sarcoplasmic reticulum in rainbow trout atrial myocytes

Using the whole-cell configuration of the patch-clamp technique, we quantified calcium release from the sarcoplasmic reticulum (SR) elicited by short depolarization pulses before and after clearance of the SR Ca2+ content with 10 mM caffeine (CAF). With a loaded SR, the first detectable contraction occurred with a pulse duration of 5 ms. CAF exposure increased this pulse duration to 85 ms and slowed the inactivation of the Ca2+ current (ICa). Repolarization of the cell to -80 mV after a short depolarization elicited a tail current that was attenuated markedly after CAF exposure. The difference between the charge carried by the tail currents obtained before and after CAF exposure was taken as a measure of the Ca2+ released from the SR. SR Ca2+ release increased with increasing SR Ca2+ load over the range of loads examined. In contrast, SR Ca2+ release reached a maximum when the duration of the preceding depolarization exceeded 10 ms. Maximal Ca2+ release was 1.64 amol/pF or 62 microM and elicited a contraction that was 40 +/- 6% of the amplitude of a normal contraction. This release could account for 48 +/- 10% of the total Ca2+ required to activate contraction but only a few percent of the CAF-releasable Ca2+. Thus, contrary to the general view of excitation-contraction coupling in lower vertebrates, SR Ca2+ release in trout atrial myocytes may account for up to 50% of the total Ca2+ transient at room temperature.     

An inward calcium current underlying regenerative calcium potentials in rat striatal neurons in vitro enhanced by Bay K 8644

The single electrode voltage clamp technique was used to characterize the currents underlying the calcium potentials in rat caudate neurons in vitro. In current clamp experiments, long depolarizing current pulses evoked repetitive firing of fast somatic action potentials. These were abolished by tetrodotoxin (1 microM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 microM) abolished the hyperpolarizing notch, enhanced the slow graded depolarizing response and induced the appearance of a slow all-or-nothing action potential. Both the slow graded response and the all-or-nothing action potential were abolished by cobalt (2 mM), suggesting the involvement of voltage-dependent calcium conductances. When the neurons were loaded intracellularly with caesium the action potential duration increased. Substitution of the extracellular calcium by barium (1-3 mM) or external addition of tetraethylammonium (5 mM) further prolonged spike duration and induced the appearance of long-lasting plateau potentials. These were insensitive to tetrodotoxin and were reversibly blocked by the calcium antagonists cobalt (2 mM), manganese (2 mM) or cadmium (500 microM). The calcium potentials were enhanced by the calcium 'agonist' BAY K 8644 (1-5 microM). In voltage clamp experiments when intracellular caesium was used to reduce outward currents and tetrodotoxin to block fast regenerative sodium currents, depolarizing voltage steps from a holding potential of -50, -40 mV activated an inward current. This current peaked in 50-80 ms and inactivated in two phases: an initial one at 150-200 ms followed by a second one after several hundred ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of activation of the tonic component of contraction in myocytes of guinea pig heart

AIM: Contractions of myocytes of guinea pig heart consist of a phasic component relaxing independently on the voltage and a tonic component relaxing upon repolarization. We found previously that Ca(2+) activating the tonic component is released from the sarcoplasmic reticulum. In this study, we analysed the mechanism of activation and maintenance of this release. METHODS: Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses of the duration of 300 ms to 15-45 s from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo-1 and contractions were recorded with the TV edge-tracking system. RESULTS: Myocytes responded to the short and long pulses with phasic contraction or Ca(2+) transient followed by the sustained contraction or increase in [Ca(2+)](i). Repolarization brought about relaxation. 10 mmol L(-1) Ni(2+) blocking Na(+)/Ca(2+) exchange superfused during the tonic component increased its amplitude. Superfusion of Ca(2+)-free solution during sustained contraction brought about relaxation both in normal cells and in cells superfused with Ni(2+) despite preserved sarcoplasmic reticulum Ca(2+) content assessed with caffeine spritz. Relaxing effect of Ca(2+)-free solution was not affected by carboxyeosin, a blocker of sarcolemmal Ca(2+)-ATPase. Tonic component of contraction and of Ca(2+) transient was inhibited by 200 micromol L(-1) ryanodine, a blocker of Ca(2+) release channels of the sarcoplasmic reticulum and by 20 micromol L(-1) nifedipine, a blocker of L-type I(Ca). CONCLUSION: Tonic component of contraction results from Ca(2+) release via the sarcoplasmic reticulum Ca(2+) channels activated by sustained, nifedipine-sensitive and Ni(2+)-insensitive Ca(2+) influx. Alternatively, the SR Ca(2+) release is activated by voltage, the dihydropyridine receptors acting like voltage sensors.     

Mechanical restitution of the rat papillary muscle

The experiments were performed on isometrically contracting rat papillary muscles paced at 1.0 Hz and at a temperature of 37°C. The contractile response of a test contraction continuously rose when the duration of the preceding stimulus interval was gradually increased from 0.2 s. A maximum value was seen at intervals of 60–120 s. This phenomenon was called mechanical restitution of the papillary muscle. The insertion of a priming beat before the test contraction in order to increase the amount of contractile calcium elevated the mechanical restitution curve but it did not change the maximum contractile force seen after 60–120 s intervals. Lowering the extracellular calcium concentration from 2.0 mM to 1.0 mM, however, depressed this maximum contractile force to about 50%. The mechanical restitution is thought to reflect inflow of activator calcium to a cellular store, from which it is later released in response to the action potential. By using two test contractions a simple method is described to estimate the recirculating fraction of activator calcium between beats in this preparation. In 11 preparations the recirculating fraction of activator calcium was 0.72±0.03 (mean ± SE). The results are consistent with the view that the same model of metabolism of activator calcium as previously proposed for rabbit papillary muscles is also applicable to the rat heart.     

Charybdotoxin and apamin sensitivity of the calcium-dependent repolarization and the afterhyperpolarization in neostriatal neurons.

1. Intracellular recordings from neostriatal neurons in an in vitro slice preparation of the rat brain were used to analyze the pharmacological sensitivity of the action potential (AP) repolarization and the afterhyperpolarization (AHP) that follows a single action potential. The interspike voltage trajectory and the AHP could be divided into two main parts: a fast component lasting a few milliseconds and better observed during a train of spikes, and a slow component lasting approximately 250 ms and that comprises the main portion of the AHP. In some cells, a slow (up to 1 s) component of low amplitude was also detected. 2. Single APs were elicited at two imposed membrane potentials (around -60 and around -80 mV). The AP amplitude was larger, the repolarization rate was faster, and the duration was shorter when spikes were evoked at -80 mV. When measured from the -60 mV holding potential, the afterpotential was an AHP with peak amplitude of -5 mV. The afterpotential became a delayed depolarization (DD) at -80 mV. 3. Firing frequency adaptation was voltage sensitive. The firing of APs induced by long intracellular current pulses from a holding potential of -80 mV exhibited only a slow-frequency adaptation (time constant of seconds). However, at -60 mV, an initial and faster frequency adaptation was evident (time constant of tens of milliseconds). 4. The Ca2+ channel blocker Cd2+ retarded AP repolarization rate. This effect correlated with a significant block of the fast and slow components of the AHP. In contrast, Ni2+ had no significant effects on the same parameters.(ABSTRACT TRUNCATED AT 250 WORDS)