Intramembrane charge movement and L-type calcium current in skeletal muscle fibers isolated from control and mdx mice.

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 +/- 2 nC/ micro F, -37 +/- 3 mV, and 13 +/- 1 mV (n = 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations (tau approximately 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms-long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 +/- 15 S/F, 65.5 +/- 2.9 mV, -0.76 +/- 1.2 mV, and 6.2 +/- 0.5 mV (n = 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (-6.2 +/- 1.2 mV, n = 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5-2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.

Effects of dantrolene on steps of excitation-contraction coupling in mammalian skeletal muscle fibers.

The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 microM and a 53 +/- 8% (mean +/- SEM, n = 9, cut fibers) attenuation at 0 mV with 25 microM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 +/- 8%) and of the early peak component (46 +/- 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (I(Ca)) were left, essentially, unaltered. However, the inactivation of I(Ca) was slowed fourfold, and the conductance was reduced from 200 +/- 16 to 143 +/- 8 SF(-1) (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 +/- 10% (n = 3) at 12 microM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30-50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.

Intracellular Mg2+ diffusion within isolated rat skeletal muscle fibers.

Intracellular free magnesium concentration ([Mg2+]i) was measured in enzymatically isolated rat skeletal muscle fibers using the fluorescent dye mag-indo-1. The change in [Mg2+]i produced by a local intracellular microinjection of magnesium pidolate (magnesium pyrrolidone carboxylate) was measured at a given distance from the injection site. In one series of experiments this protocol was tested on isolated fibers that were completely embedded into silicone grease: under these conditions, the injection produced an increase in [Mg2+]i that reached a steady level some time following the injection. The time-course of the [Mg2+]i change could be well accounted for by a model of longitudinal diffusion. The mean apparent Mg2+ diffusion coefficient (D(app)) was 188+/-9 microm2 s(-1) (n = 16), approximately four times lower than the value measured in vitro. This reduction likely results from the effects of cytoplasmic viscosity and of Mg2+ binding to low affinity static sites. Another series of measurements was performed on fibers that were either partially or completely free of silicone: under these conditions, the time course of the change in [Mg2+]i was in many cases more complex than predicted by simple diffusion.

Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels.

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca(2+) is increased in dystrophic muscle, leading to Ca(2+) accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca(2+) in muscle from mdx mice, an animal model of the human disease. In the present study, using plasma membrane Ca(2+)-activated K(+) channels as subsarcolemmal Ca(2+) probe, we tested the possibility of a Ca(2+) accumulation at the restricted subsarcolemmal level in mdx skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca(2+)-activated K(+) channels is significantly lower in mdx than in control muscle, suggesting a higher subsarcolemmal [Ca(2+)]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca(2+)-activated K(+) channel activation could correspond to a approximately 3-fold increase in the subsarcolemmal Ca(2+) concentration in mdx muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in mdx skeletal muscle, leading to Ca(2+) overload at the subsarcolemmal level.

Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice.

1. Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2. The mean (+/- s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 +/- 5 nM (n = 72) and 48 +/- 7 nM (n = 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than approximately 35 weeks, resting [Ca2+]i was significantly lower in mdx (16 +/- 4 nM, n = 11) than in control fibres (71 +/- 10 nM, n = 14). 3. Changes in [Ca2+]i elicited by short (5-35 ms) depolarizing pulses from -80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5-2. For longer pulses, no significant difference could be detected. 4. In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5. Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.

Measurements of intracellular Mg2+ concentration in mouse skeletal muscle fibers with the fluorescent indicator mag-indo-1.

Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.

Activation of Ca2+-activated K+ channels by an increase in intracellular Ca2+ induced by depolarization of mouse skeletal muscle fibres.

1. Ionic currents were simultaneously recorded at macroscopic and unitary level using the whole-cell and cell-attached patch-clamp procedures together on the same portion of isolated mouse skeletal muscle fibres. 2. In the presence of Tyrode solution in the patch pipette and Tyrode-TTX solution in the bath, macroscopic and unitary currents through delayed rectifier K+ channels were simultaneously recorded in response to depolarizing pulses of 1 s duration. 3. In five fibres, successive long-lasting incremental depolarizing levels induced, at -40 mV or -30 mV, the opening of a high conductance channel carrying an outward current superimposed on delayed rectifier K+ channel activity. Opening of this high conductance channel was not observed when the depolarization steps were applied in the patch pipette. 4. Using the same depolarizing protocol, activation of a high conductance channel was also observed in two fibres in the presence of a K+-rich solution in the pipette (145 mM K+) . 5. With either Tyrode or K+-rich solution in the pipette, unitary current amplitudes of the high conductance channel matched well with the values obtained for Ca2+-activated K+ (KCa) channels in inside-out patches under similar ionic conditions. 6. Indo-1 fluorescence measurements showed that the stimulation protocol that led to KCa channel opening induced stepwise increases in intracellular [Ca2+] in the submicromolar range. 7. Our results provide evidence that activation of sarcolemmal KCa channels can be induced by a rise in intracellular [Ca2+] following voltage-activated sarcoplasmic reticulum Ca2+ release.

Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers.

Indo-1 fluorescence signals were measured from one extremity of enzymatically isolated skeletal muscle fibers of mice. An original and simple method was developed to allow the measurements to be made under voltage-clamp control: the major part of a single fiber was embedded in silicone grease, so that only a short portion of one end of the fiber, from which the fluorescence measurements were taken, was in contact with the external solution. Membrane potential was held and varied by using a patch-clamp amplifier in whole-cell configuration with a single microelectrode, the tip of which was inserted across the silicone grease within the insulated portion of the fiber. In response to 100-ms depolarizing command pulses to voltages more positive than -40 mV (from a holding potential of -80 mV), clear changes in fluorescence were qualitatively observed to feature a time course of rise and decay expected from a change in intracellular calcium concentration ([Ca2+]i) due to voltage-dependent sarcoplasmic reticulum (SR) calcium release. Although the peak [Ca2+]i elicited by a 100-ms depolarization at 0 or +10 mV varied from fiber to fiber, it could clearly reach a value high enough to saturate Indo-1. The overall results show that this method represents an efficient way of measuring depolarization-induced [Ca2+]i changes in enzymatically dissociated skeletal muscle fibers.

Intramembrane charge movement and sarcoplasmic calcium release in enzymatically isolated mammalian skeletal muscle fibres.

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea-pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions. 2. The voltage dependence of intramembrane charge movement followed a two-state Boltzmann distribution with maximal available charge of 26.1 +/- 1.5 and 26.1 +/- 1.3 nC microF-1, mid-point voltage of -35.1 +/- 5.0 and -42.2 +/- 1.2 mV and steepness of 16.7 +/- 2.2 and 17.0 +/- 1.9 mV (means +/- S.E.M., n = 7 and 4) in rats and guinea-pigs, respectively. 3. Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium-sensitive dyes antipyrylazo III, fura-2 and mag-fura-5. Resting [Ca2+]i was similar in rats and guinea-pigs with 125 +/- 18 and 115 +/- 8 nM (n = 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 +/- 1.0 microM; n = 7), than in guinea-pigs (2.8 +/- 0.3; n = 4). 4. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 +/- 0.1 and 0.9 +/- 0.1 mM in rats and guinea-pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 +/- 0.08 and 0.81 +/- 0.06% ms-1 in rats and 2.43 +/- 0.25 and 0.88 +/- 0.01% ms-1 in guinea-pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians. 5. These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.

Intracellular Ca2+ changes and Ca2+-activated K+ channel activation induced by acetylcholine at the endplate of mouse skeletal muscle fibres.

1. Enzymatically isolated skeletal muscle fibres were used to investigate the effects of applying acetylcholine (ACh) onto the endplate area on intracellular free calcium concentration ([Ca2+]i) measured using the indicator indo-1 and single channel activity using the patch clamp technique. 2. Using a Tyrode solution containing 5 microM tetrodotoxin (TTX) as extracellular solution, ACh applications (at 0.1 or 1 mM) onto the endplate induced intracellular free calcium transients the mean maximal amplitude of which was 360 +/- 30 nM from a mean resting value of 72 +/- 7 nM (n = 13). In cells bathed with a K(+)-rich solution (145 mM K+), applications of ACh (0.1 mM) induced transient rises in [Ca2+]i from a mean resting value of 53 +/- 7 nM to a maximum of 222 +/- 24 nM (n = 33). 3. In cell-attached membrane patches at the endplate membrane of muscle fibres bathed in a K(+)-rich external solution, using a pipette filled with Tyrode solution, external application of 0.1 mM ACh could induce a transient burst opening of channels carrying an outward current of an average amplitude of 4.6 +/- 0.2 pA at 0 mV (n = 8). 4. These channels were characterized as Ca2(+)-activated K+ channels. At 0 mV, in inside-out patches excised from the endplate membrane area, they displayed a conductance of 60 and 224 pS in the presence of Tyrode and K(+)-rich solution in the pipette, respectively. Half-maximum activation was found for a [Ca2+]i close to 4 microM. The channels showed a typical voltage dependence. In outside-out patches these channels were shown to be blocked by 100 nM charybdotoxin (CTX). 5. In fibres bathed in a Tyrode solution containing TTX (5 microM), CTX had no clear effect on the change in membrane voltage, recorded near the endplate with a single intracellular microelectrode, in response to the application of ACh. 6. Although the physiological relevance of this ACh-induced K+ channel activation remains unclear, results suggest that, in the presence of a physiological extracellular [Ca2+], Ca2+ entry through the endplate nicotinic receptors can produce a local increase in [Ca2+]i, sufficient to trigger the opening of Ca2+-activated K+ channels in the adjacent surface membrane.

Picrotoxin potentiates contraction while inhibiting Ca current but increasing birefringence signal in frog skeletal muscle fibers.

In single frog skeletal muscle fibers, picrotoxin (5 mM) potentiated the voltage-dependent component of contractions in response to 2-s depolarizing pulses while greatly inhibiting the simultaneously recorded tubular Ca current in a normal-Ca, Na- and CI-deficient solution, provided the contractions were generated at long time intervals (2 min). In normal Ringer's solution, picrotoxin reversibly increased the amplitude of the early large birefringence signal and the amplitude and duration of the simultaneously recorded twitch tension, suggesting that the drug may increase, directly or indirectly, the release of Ca from the SR.

Measurements of sarcomere dynamics simultaneously with auxotonic force in isolated cardiac cells.

We developed an easy to use and non-invasive method to study sarcomere motion of enzymatically isolated myocytes which can be simultaneously combined with auxotonic force detection, thus being very useful when studying the contractile performance of cardiac cells. This method basically consists in analyzing the periodicity of the cell striation pattern using the Cooley-Tukey fast Fourier transform (FFT) algorithm on a video image of the cell during the course of the experiment. A longitudinal fraction of the cell image is recorded with a CCD TV camera, digitized, then transiently stored on a computer and used to calculate the spectrum corresponding to the distribution of the sarcomere lengths (SL). The method gives a real-time measurement of the most probable value of sarcomere length in one isolated cell with a temporal resolution of 20 ms. When used on a cell attached between two carbon fibers, the auxotonic force developed by the cell upon electrical stimulation can be simultaneously measured together with the SL in various conditions of stretch. Preliminary results have been presented in abstract form (Gannier et al., vol 24, pp. S47, 1992).

Inward barium current and excitation-contraction coupling in frog twitch muscle fibres.

The role of barium ions in excitation-contraction coupling was studied in single isolated frog semitendinosus fibres. Simultaneous recordings of membrane currents and contraction under voltage-clamp conditions in a sucrose-vaseline gap device show that barium ions have a reversible inhibiting effect on contraction. This inhibiting action was correlated to the entry of barium ions via the DHP-sensitive tubular calcium channel. Cytological observations and X-ray microanalysis performed on the fibres used in the electrophysiological experiments indicate that barium ions do not accumulate in the junctional sarcoplasmic reticulum; they can freely diffuse in the intermyofibrillar space and they accumulate in mitochondria. Calcium release experiments performed on isolated sarcoplasmic reticulum vesicles show that barium ions are not able to induce calcium release from calcium-loaded vesicles, they behave as calcium release inhibitors. These results are discussed in relation with the possible role of the slow Ca current in excitation-contraction coupling.

Microinjection of strong calcium buffers suppresses the peak of calcium release during depolarization in frog skeletal muscle fibers.

The effects of high intracellular concentrations of various calcium buffers on the myoplasmic calcium transient and on the rate of release of calcium (Rrel) from the sarcoplasmic reticulum (SR) were studied in voltage-clamped frog skeletal muscle fibers. The changes in intracellular calcium concentration (delta[Ca2+]) for 200-ms pulses to 0-20 mV were recorded before and after the injection of the calcium buffer and the underlying Rrel was calculated. If the buffer concentration after the injection was high, the initial rate of rise of the calcium transient was slower after injection than before and was followed by a slow increase of [Ca2+] that resembled a ramp. The increase in myoplasmic [Mg2+] that accompanies the calcium transient in control was suppressed after the injection and a slight decrease was observed instead. After the injection the buffer concentration in the voltage-clamped segment of the fiber decreased as the buffer diffused away toward the open ends. The calculated apparent diffusion coefficient for fura-2 (Dapp = 0.40 +/- 0.03 x 10(-6) cm2/s, mean +/- SEM, n = 6) suggests that approximately 65-70% of the indicator was bound to relatively immobile intracellular constituents. As the concentration of the injected buffer decreased, the above effects were reversed. The changes in delta[Ca2+] were underlined by characteristic modification of Rrel. The early peak component was suppressed or completely eliminated; thus, Rrel rose monotonically to a maintained steady level if corrected for depletion. If Rrel was expressed as percentage of SR calcium content, the steady level after injection did not differ significantly from that before. Control injections of anisidine, to the concentration that eliminated the peak of Rrel when high affinity buffers were used, had only a minor effect on Rrel, the peak was suppressed by 26 +/- 5% (mean +/- SE, n = 6), and the steady level remained unchanged. Thus, the peak component of Rrel is dependent on a rise in myoplasmic [Ca2+], consistent with calcium-induced calcium release, whereas the steady component of Rrel is independent of myoplasmic [Ca2+].

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.

Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.

The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.

Action of heptaminol hydrochloride on contractile properties in frog isolated twitch muscle fibre.

1. Heptaminol stopped or delayed the progressive decline in tension which characterizes the phenomenon of fatigue in frog isolated twitch muscle fibre. 2. Heptaminol had no action on the sodium, potassium and calcium voltage-dependent ionic conductances. 3. The hypothesis of an action via an internal alkalinization was tested by comparison with the action of NH4Cl. Both substances increased the tension. 4. The action of heptaminol was suppressed in sodium-free (TRIS) solution or in the presence of amiloride while the action of NH4Cl was always observed. 5. These results could be explained by a stimulation of the Na/H antiport by heptaminol.

Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibers.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.

Nifedipine and BAY K inhibit contraction independently from their action on calcium channels.

Voltage-clamp experiments were performed on twitch skeletal muscle fibres in a double sucrose-gap device in order to investigate the effect of 1,4-dihydropyridines (DHP) on excitation-contraction coupling during 50 ms step depolarizations. External solution used was free of permeant anions and contained only Ca++ as permeant cation. It is shown that in these conditions Nifedipine (a Ca++ channel antagonist) and BAY K 8644 (a Ca++ channel agonist) inhibit contraction in a way independent of their action on the gating of tubular calcium channels. These results indicate also that a close relation between DHP receptors and calcium channels must be taken with caution.

Excitation contraction coupling in skeletal muscle: evidence for a role of slow Ca2+ channels using Ca2+ channel activators and inhibitors in the dihydropyridine series.

Ca2+ current and tension have been simultaneously recorded from single twitch fibres of the semi-tendinosus of Rana esculenta in a medium containing a physiological Ca2+ concentration (1.8 mM). Under appropriate conditions it can be shown that tension develops in two phases. The first is rapid and reaches its maximum before activation of the inward Ca2+ current. The second phase is slower and with a time course which appears to be correlated with that of the inward current. Nifedipine, a specific Ca2+ channel inhibitor greatly reduced ICa2+ and the slower component of tension. Bay K8644 a Ca2+ channel activator, which has receptors on T-tubule, increased ICa2+ and the slow component of tension. These results indicate that a slow component of skeletal muscle contraction is related to the inward Ca2+ current flowing through dihydropyridine sensitive voltage-dependent Ca2+ channels.