Effects of fluvastatin and coenzyme Q10 on skeletal muscle in normo- and hypercholesterolaemic rats.

Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium homeostasis and reduced coenzyme Q10 levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q10 was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q10 supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q10 supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q10 to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q10 in statin treated patients symptomatic of this condition.

Alteration of sarcoplasmic reticulum Ca2+ ATPase expression in lower limb ischemia caused by atherosclerosis obliterans.

Atherosclerosis is a disease caused by a build-up of fatty plaques and cholesterol in the arteries. The lumen of the vessels is obliterated resulting in restricted blood supply to tissues. In ischemic conditions, the cytosolic Ca2+ level of skeletal muscle may increase, indicating the alteration of Ca2+ removal mechanisms. Ca2+ is transported from cytosol into the sarcoplasmic reticulum by Ca2+ ATPase (SERCA), with its 1a isoform expressed in adult, while its 1b isoform in neonatal and regenerating fast-twitch skeletal muscle. To investigate the role of these isoforms in ischemic skeletal muscle, biopsies from musculus biceps femoris of patients who underwent amputation due to atherosclerosis were examined. Samples were removed from the visibly healthy and hypoxia-affected tissue. Significantly increased SERCA1a expression was detected under the ischemic conditions (246 ± 69%; p < 0.05) compared with the healthy tissue. Furthermore, the ratio of SERCA1a-positive fibers was slightly increased (46 ± 4% in healthy tissue and 60 ± 5% in ischemic tissue; p > 0.05), whereas SERCA2a did not change. In addition, in primary cultures derived from hypoxia-affected tissue, the diameter and fusion index of myotubes were significantly increased (30 ± 1.6 µm vs. 41 ± 2.4 µm and 31 ± 4% vs. 45 ± 3%; p < 0.05). We propose that the increased SERCA1a expression indicates the existence and location of compensating mechanisms in ischemic muscle.

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+].

Contraction threshold and the "hump" component of charge movement in frog skeletal muscle.

The delayed component of intramembranous charge movement (hump, I gamma) was studied around the contraction threshold in cut skeletal muscle fibers of the frog (Rana esculenta) in a single Vaseline-gap voltage clamp. Charges (Q) were computed as 50-ms integrals of the ON (QON) and OFF (QOFF) of the asymmetric currents after subtracting a baseline. The hump appeared in parallel with an excess of QON over QOFF by approximately 2.5 nC/mu F. Caffeine (0.75 mM) not only shifted the contraction threshold but moved both the hump and the difference between the ON and OFF charges to more negative membrane potentials. When using 10-mV voltage steps on top of different prepulse levels, the delayed component, if present, was more readily observable. The voltage dependences of the ON and OFF charges measured with these pulses were clearly different: QON had a maximum at or slightly above the contraction threshold, while QOFF increased monotonically in the voltage range examined. Caffeine (0.75 mM) shifted this voltage dependence of QON toward more negative membrane potentials, while that of QOFF was hardly influenced. These results show that the delayed component of intramembranous charge movement either is much slower during the OFF than during the ON, or returns to the OFF position during the pulse. Tetracaine (25 microM) had similar effects on the charge movement currents, shifting the voltage dependence on the ON charge in parallel with the contraction threshold, but to more positive membrane potentials, and leaving QOFF essentially unchanged. The direct difference between the charge movement measured in the presence of caffeine and in control solution was either biphasic or resembled the component isolated by tetracaine, suggesting a common site of caffeine and tetracaine action. The results can be understood if the released Ca plays a direct role in the generation of the hump, as proposed in the first paper of this series (Csernoch et al. 1991. J. Gen. Physiol. 97:845-884).

Kinetic properties of intramembrane charge movement under depolarized conditions in frog skeletal muscle fibers.

Intramembrane charge movement was measured on skeletal muscle fibers of the frog in a single Vaseline-gap voltage clamp. Charge movements determined both under polarized conditions (holding potential, VH = -100 mV; Qmax = 30.4 +/- 4.7 nC/micro(F), V = -44.4 mV, k = 14.1 mV; charge 1) and in depolarized states (VH = 0 mV; Qmax = 50.0 +/- 6.7 nC/micro(F), V = -109.1 mV, k = 26.6 mV; charge 2) had properties as reported earlier. Linear capacitance (LC) of the polarized fibers was increased by 8.8 +/- 4.0% compared with that of the depolarized fibers. Using control pulses measured under depolarized conditions to calculate charge 1, a minor change in the voltage dependence (to V = -44.6 mV and k = 14.5 mV) and a small increase in the maximal charge (to Qmax = 31.4 +/- 5.5 nC/micro(F] were observed. While in most cases charge 1 transients seemed to decay with a single exponential time course, charge 2 currents showed a characteristic biexponential behavior at membrane potentials between -90 and -180 mV. The voltage dependence of the rate constant of the slower component was fitted with a simple constant field diffusion model (alpha m = 28.7 s-1, V = -124.0 mV, and k = 15.6 mV). The midpoint voltage (V) was similar to that obtained from the Q-V fit of charge 2, while the steepness factor (k) resembled that of charge 1. This slow component could also be isolated using a stepped OFF protocol; that is, by hyperpolarizing the membrane to -190 mV for 200 ms and then coming back to 0 mV in two steps. The faster component was identified as an ionic current insensitive to 20 mM Co2+ but blocked by large hyperpolarizing pulses. These findings are consistent with the model implying that charge 1 and the slower component of charge 2 interconvert when the holding potential is changed. They also explain the difference previously found when comparing the steepness factors of the voltage dependence of charge 1 and charge 2.

The relationship between Q gamma and Ca release from the sarcoplasmic reticulum in skeletal muscle.

Asymmetric membrane currents and fluxes of Ca2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca2+ release and the "hump" component of charge movement current (I gamma), described in the first paper of this series, was applied at different test pulse voltages. The amplitude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages (-50 to -40 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 microM tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property of I gamma, defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump", (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and (f) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the amplitude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca2+ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local transmembrane potential, thus driving additional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic properties of I gamma described in the previous papers. It also accounts for the inward phase in total asymmetric current and in the current suppressed by protocol 1. According to this model, I gamma accompanies activating transitions at the same set of voltage sensors as I beta. Therefore it should open additional release channels, which in turn should cause more I gamma, providing a positive feedback mechanism in the regulation of calcium release.

Interfering with calcium release suppresses I gamma, the "hump" component of intramembranous charge movement in skeletal muscle.

Four manifestations of excitation-contraction (E-C) coupling were derived from measurements in cut skeletal muscle fibers of the frog, voltage clamped in a Vaseline-gap chamber: intramembranous charge movement currents, myoplasmic [Ca2+] transients, flux of calcium release from the sarcoplasmic reticulum (SR), and the intrinsic optical transparency change that accompanies calcium release. In attempts to suppress Ca release by direct effects on the SR, three interventions were applied: (a) a conditioning pulse that causes calcium release and inhibits release in subsequent pulses by Ca-dependent inactivation; (b) a series of brief, large pulses, separated by long intervals (greater than 700 ms), which deplete Ca2+ in the SR; and (c) intracellular application of the release channel blocker ruthenium red. All these reduced calcium release flux. None was expected to affect directly the voltage sensor of the T-tubule; however, all of them reduced or eliminated a component of charge movement current with the following characteristics: (a) delayed onset, peaking 10-20 ms into the pulse; (b) current reversal during the pulse, with an inward phase after the outward peak; and (c) OFF transient of smaller magnitude than the ON, of variable polarity, and sometimes biphasic. When the total charge movement current had a visible hump, the positive phase of the current eliminated by the interventions agreed with the hump in timing and size. The component of charge movement current blocked by the interventions was greater and had a greater inward phase in slack fibers with high [EGTA] inside than in stretched fibers with no EGTA. Its amplitude at -40 mV was on average 0.26 A/F (SEM 0.03) in slack fibers. The waveform of release flux determined from the Ca transients measured simultaneously with the membrane currents had, as described previously (Melzer, W., E. Ríos, and M. F. Schneider. 1984. Biophysical Journal. 45:637-641), an early peak followed by a descent to a steady level during the pulse. The time at which this peak occurred was highly correlated with the time to peak of the current suppressed, occurring on average 6.9 ms later (SEM 0.73 ms). The current suppressed by the above interventions in all cases had a time course similar to the time derivative of the release flux; specifically, the peak of the time derivative of release flux preceded the peak of the current suppressed by 0.7 ms (SEM 0.6 ms). The magnitude of the current blocked was highly correlated with the inhibitory effect of the interventions on Ca2+ release flux.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of tetracaine on charge movements and Ca2+ signals in frog skeletal muscle.

The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions.

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.

Biphasic effect of bimoclomol on calcium handling in mammalian ventricular myocardium.

1. Concentration-dependent effects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea-pig hearts loaded with the fluorescent calcium indicator dye Fura-2. Bimoclomol had a biphasic effect on contractility: both peak left ventricular pressure and the rate of force development significantly increased at a concentration of 10 nM or higher. The maximal effect was observed between 0.1 and 1 microM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentration-dependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10 - 100 nM) increased, whereas high concentrations (10 microM) decreased the amplitude of intracellular calcium transients. 2. Effects of bimoclomol on action potential configuration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaffected at intermediate (0.1 - 1 microM) and decreased at high (10 - 100 microM) concentrations of the drug. 3. In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into artificial lipid bilayer, bimoclomol significantly increased the open probability of the channel in the concentration range of 1 - 10 microM. The increased open probability was associated with increased mean open time. The effect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol. 4. Bimoclomol (10 microM - 1 mM) had no significant effect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be affected by the drug. 5. In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium.

Interacting effects of capsaicin and anandamide on intracellular calcium in sensory neurones.

Capsaicin (100 nM to 1 microM) and anandamide (200 nM to 10 microM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurones of rats measured with a ratiometric technique. The percentage of cells responding to capsaicin at 100 nM, 330 nM and 1 microM was 47.4%, 45.3%, and 70.4%, respectively. Averaged peak value of fluorescense ratio (R) at 340 and 380 nm excitation was slightly dose dependent. Peaks of anandamide-induced transients were R = 0.2 at 200 nM and 0.16 at 10 microM. Near 40% of capsaicin-sensitive cells responded also to anandamide. Anandamide (200 nM) inhibited the capsaicin-induced calcium influx. The results suggest that anandamide increases intracellular calcium and inhibits capsaicin-evoked calcium transients.

Distinct subpopulations in HaCaT cells as revealed by the characteristics of intracellular calcium release induced by phosphoinositide-coupled agonists.

Intracellular calcium release induced by transient applications of phosphoinositide agonists was measured using adherent single HaCaT keratinocytes loaded with the acetoxymethyl derivative of fura-2. Application of ATP, bradykinin and formyl-Met-Leu-Phe (fMLP) resulted in a transient increase in intracellular calcium concentration ([Ca2+]i) with an average half-width of 40+/-21 s and a decay time constant of 15+/-10 s (mean +/- SD, n = 108), irrespective of the agonist applied. The cells could be classified into two groups: in 53% of the cells repeated stimulation brought about a progressively smaller change in [Ca2+]i (type 1 cells), whereas in the remaining cells the amplitude of the calcium transients was essentially unchanged (type 2 cells). Furthermore, calcium transients in type 1 cells had broader half-widths and slower decays. No difference was found between the agonists in respect of the characteristics of the evoked calcium transient within each subpopulation. However, bradykinin and fMLP desensitized some cells. These results indicate that the activation of the inositol trisphospate transduction pathway by different agonists induces a characteristic elevation of [Ca2+]i within a given cell. Our results demonstrate that cultured HaCaT keratinocytes are heterogeneous in respect of the calcium transients evoked by the activators of this second messenger system.

Caffeine and the myoplasmic calcium removal mechanisms in cut frog skeletal muscle fibres.

Antipyrylazo III myoplasmic calcium transients were recorded in cut skeletal muscle fibres of the frog (Rana esculenta), using the double vaseline-gap voltage-clamp system. Intracellular calcium removal mechanisms were analysed, using a slightly modified model taken from the literature. Parameter values reported here are generally consistent with those obtained by the original model. Caffeine (0.5 mmol.l-1) moderately enhanced the overall myoplasmic calcium removal. In particular, the rate constant of the non-saturable uptake increased by 51% on the average, but there was a considerable fiber-to-fiber variation. The kinetic features of the binding sites representing the saturable uptake did not change significantly while the concentration of the available sites decreased by 36%. It is concluded that the caffeine-induced changes of the calcium removal components can be explained by supposing an increased resting myoplasmatic Ca2+ concentration in the presence of the drug.

The role of the different neoglottis forms in the development of esophageal voice.

UNLABELLED: Evaluation of voice quality parameters of esophageal speech in different neoglottis forms after total laryngectomy. METHODS: Presentation of voice analysis of 20 patients, who underwent total laryngectomy. The success of acquiring this technique was estimated by means of a voice analyzing program (pitch, sound-holding, loudness, spectrogram),and by the intelligibility via the telephone. Shape of the different types of neoglottis that developed and its functioning during vocalization and continuous speech were observed by nasal endoscopy. Data obtained from the voice analysis were compared among the observed three different neoglottis forms. RESULTS: The average dysphonia index of the 20 patients was 1.67 ± 0.38 (mean ± SD). Nasal fiberoscopic examination revealed three different types of neoglottis forms ­ a small mucosal button, two mucosal battens, and a mucosal lip. Voice quality of the esophageal speech of the patients with the mucosal button was found to be the closest to normal by subjective and objective acoustical evaluation. CONCLUSIONS: These findings emphasize the importance of the proper wound closure technique which can facilitate the development of a special button shape neoglottis form and help to acquire esophageal speech with the best quality parameters shortly after total laryngectomy.

Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture.

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

Regulation of the ryanodine receptor calcium release channel of the sarcoplasmic reticulum in skeletal muscle.

In striated muscle contraction is under the tight control of myoplasmic calcium concentration ([Ca2+]i): the elevation in [Ca2+]i and the consequent binding of calcium to troponin C enables, while the decrease in [Ca2+]i prevents the actin-myosin interaction. Calcium ions at rest are stored in the sarcoplasmic reticulum (SR) from which they are rapidly released upon the depolarisation of the sarcolemmal and transverse (T-) tubular membranes of the muscle cell. The protein responsible for this controlled and fast release of calcium is the calcium release channel found in the membrane of the terminal cisternae of the SR. This review focuses on the physiological and pharmacological modulators of the calcium release channel and tries to draw an up-to-date picture of the events that occur between T-tubular depolarisation and the release of calcium from the SR.

Calcium transients and calcium binding to troponin at the contraction threshold in skeletal muscle.

Antipyrylazo III calcium transients from voltage-clamped, cut skeletal muscle fibers of the frog were recorded, and the calcium binding to the regulatory sites of troponin C was calculated. The strength-duration curve for the contraction threshold was determined. It was found that the increase in myoplasmic calcium concentration necessary to produce the same level of contractile activation, i.e., the just visible movement, was approximately 60% higher at more positive membrane potentials resulting from short depolarizing pulses than at rheobase. However, using biochemical data for the kON and kOFF rate coefficients of the binding sites, the calculated maximums of the calcium binding curves were about the same at different voltages, and the time to maximum saturation was roughly equal to the latency of the contractions. To characterize the calcium binding in intact fibers more accurately, those values of the kON and kOFF rate coefficients that gave equal peak saturations during threshold movement at different membrane potentials were determined.

Perchlorate and the relationship between charge movement and contractile activation in frog skeletal muscle fibres.

1. The effects of perchlorate ions (1-8 mM) on intramembrane charge movement, myoplasmic Antipyrylazo III Ca2+ transients and contractile activation were examined in voltage-clamped cut skeletal muscle fibres of the frog. 2. Perchlorate shifted both the voltage dependence of charge movement and the rheobase of the strength-duration relation for contraction threshold towards more negative membrane potentials. 3. Both charge movements and myoplasmic Ca2+ transients were much slower at the new rheobase in the presence of perchlorate than in the control solution but there was no change in the threshold amount of charge or in the calculated peak binding of Ca2+ to troponin C. 4. The peak release rate had a steeper voltage dependence than the non-linear charge, but a lower concentration (2 mM) of perchlorate shifted both voltage dependences equally without altering the maxima in the amount of charge and in the rate of Ca2+ release. 5. The voltage dependence of the difference between total charge and charge at the threshold of Ca2+ transients agreed well with the voltage dependence of the rate of Ca2+ release in both the presence and absence of perchlorate. 6. It is concluded that the effect of perchlorate on contractile activation can be accounted for by its action on the intramembrane charge movement responsible for contraction, without significant effects on subsequent Ca2+ release from the sarcoplasmic reticulum or on Ca2+ binding to regulatory sites of troponin C.

Deterministic inactivation of calcium release channels in mammalian skeletal muscle.

Enzymatically dissociated fibres from the extensor digitorum communis muscle of rats were mounted into a double Vaseline gap chamber. The rate of calcium release (R(rel)) from the sarcoplasmic reticulum (SR) and changes in SR permeability to Ca2+ (PSR) were calculated from measured changes in intracellular calcium concentration. Calcium release during a prepulse attenuated the inactivating component of PSR of the subsequent test pulse. The suppression was graded, larger release causing greater suppression, as expected from a calcium-dependent inactivation process. However, if the dissociation constant of the putative inhibitory calcium binding site (Kd) was estimated using different test pulses different affinities were obtained: a smaller test pulse yielded a smaller Kd. Comparing the suppression of the inactivatable component of PSR during the test pulse (suppression) with the inactivatable component during the prepulse (pre-inactivation) revealed a linear relationship with a regression coefficient close to unity. Lowering intracellular magnesium by decreasing its concentration to 25 microM in the internal solution altered the time course of PSR. The maximal peak-to-steady-level ratio was increased to 6.3 +/- 0.4 (n = 10, mean +/- s.e.m.) from a control value of 3.0 +/- 0.2 (n = 19). Despite the apparent change in steady-state inactivation, suppression remained equal to that pre-inactivation. Our results support the view that a depolarizing pulse always recruits the same set of calcium release channels and a portion of these channels undergoes a deterministic inactivation process.