Effects of high myoplasmic L-lactate concentration on E-C coupling in mammalian skeletal muscle.

The effects of high myoplasmic L-lactate concentrations (20-40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca(2+) release, and passive Ca(2+) leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. L-Lactate (20 mM) significantly reduced maximum Ca(2+)-activated force by 4 +/- 0.5% (n = 5, P < 0.05) and 5 +/- 0.4% (n = 6, P < 0.05) for EDL and soleus, respectively. The Ca(2+) sensitivity was also significantly decreased by 0.06 +/- 0. 002 (n = 5, P < 0.05) and 0.13 +/- 0.01 (n = 6, P < 0.001) pCa units, respectively. Exposure to L-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 +/- 3% (n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 microM), or the chloride channel blocker anthracene-9-carboxylic acid (100 microM). L-Lactate (20 mM) increased passive Ca(2+) leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 +/- 5%, n = 9, P < 0.05) with no apparent effect in soleus fibers (100 +/- 2%, n = 3). These results indicate that the L-lactate ion per se has negligible effects on either voltage-dependent Ca(2+) release or SR Ca(2+) handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.

Effects of terbutaline on force and intracellular calcium in slow-twitch skeletal muscle fibres of the rat.

1. The effect of the alpha2-adrenoceptor agonist, terbutaline, was investigated on simultaneously measured force and intracellular free calcium ([Ca2+]i) in intact rat soleus muscle fibres, and on contractile protein function and Ca2+ content of the sarcoplasmic reticulum (SR) in skinned fibres. 2. Terbutaline (10 microM) had no significant effect on either resting force or [Ca2+]i. Exposure to terbutaline increased both the integral of the indo-1 ratio transient and peak twitch force by 37%. 3. At sub-maximal (10 Hz) stimulation frequencies, terbutaline accelerated force relaxation but had highly variable effects on tetanic force amplitude. The corresponding indo-1 ratio transients were significantly larger, and faster to decay than the controls. 4. Terbutaline increased tetanic force at near maximal stimulation frequencies (50 Hz) by increasing tetanic [Ca2+]i. Force relaxation was accelerated at this frequency with no significant change in the indo-1 ratio transient decay rate. 5. All of terbutaline's effects on force and indo-1 ratio transients in intact fibres were completely blocked and reversed by ICI 118551 (1 microM). 6. Mechanically skinned fibres isolated from intact muscles pre-treated with terbutaline showed no significant changes in SR Ca2+ content, myofilament [Ca2+]i-sensitivity or maximum force generating capacity. 7. The results suggest that terbutaline primarily modulates force by altering the amplitude and decay rate of the [Ca2+]i transient via phosphorylation of both the ryanodine receptor (RR) and the SR pump regulatory protein, phospholamban (PLB). The high variability of responses of slow-twitch muscles to beta2-agonists probably reflects individual differences in basal phosphorylation levels of PLB relative to that of RR.

Mechanisms underlying phosphate-induced failure of Ca2+ release in single skinned skeletal muscle fibres of the rat.

1. Single mechanically skinned fibres from rat extensor digitorum longus (EDL) muscles were used to investigate the mechanisms underlying inorganic phosphate (Pi) movements between the myoplasm and the sarcoplasmic reticulum (SR). Force transients elicited by caffeine/low Mg2+ application were used to assess the rate of Pi-induced inhibition of SR Ca2+ release and the subsequent recovery of Ca2+ release following removal of myoplasmic Pi. 2. Myoplasmic Pi reduced SR Ca2+ release in a concentration- and time-dependent manner. A 10 s exposure to 10, 20 and 50 mM myoplasmic Pi reduced SR Ca2+ release by 12 +/- 9, 29 +/- 5 and 82 +/- 5 %, respectively. 3. Removal of myoplasmic ATP at the time of Pi exposure significantly increased the rate and extent of SR Ca2+ release inhibition. For example, Ca2+ release was reduced by 86 +/- 6 % (n = 6) after 20 s exposure to 20 mM Pi in the absence of ATP compared with only 47 +/- 5 % (n = 5) in the presence of ATP. 4. The half and full recovery times for SR Ca2+ release following washout of myoplasmic Pi were 35 s and approximately 7 min, respectively. Recovery of Ca2+ release was unaffected by the absence of ATP during washout of Pi but was prevented when fibres were washed in the presence of high myoplasmic Pi (30 mM). Neither the Pi transporter blocker phenylphosphonic acid (PHPA) nor the anion channel blockers anthracene-9-carboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) affected the rate of recovery of SR Ca2+ release. 5. These results show that Pi entry and exit from the SR occur primarily through a passive pathway that is insensitive to well-known anion channel blockers. Pi inhibition of SR Ca2+ release appears to be a complicated phenomenon influenced by the rate of Pi movement across the SR as well as by the rate, extent and species of Ca2+-Pi precipitate formation in the SR lumen. The more rapid inhibitory effect of Pi in the absence of myoplasmic ATP suggests that Pi may inhibit SR Ca2+ release more efficiently during the later stages of fatigue.

Effects of 4-methyl-2-aminopyridine on [3H]-noradrenaline overflow and contractility of isolated rabbit arteries.

1. The effects of 4-methyl-2-aminopyridine (4M2AP) and 4-aminopyridine (4AP) on spontaneous and evoked [3H]-noradrenaline overflow were compared in rabbit ear artery strips. The effects of 4M2AP on smooth muscle contractility were also investigated in isolated perfused ear arteries. 2. Both 4M2AP and 4AP enhanced spontaneous [3H] overflow from arterial strips in a concentration-dependent manner (10-1000 microM). A bell-shaped dose-response relation was obtained for evoked [3H] overflow over the same concentration range, with maximum effects occurring at 10 microM for 4M2AP (163 +/- 31% increase) and 100 microM for 4AP (154 +/- 16% increase). 3. 4M2AP did not significantly affect evoked tension in the 1-to 100-microM range but clearly depressed it at 1,000 microM (by 65 +/- 11%). In contrast, 4AP enhanced evoked tension in the 10- to 100-microM range (by 30-50%). 4. 4M2AP (10-100 microM) enhanced vasoconstrictor responses to exogenous noradrenaline injections in isolated perfused rabbit ear arteries, whereas higher concentrations (1,000 microM) caused significant depression. 5. 4M2AP (1,000 microM) markedly potentiated vasoconstrictor responses induced by perfusion with a high extracellular K+ solution. When 4M2AP was present during the reloading of noradrenaline-sensitive Ca2+ stores, it enhanced the subsequent vasoconstrictor responses to noradrenaline obtained in a Ca(2+)-free medium. 6. The results show that 4M2AP, like 4AP, enhances [3H] overflow from sympathetic nerve terminals and has complex effects on vascular smooth muscle contractility, indicating the ability of these compounds to affect the Ca2+ permeability of both extracellular and intracellular membrane systems.

Inhibitory effects of (+/-)-propranolol on excitation-contraction coupling in isolated soleus muscles of the rat.

1. The effect of a beta-adrenoceptor antagonist, propranolol, was investigated on excitation-contraction coupling in small, intact bundles of soleus muscle fibres from the rat. 2. (+/-)-Propranolol significantly inhibited twitch and tetanic tension with IC50 values of 6.7 microM and 3.5 microM, respectively. 3. (+)-Propranolol (which has 100 times less beta-blocking activity than the (+/-) form) was approximately one third as effective as the (+/-) form at inhibiting isometric tension. 4. (+/-)-Propranolol (20 microM) had no significant effect on the amplitude of caffeine contractures, suggesting that it did not directly inhibit Ca2+ release from the sarcoplasmic reticulum. 5. The resting membrane potential measured after 15 min perfusion with 20 microM (+/-)-propranolol was not significantly different from control. However, this concentration of (+/-)-propranolol significantly reduced both the peak amplitude and the maximum rate of rise of the action potential. Both effects were only partially reversible after extensive washing. 6. (+/-)-Propranolol perfusion caused a modest reduction in the amplitude of sub-maximal K+ contractures at concentrations (5 microM) that markedly depressed tetanic tension. 7. The results indicate that (+/-)-propranolol can decrease isometric tension independently of beta-receptor occupation by (i) reducing the amplitude and rate of rise of the action potential and (ii) by directly inhibiting excitation-contraction coupling. The relatively low IC50 for the 'membrane-stabilizing' action of propranolol on tetanic tension (3.5 microM), combined with the ability of the drug to accumulate gradually in biological membranes, may contribute to a peripheral component of the tremorolytic and fatigue-inducing actions of propranolol on skeletal muscle.

Phosphate transport into the sarcoplasmic reticulum of skinned fibres from rat skeletal muscle.

The rate, magnitude and pharmacology of inorganic phosphate (Pi) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and Pi transport into the sarcoplasmic reticulum lumen during Ca-Pi precipitation-induced Ca2+ loading. The apparent rate of Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [Pi] in the 10 mM-50 mM range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca-Pi precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mM myoplasmic [Pi] the rate of Pi entry was calculated to be at least 51 microM s-1 while the amount of Pi loaded appeared to saturate at around 3.5 mM (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca-Pi precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mM) inhibited Pi transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mM) appeared to enhance both the degree of Pi entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+. Thus, results from these studies indicate the presence of a Pi transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic Pi and Ca2+ and (2) partially inhibited by PhPA. This Pi transporter represents a link between changes in myoplasmic [Pi] and subsequent changes in sarcoplasmic reticulum luminal [Pi]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca-Pi solubility product is exceeded in the sarcoplasmic reticulum lumen.

Relationship between depolarization-induced force responses and Ca2+ content in skeletal muscle fibres of rat and toad.

1. The relationship between the total Ca2+ content of a muscle fibre and the magnitude of the force response to depolarization was examined in mechanically skinned fibres from the iliofibularis muscle of the toad and the extensor digitorum longus muscle of the rat. The response to depolarization in each skinned fibre was assessed either at the endogenous level of Ca2+ content or after depleting the fibre of Ca2+ to some degree. Ca2+ content was determined by a fibre lysing technique. 2. In both muscle types, the total Ca2+ content could be reduced from the endogenous level of approximately 1.3 mmol l-1 (expressed relative to intact fibre volume) to approximately 0.25 mmol l-1 by either depolarization or caffeine application in the presence of Ca2+ chelators, showing that the great majority of the Ca2+ was stored in the sarcoplasmic reticulum (SR). Chelation of Ca2+ in the transverse tubular (T-) system, either by exposure of fibres to EGTA before skinning or by permeabilizing the T-system with saponin after skinning, reduced the lower limit of Ca2+ content to < or = 0.12 mmol l-1, indicating that 10-20% of the total fibre Ca2+ resided in the T-system. 3. In toad fibres, both the peak and the area (i.e. time integral) of the force response to depolarization were reduced by any reduction in SR Ca2+ content, with both decreasing to zero in an approximately linear manner as the SR Ca2+ content was reduced to < 15% of the endogenous level. In rat fibres, the peak size of the force response was less affected by small decreases in SR content, but both the peak and area of the response decreased to zero with greater depletion. In partially depleted toad fibres, inhibition of SR Ca2+ uptake potentiated the force response to depolarization almost 2-fold. 4. The results show that in this skinned fibre preparation: (a) T-system depolarization and caffeine application can each virtually fully deplete the SR of Ca2+, irrespective of any putative inhibitory effect of SR depletion on channel activation; (b) all of the endogenous level of SR Ca2+ must be released in order to produce a maximal response to depolarization; and (c) a substantial part (approximately 40%) of the Ca2+ released by a depolarization is normally taken back into the SR before it can contribute to force production.

Total and sarcoplasmic reticulum calcium contents of skinned fibres from rat skeletal muscle.

1. The Ca2+ content of single mammalian skeletal muscle fibres was determined using a novel technique. Mechanically skinned fibres were equilibrated with varying amounts of the Ca2+ buffer BAPTA and were then lysed in a detergent-paraffin oil emulsion. The subsequent myofilament force response was used to estimate the additional amount of Ca2+ bound to BAPTA following lysis of intracellular membranes. 2. The total endogenous Ca2+ content (corrected for endogenous Ca2+ buffering) of fast-twitch (FT) and slow-twitch (ST) fibres at a myoplasmic pCa (-log[Ca2+]) of 7.15 was 1.32 +/- 0.02 and 1.35 +/- 0.08 mM per fibre volume, respectively. The sarcoplasmic reticulum (SR) component of these estimates was calculated as 1.01 and 1.14 mM, respectively, which normalized to SR volume corresponds to resting SR Ca2+ contents of 11 and 21 mM, respectively. 3. Equilibration of 'resting' fibres with low myoplasmic [Ca2+] (pCa 7.67-9.00) elicited a time-dependent decrease in Ca2+ content in both fibre types. Equilibration of resting fibres with higher myoplasmic [Ca2+] (pCa 5.96-6.32) had no effect on the Ca2+ content of ST fibres but increased the Ca2+ content of FT fibres. The maximum steady-state total Ca2+ content (3.85 mM) was achieved in FT fibres after 3 min equilibration at pCa 5.96. Equilibration at higher myoplasmic [Ca2+] was less effective, probably due to Ca2(+)-induced Ca2+ release from the SR. 4. Exposure of fibres to either caffeine (30 mM, pCa approximately 8, 2 min) or low myoplasmic [Mg2+] (0.05 mM, pCa approximately 9, 1 min) released approximately 85% of the resting SR Ca2+ content. The ability of caffeine to release SR Ca2+ was dependent on the myoplasmic Ca2+ buffering conditions. 5. The results demonstrate that the SR of ST fibres is saturated with Ca2+ at resting myoplasmic [Ca2+] while the SR of FT fibres is only about one-third saturated with Ca2+ under equivalent conditions. These differences suggest that the rate of SR Ca2+ uptake in FT fibres is predominantly controlled by myoplasmic [Ca2+] while that of ST fibres is more likely to be limited by the [Ca2+] within the SR lumen.

Effects of creatine phosphate and P(i) on Ca2+ movements and tension development in rat skinned skeletal muscle fibres.

1. Mechanically skinned fast-twitch (FT) and slow-twitch (ST) muscle fibres of the rat were used to investigate the effects of fatigue-like changes in creatine phosphate (CP) and inorganic phosphate (P(i)) concentration on Ca(2+)-activation properties of the myofilaments as well as Ca2+ movements into and out of the sarcoplasmic reticulum (SR). 2. Decreasing CP from 50 mM to zero in FT fibres increased maximum Ca(2+)-activated tension (Tmax) by 16 +/- 2% and shifted the mid-point of the tension-pCa relation (pCa50) to the left by 0.28 +/- 0.03 pCa units. In ST fibres, a decrease of CP from 25 mM to zero increased Tmax by 9 +/- 1% and increased the pCa50 by 0.16 +/- 0.01 pCa units. The effect of CP on Tmax was suppressed in both fibre types by prior treatment with 0.3 mM FDNB (1-fluoro-2,4-dinitrobenzene), suggesting that these effects may occur via changes in creatine kinase activity. 3. Increases of P(i) in the range 0-50 mM reduced the pCa50 and Tmax in both fibre types. These effects were more pronounced in ST fibres than in FT fibres in absolute terms. However, normalization of the results to resting P(i) levels appropriate to both fibre types (1 mM for FT and 5 mM for ST fibres) revealed similar decreases in Tmax (approximately 39% at 25 mM P(i) and approximately 48% at 50 mM P(i)) and pCa50 (0.25 pCa units at 25-50 mM P(i)). The depressant action of P(i) on both parameters was considerably reduced when the rise in P(i) was accompanied by an equivalent reduction in [CP]. 4. Tension development in the presence of complex, fatigue-like milieu changes (40 mM P(i) for FT; 20 mM P(i) for ST) was decreased by 35-40% at a constant myoplasmic [Ca2+] of 6 microM in both fibre types. 5. SR Ca2+ loading at a myoplasmic [Ca2+] of 100 nM was found to increase abruptly when the [P(i)] during loading was increased to near 9 mM. At a myoplasmic [Ca2+] of 300 nM, the threshold P(i) for this effect dropped to approximately 3 mM. 6. Tension responses evoked by caffeine in the absence of P(i) were smaller and slower to peak if fibres were exposed to P(i) in a restricted myoplasmic Ca2+ pool after SR Ca2+ loading. This indicated that myoplasmic P(i) can decrease and prolong the rate of Ca2+ release from the SR.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca(2+)-dependent inactivation of Ca2+ current in Aplysia neurons: kinetic studies using photolabile Ca2+ chelators.

1. The kinetics and sensitivity of the Ca(2+)-dependent inactivation of calcium current (ICa) were examined in intact cell bodies from the abdominal ganglion of Aplysia californica under two-electrode voltage clamp. 2. Rapid changes in the level of intracellular free calcium ([Ca2+]i) were generated at the cell surface by photolytic release of Ca2+ (nitr-5 and dimethoxy nitrophen) or Ca2+ buffer (diazo-4). 3. Diazo-4 increased ICa by 10-15% and slowed the rate of ICa decay when photolysed before a test pulse or between a prepulse and a test pulse. The predominant effect of further light flashes was to increase the amount of non-inactivating current (I infinity) remaining at the end of long (> 1 s) depolarizing pulses. 4. A rapid increase in [Ca2+]i buffering during ICa inactivation did not cause a rapid recovery of current but merely reduced the rate and extent of subsequent inactivation. This effect was not seen when Ba2+ was the charge carrier. 5. Photolytic release of Ca2+ from nitr-5 produced estimated Ca2+ jumps of 3-4 microM at the front surface of the cell but failed to augment inactivation either before or during ICa. In contrast, photolysis of DM-nitrophen 10-90 ms before the test pulse decreased peak ICa by about 30%. A flash given during ICa rapidly blocked 41 +/- 3% of peak current with a time constant of 3-4 ms at 17 degrees C. Similar results were seen with the barium current (IBa). 6. Microinjection of the potent phosphatase inhibitor microcystin-LR (5 microM) had variable effects on ICa inactivation and augmented the cyclic AMP-induced depression of the delayed rectifier (IK(V) by forskolin (100 microM) and 3-isobutyl-1-methylxanthine (IBMX; 200 microM). 7. Full recovery from inactivation measured in two-pulse experiments took at least 20 s. This slow recovery process was unaffected by increases in intracellular cyclic AMP elicited by direct injection or by bath application of forskolin and IBMX. It was also unaffected by decreases in cyclic AMP induced by injecting 2',5'-dideoxyadenosine (1 mM) or bath application of the Rp isomer of cyclic adenosine 3',5'-monophosphothioate (Rp-cAMPS; 200 microM). 8. A 'shell' model relating submembrane Ca2+ to inactivation was inconsistent with the experimental results since it greatly overestimated the effects of diazo-4 and predicted significant inactivation by nitr-5 photolysis. 9. A model linearly relating [Ca2+]i in a single Ca2+ channel 'domain' to inactivation more closely matched the experimental results with diazo-4 and DM-(ABSTRACT TRUNCATED AT 400 WORDS)

An N-ethylmaleimide-sensitive G-protein modulates Aplysia Ca2+ channels.

The sulfhydryl alkylating agent N-ethylmaleimide (NEM) was used to probe the possible modulation of calcium current (ICa) by G-proteins in identified neurons of Aplysia californica. ICa recorded with conventional two-electrode voltage clamp was irreversibly suppressed by bath applied NEM in a concentration-dependent manner. This effect was fully blocked by addition of dithiothreitol or intracellular pressure injection of GTP gamma S but was unaffected by pre-treatment with pertussis toxin. These findings suggest that NEM inhibits ICa by causing persistent activation of an inhibitory G-protein in the absence of applied agonist. It appears that alkylation of key cysteine residues involved in G-protein deactivation underlie this effect.

Actions of caffeine on fast- and slow-twitch muscles of the rat.

1. The effects of caffeine (0.2-20 mmol l-1) have been examined on calcium transients (measured with aequorin) and isometric force in intact bundles of fibres from soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles of the rat. 2. At 25 degrees C, threshold caffeine concentration for an observable increase in resting [Ca2+]i was 0.2 and 1.0 mmol l-1 for soleus and EDL muscles respectively. Increases in resting force were first detectable at about 0.5 mmol l-1 caffeine for soleus muscles and 5.0 mmol l-1 caffeine for EDL muscles and occurred in the range 0.2-0.4 mumol l-1 [Ca2+]i for soleus and 0.7-0.9 mumol l-1 for EDL. 3. Caffeine potentiated the twitch responses of soleus and EDL in a dose-related manner. The soleus was more sensitive in this respect, with 50% potentiation occurring at 1 mmol l-1 caffeine compared with 3.5 mmol l-1 for the EDL. Concentrations of caffeine below 2 mmol l-1 potentiated Ca2+ transients associated with twitches in both soleus and EDL muscles with no apparent change in the decay rate constant. 4. High concentrations of caffeine (greater than 2 mmol l-1) further potentiated peak Ca2+ in the EDL but depressed it in the soleus. The rate of decay of the Ca2+ transient in high caffeine was significantly prolonged in the soleus but remained unaffected in the EDL. 5. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) had little effect on force or [Ca2+]i at concentrations known to significantly increase intracellular cyclic AMP levels. 6. The Ca2+ transient during fused tetani was characterized by an initial peak, a decline to a plateau level and sometimes a gradual rise towards the end of the stimulus train. Peak [Ca2+]i during normal tetani ranged between 1.1 and 2.4 mumol l-1 in the soleus and 1.9 and 4.0 mumol l-1 in the EDL. 7. Caffeine potentiated both force and [Ca2+]i during tetanus. Since the increase of the Ca2+ transient was significantly greater than potentiation of force, it is likely that saturation of myofilaments occurs. The primary effect of caffeine on the Ca2+ transient was an elevation of the plateau phase. 8. Caffeine concentrations below 5 mmol l-1 potentiate twitch and tetanic force in both fast- and slow-twitch mammalian skeletal muscles primarily by increasing both the basal and stimulus-evoked release of Ca2+ from the sarcoplasmic reticulum.

The action of ryanodine on rat fast and slow intact skeletal muscles.

1. The action of ryanodine on force development of bundles dissected from rat extensor digitorum longus (EDL) and soleus muscles has been examined. 2. Ryanodine (100-5000 nM) irreversibly depressed twitch and tetanic tension of both muscle types in a dose-related manner. 3. At concentrations above 250 nM, ryanodine induced a slowly developing, dose-dependent contracture which could not be blocked by 5 mM-Co2+. Increasing the stimulation rate or decreasing the oxygenation of the preparation accelerated the rate of contracture development while the total removal of extracellular Ca2+ was required to prevent it. 4. Following the relaxation of the initial contracture (IC) in Ca2+-free solution, a second type of contracture (SC) could be induced by the readdition of Ca2+. This contracture differed from IC in that it was dependent on Ca2+ in the millimolar range and was prevented by 5 mM-Co2+. Both IC and SC were relaxed by perfusion with Ca2+-free, EGTA-containing solution. 5. Subcontracture doses of ryanodine (100 nM) markedly potentiated caffeine contractures of both muscle types. 6. Asymmetric charge movement in EDL fibres was recorded with the Vaseline-gap technique. The amount of charge moved near threshold was virtually unaffected by the presence of 10 microM-ryanodine over the time examined. 7. The results are consistent with the suggestion that ryanodine locks the calcium release channels of the sarcoplasmic reticulum (SR) in an open subconductance state with reduced conductance. It appears that lowering the external calcium concentration might still inactivate the release channels after they have been blocked open by ryanodine, possibly by an effect on the T-tubular voltage sensor.

Effects of 2,3-butanedione monoxime on the contractile activation properties of fast- and slow-twitch rat muscle fibres.

1. The effects of 2,3-butanedione monoxime (BDM, 0.2-10 mmol/l) have been examined at different temperatures on calcium transients (measured with aequorin) and isometric force in intact bundles of fibres from soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles of the rat and on Ca2+-activated isometric force of mechanically skinned soleus and EDL fibres. Ca2+ release and uptake by the sarcoplasmic reticulum has also been investigated directly on skinned EDL fibres at 1 mmol/l BDM at 23 degrees C. 2. BDM bound calcium with low affinity (KCaBDMapp = 55.9 +/- 4.3 M-1 at 22 degrees C, pH 7.10, ionic strength approximately 155 mmol/l) and did not affect the kinetics and quantum yield of the Ca2+-induced aequorin luminescence. 3. BDM reversibly reduced both force- and Ca2+-dependent aequorin light during the twitch and tetanus of all intact-muscle preparations in a dose-related manner. Twitch responses of soleus fibres were more sensitive to BDM than were EDL fibres. Higher concentrations of BDM were needed to abolish the tetanic force response than the twitch response in both muscle types. 4. The initial rate of rise and the decay rate constants of twitches in both soleus and EDL muscles were increased in the presence of BDM. Accordingly, a higher frequency of stimulation was required to produce fusion of the tetanus. This phenomenon could be explained by the reduced magnitude of the Ca2+ transient. 5. BDM reduced maximal Ca2+-activated force development in mechanically skinned soleus and EDL muscle fibres in a dose-dependent fashion. This depression of force was less sensitive to BDM than were the light transients in intact fibres. Furthermore, BDM affected EDL skinned fibres to a greater extent than soleus skinned fibres, in contrast with its effects on intact fibres. 6. At concentrations of BDM greater than 2.5 mmol/l, BDM significantly decreased the sensitivity of the contractile apparatus to calcium. The relative force-pCa (= -log [Ca2+]) curves for both soleus and EDL skinned fibres were shifted to the right (i.e. to higher calcium concentrations) in a dose-related manner. 7. BDM (10 mmol/l) slowed maximal Ca2+-activated force development of skinned preparations of both fibre types at all temperatures investigated (4-24 degrees C). 8. BDM reduced stiffness in skinned preparations maximally activated by calcium in proportion to the reduction in the isometric force response. BDM also inhibited rigor force of all fibres but only if it was present before the onset of rigor.(ABSTRACT TRUNCATED AT 400 WORDS)

Paralysis of skeletal muscle by butanedione monoxime, a chemical phosphatase.

The chemical phosphatase butanedione monoxime (BDM) reversibly inhibited twitches and tetanic contractions in bundles of rat soleus fibres in a dose-dependent manner (2-20 mM) but had no effect on the amplitude or time course of action potentials. In addition, BDM reversibly reduced the amplitude of potassium contractures demonstrating a depressant effect on contraction not mediated by action potentials. BDM had no effect on asymmetric charge movement but depressed calcium currents across the surface membrane in voltage-clamped fibres. The most significant effect of BDM on excitation-contraction coupling was a reduction in the amplitude of the calcium transient associated with contraction in aequorin-injected fibres. While these experiments do not eliminate the possibility of a direct effect of BDM on contractile filaments, reduction of calcium release from the sarcoplasmic reticulum, at least at low concentrations of BDM (below 2 mM), would seem to be the main mechanism for the inhibition of contractions in rat skeletal muscle.

The effect of alcohols on aequorin luminescence.

The effect of alcohols from ethanol to octanol on aequorin luminescence was biphasic; enhancement of both peak light and total photon yield at low concentrations, and inhibition of these parameters at high concentrations. The potency of alcohols to exert these effects was in the same order as the oil/water partition coefficients of the alcohols. It is argued that neither of these effects is related to calcium binding by aequorin, since alcohols enhanced calcium-independent luminescence, and the inhibition of responses is not associated with a reduction in the rate of aequorin 'consumption' on binding of calcium.

Relationship between intracellular calcium concentration and relaxation of rat fast and slow muscles.

Following injection of aequorin into small bundles of rat soleus and extensor digitorum longus muscles, simultaneous measurements of light (proportional to the intracellular free calcium ion concentration ([Ca2+]i)) and force were recorded during muscle twitches over a range of temperatures from 10 to 37 degrees C. The Arrhenius plots for rate of decline of [Ca2+]i following a twitch were linear and yielded the same activation enthalpy (Ea) for both muscle types, indicating that the same process controls Ca2+ uptake in both fast and slow muscles. Arrhenius plots of force decay rate constants showed a break around 20 to 25 degrees C with the Ea's above the break being similar to the Ea's for decline of [Ca2+]i and indicating a close relationship between rate of relaxation and decline of [CA2+]i above this temperature.