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1. In skeletal muscle the presence of a positive inotropic effect induced by adrenaline has been a matter of controversy. If it exists, it could be due to catecholamines acting on the actomyosin system, on the sarcoplasmic reticulum (SR) Ca2+ pump or on the release or influx of Ca2+. We investigated these possibilities by using intact, split and skinned skeletal muscle fibres. We also investigated whether adrenaline acts directly or through cyclic AMP. 2. Catecholamines produced an increase in twitch tension and in maximum rates of tension development and tension decay. The inotropic effect took 3 min to appear and 8 min to reach its maximum level. With tetanic stimulations the extra force appeared only at the beginning of the tetanus while approaching the same maximum level, and tended to disappear faster, the higher the frequency of stimulation. At 4 shocks/sec the peak twitch tension with catecholamines decreased during the first seven to ten twitches and became steady afterwards at a level that was still greater than the control. 3. Resting and action potentials showed no important changes in the presence of adrenaline that could explain the inotropic effect. 4. In split fibres the force produced with the release of Ca2+ from the SR by caffeine was 60‐100% larger when cyclic AMP was added to the previous loading solution. In skinned fibres adrenaline given directly to the interior of the cell produced no changes in contraction‐‐relaxation cycles induced by fixed amounts of Ca2+ applied with a pipette. 5. These results strongly suggest that catecholamines through cyclic AMP stimulate the SR Ca2+ pump, increasing thereby the concentration of Ca2+ within the SR. This extra Ca2+ when released during subsequent activation may produce the increase in twitch tension.

An extracellular Na+ (Nao)-dependent Mg2+ efflux process that requires intracellular ATP has been proposed as the sole mechanism responsible for Mg2+ extrusion in internally dialyzed squid axons (12). We have shown that this exchanger can also “reverse” and mediate an extracellular Mg2+ (Mgo)-dependent Na+ efflux (16). We have extended these studies and found that, in the presence of ouabain, bumetanide, tetrodotoxin, and K+ channel blockers and in the absence of extracellular Na+, K+, and bicarbonate, intracellular K+ and Cl- are also involved in the Mgo-dependent Na+ efflux process. Two main observations support this view: 1) operation of the Mgo-dependent Na+ efflux requires the presence of intracellular K+ and Cl-, and 2) Mgo removal produces a reversible and nearly identical reduction in the magnitude of the simultaneous efflux of the ionic pairs K(+)-Na+ and Cl(-)-Na+. These results suggest that the putative bumetanide-insensitive Na-Mg exchanger also transports K+ and Cl-.

If the space constant of the T‐system (lambdaT) its not large in comparison with the radius (a) of a muscle fibre, different levels of depolarization should activate different proportions of the cross‐section. This possibility was tested in isolated muscle fibres with isotonic and isometric K contractures. 2. During isonic contractures with more than 40 mM‐K, wavy myofibrils appeared in the centre of the fibre. The sarcomere spacings (s) of the wavy myofibrils, measured parallel to the long axis of the myofibrils, were 1‐9‐1‐95 mum. However, the superficial myofibrils could shorten to or below s=1‐5 mum without becoming wavy. 3. In the same muscle fibre where myofibrils became wavy during K contractures, no waviness appeared during repetitive electric stimulation in normal Ringer (50 shocks/sec, 12 degrees C), although s decreased below 1‐5 mum. Wavy myofibrils were interpreted as not activated. 4. With isometric contractures it was found that the amount of depolarization needed to obtain maximal tension was smaller for fibres of shorter radius. The degree of depolarization for producing maximal tension is related to a by 6 mV/10mum. 5. These results strongly suggest that in K contractures lambdaT is not large in comparison with a.

Summary In fatigued muscles the T-system is swollen; thus the action potential may fail to travel along the T-system or the T-tubule terminal cisternae signal may fail to bring about TC Ca2+ release. This would lead to a decrease in the number of myofibrils activated and in force development, but if fatigue is the result of a generalized process, all the myofibrils would be affected equally leading to a lower activation of all of them. We have investigated this possibility in isolated twitch muscle fibres by giving them repetitive tetanic stimulations until fatigue developed. The behaviour of myofibrils was followed with cinémicrophotography. Before fatigue, no lack of shortening of myofibrils could be found. During fatigue groups of myofibrils became wavy. When exposed to caffeine, the wavy myofibrils disappeared and tension similar to the control developed. The tension-caffeine concentration relationship was shifted to the left after development of fatigue. In low Na+ solution fatigue developed faster and after reintroducing normal Ringer, tension recovered substantially. K-contractures were smaller during fatigue. These results indicate that in this type of fatigue, a step in the EC coupling chain of events is involved in its development.

Ca2+ and Mg2+ movements across the sarcoplasmic reticulum (SR) of frog skeletal muscle fibers were measured in situ by electron probe microanalysis of muscles rapidly frozen following a tetanus. At 400 ms following a 1.2-s tetanus at room temperature, the force had relaxed to base-line, and 0.3 mmol of Ca2+/liter of cytoplasmic H2O had been pumped by the SR, indicating that the in situ pumping of the SR Ca-ATPase is sufficiently high to account for the removal of Ca2+ from the Ca2+-specific sites of troponin (0.18 mmol of Ca2+-specific sites/liter of cytoplasmic H2O) and for the rate of relaxation from a tetanus at room temperature. The half-time of the return of the total 1.0 mmol of Ca2+/liter of cytoplasmic H2O released during a tetanus was 1.1 s, comparable to the slow Koff rate of Ca2+ from (carp) parvalbumin (1.0 s-1) and consistent with the hypothesis that the return of this Ca2+ to the terminal cisternae is rate-limited by the Ca2+ off-rate from parvalbumin. The return of the Mg2+ taken up by the terminal cisternae during a tetanus to resting levels was significantly slower than the time course of the Ca2+ movements, suggesting that the Mg2+ permeability of the SR in situ is low and may be transiently increased during tetanic stimulation.

Approximately 60-70% of the total fiber calcium was localized in the terminal cisternae (TC) in resting frog muscle as determined by electron-probe analysis of ultrathin cryosections. During a 1.2 s tetanus, 59% (69 mmol/kg dry TC) of the calcium content of the TC was released, enough to raise total cytoplasmic calcium concentration by approximately 1 mM. This is equivalent to the concentration of binding sites on the calcium-binding proteins (troponin and parvalbumin) in frog muscle. Calcium release was associated with a significant uptake of magnesium and potassium into the TC, but the amount of calcium released exceeded the total measured cation accumulation by 62 mEq/kg dry weight. It is suggested that most of the charge deficit is apparent, and charge compensation is achieved by movement of protons into the sarcoplasmic reticulum (SR) and/or by the movement of organic co- or counterions not measured by energy dispersive electron-probe analysis. There was no significant change in the sodium or chlorine content of the TC during tetanus. The unchanged distribution of a permeant anion, chloride, argues against the existence of a large and sustained transSR potential during tetanus, if the chloride permeability of the in situ SR is as high as suggested by measurements on fractionated SR. The calcium content of the longitudinal SR (LSR) during tetanus did not show the LSR to be a major site of calcium storage and delayed return to the TC. The potassium concentration in the LSR was not significantly different from the adjacent cytoplasmic concentration. Analysis of small areas of I-band and large areas, including several sarcomeres, suggested that chloride is anisotropically distributed, with some of it probably bound to myosin. In contrast, the distribution of potassium in the fiber cytoplasm followed the water distribution. The mitochondrial concentration of calcium was low and did not change significantly during a tetanus. The TC of both tetanized and resting freeze-substituted muscles contained electron-lucent circular areas. The appearance of the TC showed no evidence of major volume changes during tetanus, in agreement with the estimates of unchanged (approximately 72%) water content of the TC obtained with electron-probe analysis.

In single isolated skeletal muscle fibers of the frog, we studied (i) the recovery from large sarcolemmal mechanical injuries of the response to electric stimulation and (ii) the integrity of the sarcolemma under the light microscope. In Ringer's solution, the damaged cells stopped contracting and deteriorated completely within 1 hr. In the presence of phosphatidylcholine (0.025 g/ml in Ringer's solution), the injured cells initially responded with local twitches. Within 0.5 hr, contractility and membrane integrity started to recover and both were back to control levels within 3 hr. When these cells were placed back in normal Ringer's solution, they remained viable and active for several hours. Our results suggest that phosphatidylcholine can protect muscle fibers from the effects of sarcolemmal injury.

Electron probe analysis, cryo-ultramicrotomy, and freeze-substitution were used to determine the nature of vacuolation and the subcellular composition in fatigued frog skeletal muscle fibers. The vacuoles caused by fatigue were part of the T-tubule system and contained high concentrations of NaCl. The calcium concentration in the terminal cisternae was higher than previously measured normal resting values. Mitochondrial calcium content was relatively low (mean +/- SEM, 2 +/- 2 mmol/kg dry weight). Fiber NaCl was increased. It is concluded that fatigue is not due to the depletion of calcium stores from the terminal cisternae or to uncoupling of mitochondria due to calcium loading but may be caused by multiple mechanisms including failure of the T-tubule action potential.

Experiments were designed to determine whether the putative Na(+)-Mg2+ exchanger previously demonstrated to mediate Mg2+ efflux (R. DiPolo and L. Beague. Biochim. Biophys. Acta 946: 424-428, 1988) could also mediate the efflux of Na+ (presumably a Na+ efflux-Mg2+ influx exchange) in squid giant axons. The effects of external Mg2+ (Mg(o)) on 22Na efflux were measured in internally dialyzed, ATP-fueled axons in which the contribution to Na+ efflux by other pathways was inhibited. To facilitate measurement of Mg(o)-dependent Na+ efflux, the intracellular concentration of Na+ was increased. To prevent Na(+)-Na+ exchange, external Na+ was replaced by tris(hydroxymethyl)aminomethane. To assess the effect of Mg(o) on Na+ efflux without altering the total divalent cation concentrations, Mg(o) was replaced mole-for-mole by external Ba2+ (Ba(o)). This manipulation produced reversible reductions in Na+ efflux. These reductions were neither due to membrane hyperpolarization nor to a direct effect of Bao but were due instead to the reduction in Mg(o). The Mg(o)-dependent Na+ efflux was inhibited by external amiloride but was spared by bumetanide. In the absence of external Na+, the Mgo-dependent Na+ efflux increased as a function of external Mg2+ with Michaelis-Menten kinetics. These results indicate that the Na(+)-Mg2+ exchange can mediate the efflux of Na+ (operate in Na+ efflux-Mg2+ influx mode of exchange).

Functionally skinned and electrochemically shunted myocytes were prepared by perfusing rat hearts with collagenase in order to obtain a technically improved measurement of sarcomere dynamics and to evaluate the role of sarcoplasmic reticulum in situ with respect to contractile activation. In the presence of micromolar calcium, the myocytes exhibited phasic and propagated contraction waves beginning at one end and proceeding along the myocyte. Beating rates, the propagation velocity of the activation wave, and single sarcomere shortening and relaxation velocities were obtained by manual or automated analysis of 16-mm film recorded at 170 frames/s from a camera attached to a microscope that was equipped with a temperature-controlled stage. In parallel experiments, calcium accumulation by the sarcoplasmic reticulum of the myocytes in situ was measured by direct isotopic tracer methods. The frequency (10-38 min-1) of spontaneous contractions, the velocity (1.9-7.4 microns . s-1) of sarcomere shortening, and the velocity (1.7-6.8 microns . s-1) of sarcomere relaxation displayed identical temperature dependences (Q10 = 2.2), which are similar to that of the calcium pump of sarcoplasmic reticulum and are consistent with a rate limit imposed by enzyme-catalyzed mechanisms on all these parameters. On the other hand, the velocity (77-159 microns . s-1) of sequential sarcomere activation displayed a lower temperature dependence (Q10 = 1.5), which is consistent with a diffusion-limited and self-propagating release of calcium from one sarcomere to the other. The phasic contractile activity of the dissociated myocytes was inhibited by 10(-8)-10(6) M ryanodine (and not by myolemmal calcium blockers) under conditions in which calcium accumulation by sarcoplasmic reticulum in situ was demonstrated to proceed optimally. The effect of ryanodine is attributed to an interaction of this drug with sarcotubular structures, producing inhibition of calcium release from the sarcoplasmic reticulum. The consequent lack of sarcomere activation underlines the role of sarcoplasmic reticulum uptake and release in the phasic contractile activation of the electrochemically shunted myocytes.

1. The excitatory process travelling along the T‐system may be either electrotonic or regenerative. If Na+dependent action potential is present in the tubular membranes, high frequency of stimulation might cause a Na+depletion in the tubules sufficient to abolish this process.

It has been proposed1that an influx of calcium ions into twitch muscle fibres during an action potential might initiate contraction. However, when external Ca2+is lowered to 10−8M with EGTA, the fibres can produce normal twitches for many minutes2,3. Nevertheless, a clear Ca2+influx during contraction has been demonstrated4,5, and it has been found that phasic skeletal muscle has an inward calcium current (ICa)6,7which can give rise to calcium spikes8. In certain conditions, a reduction in external Ca2+with 80–90 mM EGTA results in reversible blockade of excitation–contraction (e–c) coupling9, leading some authors to suggest7,9–11that extracellular Ca2+moved into the myoplasm due to ICamay be involved in the e–c coupling mechanism that triggers contraction. This proposition was further supported by the localization of ICain the T-system, which circumvented the problem of the delay due to calcium diffusion from the surface membrane. We have now investigated whether ICahas a clear role in initiating or sustaining contractions in twitch muscle fibres. Our approach was to decrease or eliminate ICawith the calcium-blocking agent diltiazem (Herbesser) and to see how the twitch, tetanic and potassium-contracture tensions were affected. We found that ICacould be decreased or cancelled with the calcium-blocking agent, but that the same concentration of the drug potentiated the twitch, tetanus and contractures. We conclude, therefore, that ICahas no role in e–c coupling. A preliminary report of these results has been presented elsewhere12.