Influence of changes in external potassium and chloride ions on membrane potential and intracellular potassium ion activity in rabbit ventricular muscle

1. The membrane responses of rabbit papillary muscles to rapid changes in [K](o) and [Cl](o) were measured with open-tipped micropipettes and with closed micropipettes made from K-selective glass.2. The muscle cells behaved primarily as a K electrode, and responses to changes in [K](o) with constant [Cl](o) or with constant [K](o) x [Cl](o) were substantially the same.3. When [Cl](o) was changed at a constant [K](o) the membrane potentials changed rapidly and symmetrically by a small value and remained constant for 30 min.4. Measurement of potential with K(+)-selective micro-electrodes in these experiments showed no change in intracellular K activity. In addition to permitting calculation of K permeability, these measurements reassured us that the K(+)-selective electrodes were well insulated and not influenced by electrical shunts at the impalement site.5. Although the membrane response to changes in [Cl](o) was small, it was possible to calculate that the permeability ratio (P(Cl)/P(K)), was 0.11. The Cl and K conductances were about 0.015 mmho/cm(2) and 0.09 mmho/cm(2) respectively, resulting in a conductance ratio (g(Cl)/g(K)) of about 0.17.6. The time course of depolarization by increase in [K](o) was rapid (half-time 5 sec), but repolarization on return to lower [K](o) was much slower (half-time 50 sec). The depolarization time course was easily fitted by the potential change calculated by assuming the need for K diffusion into the extracellular spaces and taking account of the logarithmic relation between membrane potential and [K](o). These calculations did not fit the time course of repolarization, which was slowed in the fashion expected from an inward-rectifying membrane.7. The influence of [K](i) on membrane potential was investigated by changes in tonicity of the external solution. Hypotonic solution produced a change in intracellular K activity close to that produced by ideal water movement. However, in hypertonic solution, intracellular K activity did not rise as much as predicted, suggesting a change in intracellular activity coefficient.     

Ionic diffusion delays in the transverse tubules of frog twitch muscle fibres

1. The possibility of retarding the diffusional processes in the transverse tubules of frog single muscle fibres was explored by increasing the viscosity of the bathing medium. The viscosity was increased by adding Dextran 15 to the experimental solutions.2. The criterion used to test this possibility was the speed of repolarization of muscle fibre membranes produced by sudden reduction in [K](o) in media of different viscosity.3. In fibres equilibrated in media containing 15% Dextran 15, and that had been depolarized with 80 mM-K for more than 5 sec, the repolarization caused by lowering the [K](o) to 40 mM proceeded more slowly than in fibres in Dextran-free media.4. On the basis of the above results, it was deduced that high viscosity solutions would delay the diffusion of Na ions in the transverse tubules. Thus the effect of sudden changes in [Na](o) on the twitch tension of single fibres could be studied.5. The recovery of twitches when the fibres were exposed from a solution containing no Na, to one that contained 46 mM-Na, occurred more slowly in high viscosity media.6. This result can be explained assuming that a Na regenerative process occurs at the level of the membrane of the transverse tubules.     

Influence of sodium ions on the membrane potential of the sino-atrial node in response to sympathetic nerve stimulation

1. Sympathetic nerve-atrial preparations isolated from rabbits were used in the present study. Transmembrane potentials were recorded from sino-atrial (S-A) nodal pace-maker fibres.2. A reduction of [Na(+)](o) (extracellular concentration of sodium ions) decreased the maximal diastolic potential, reduced the size of the overshoot, prolonged the depolarization time, increased the rate of early repolarization and decreased the rate of late repolarization.3. A Na(+)-deficiency markedly augmented the positive chronotropic effect of stimulation of post-ganglionic sympathetic nerves at a frequency of 5/sec but slightly augmented the effect of stimulation at 1 and 20/sec.4. The marked membrane effect of sympathetic nerve stimulation at low [Na(+)](o) was an increase in the slope of slow depolarization during diastole. Corresponding to an induced acceleration of S-A nodal rate, the 90% duration of the action potential was decreased but the other action potential parameters measured were not influenced.5. A reduction of [Na(+)](o) to 51.7 mM produced in the pace-maker membrane, subthreshold oscillations the rate and the amplitude of which were increased by sympathetic nerve stimulation until conducted potentials were generated.6. It is presumed that cardiac noradrenaline elicits an increase in the slope of slow depolarization during late diastole which is due to an increase in Na(+) permeability, a decrease in K(+) permeability or a non-selective increase in ion permeability, but that it does not influence the pace-maker membrane during systole.     

Development of T-tubular vacuoles in eccentrically damaged mouse muscle fibres

Single fibres were dissected from mouse flexor digitorum brevis muscles and subjected to a protocol of eccentric stretches consisting of ten tetani each with a 40 % stretch. Ten minutes later the fibres showed a reduced force, a shift in the peak of the force-length relation and a steepening of the force-frequency relation. Addition of the fluorescent dye sulforhodamine B to the extracellular space enabled the T-tubular system to be visualized. In unstimulated fibres and fibres subjected to 10 isometric tetani, the T-tubules were clearly delineated. Sulforhodamine B diffused out of the T-tubules with a half-time of 18 ± 1 s. Following the eccentric protocol, vacuoles connected to the T-tubules were detected in six out of seven fibres. Sulforhodamine B diffused out of the vacuoles of eccentrically damaged fibres extremely slowly with a half-time of 6.3 ± 2.4 min and diffused out of the T-tubules with a half-time of 39 ± 4 s. Vacuole production was eliminated by application of 1 m m ouabain to the muscle during the eccentric protocol. On removal of the ouabain, vacuoles appeared over a period of 1 h and were more numerous and more widely distributed than in the absence of ouabain. We propose that T-tubules are liable to rupture during eccentric contraction probably because of the relative movement associated with the inhomogeneity of sarcomere lengths. Such rupture raises intracellular sodium and when the sodium is pumped from the cell by the sodium pump, the volume load of Na⁺ and water exceeds the capacity of the T-tubules and causes vacuole production. The damage to the T-tubules may underlie a number of the functional changes that occur in eccentrically damaged muscle fibres.     

The time and voltage dependence of the slow outward current in cardiac Purkinje fibres

1. In normal Tyrode solution the initial inward sodium current which is produced in Purkinje fibres in response to a sudden depolarization is followed by a very slow change in current in an outward direction. The magnitude and speed of onset of this slow change both increase with the strength of the depolarization.2. On repolarization, a transient outward current is observed (Deck & Trautwein, 1964). The initial magnitude of this outward current also increases with the strength and duration of the preceding depolarization, with a time course similar to the time course of the slow change in current during depolarization.3. Evidence is presented for the view that the slow change in current during depolarization represents the onset of delayed potassium rectification and that the decline in outward current following repolarization represents its decay.4. Removal of sodium ions greatly increases the threshold of the slow outward current. In the presence of sodium ions 15 mV depolarization is sufficient to produce an appreciable current. In the absence of sodium ions at least 50 mV depolarization is required and, in some fibres, no delayed rectification is observed even when the membrane potential is made positive.5. Prolonged depolarization of the membrane by outward current changes the quiescent membrane potential in a positive direction. This change in potential is attributed to an accumulation of potassium ions in a space immediately outside the cell membrane, which equilibrates slowly with the extracellular fluid. It is shown that this effect is very small during depolarizations of the magnitude and duration of the normal action potential.6. It is concluded that the results are consistent with the view that repolarization of the Purkinje fibre action potential is initiated by a slow increase in K conductance similar to, but much smaller and slower than, that observed in nerve fibres.     

The action of caffeine on the activation of the contractile mechanism in straited muscle fibres

1. The effect of caffeine on the initiation of isometric tension in isolated twitch muscle fibres of the frog was recorded with a mechano-electrical transducer.2. In Ringer solution as well as in solutions containing 95 mM-K(2)SO(4), caffeine (6-10 mM) caused reversible contractures. Tension of maximal potassium contractures was reached with a half-time of 2-4 sec.3. Caffeine caused a shift to lower potassium concentrations of the S-shaped curve which relates peak tension to log. [K](o) or membrane potential. In subthreshold concentrations of caffeine (1.5 mM) the potassium concentration at which half of maximal tension was reached shifted from 30 to 16 mM-K (-39 to -53 mV).4. In the ;steady state' the ability of fibres to develop tension is related to log. [K](o) or membrane potential by an S-shaped curve whose half value shifted from 28 to 45 mM-K (-41 to -29 mV) when 1.5 mM caffeine was applied.5. Fibres were most sensitive to caffeine at membrane potentials between -50 and -20 mV.6. The mechanical activity caused by caffeine was ;stabilized' by an increase in [Ca](o) or [Mg](o) resembling the stabilizing action of these ions on potassium contractures or on the sodium permeability of excitable membranes.7. Tetracaine in low concentrations (0.04-0.1 mM) increased the threshold for mechanical activation and shortened the plateau of potassium contractures. Higher concentrations (1-2 mM) suppressed mechanical activity completely.8. Tetracaine, 0.04 mM, was sufficient to suppress tension caused by a 100 times stronger concentration of caffeine. With higher concentrations of caffeine the inhibitory action of tetracaine could be reversed.9. Fibres which were immersed in subthreshold concentrations of caffeine either in Ringer solution or in a solution with 95 mM-K(2)SO(4) developed a strong contracture after a sudden drop in temperature from 20 to 1-3 degrees C.10. The fast activation of the whole cross-section of the muscle fibre caused by caffeine and its dependence on membrane potential, tetracaine and external alkali earth ions favours the idea that the drug acts at some part of the sarcotubular system which is easily accessible for external ions and drugs and in close connextion with the surface membrane.     

Contractile repriming in snake twitch muscle fibres

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

The volume of the T-system and its association with the sarcoplasmic reticulum in slow muscle fibres of the frog

1. A study has been made of the T-system and sarcoplasmic reticulum (SR) in slow muscle fibres of the frog, Rana temporaria.2. The size of the T-system was measured by an autoradiographic method, using tritium-labelled albumin as a marker. Its volume, expressed as a fraction of that of the fibre, was found to be 1.8 x 10(-3), as compared with a figure of 3.9 x 10(-3) for the T-system in a twitch fibre.3. The spatial distribution of the T-tubules, and their association with the SR, was studied with the electron microscope, employing ferritin and the enzyme peroxidase as markers. The observations show (a) the tubules form a three dimensional, rather than transverse, network and (b) the area of triadic (and diadic) contact with the SR is 5-10 x smaller than in a twitch fibre.4. The possibility that the T-system and SR of the slow fibre participate in linking membrane excitation with contraction is discussed in the light of these findings.     

The relation between external potassium concentration and the relaxation rate of potassium-induced contractures in frog skeletal muscle

1. Frog toe muscles and isolated fibres from frog semitendinosus muscles were allowed to develop maximum K-contractures, and after reaching peak tension were transferred to media containing intermediate [K](0) (20-40 mM-K). Under these circumstances, relaxation displayed an early rapid phase, and a subsequent slower phase whose magnitude and rate varied with [K](0). The removal of activator (calcium ion) from the sarcoplasm appears to have a potential-dependent component.2. The relation between relaxation and [K](0) suggests that, with progressive depolarization, membrane sites from which calcium is initially released become converted into sites which are again capable of binding calcium and removing it from the sarcoplasm.3. The rate of relaxation of frog toe muscle after maximum K-contractures can be either accelerated or retarded by abrupt alterations in [Ca](0) or by sudden replacement of the major extracellular anion. These effects are attributed to shifts in the relation between calcium rebinding and membrane potential.     

Graded activation of myofibrils and the effect of diameter on tension development during contractures in isolated skeletal muscle fibres.

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.     

Changes of extracellular potassium concentration induced by neuronal activity in the sinal cord of the cat

1. Changes of extracellular K(+) concentration, [K](e), arising in the spinal cord of the cat in response to an afferent stimulation were studied by means of K(+)-specific micro-electrodes.2. In the most active areas of the spinal cord a single volley in a large afferent input like the common peroneal nerve or the posterior tibial nerve produced a transient increase in [K](e) of 0.05-0.1 mM, which reached its peak in 0.2-0.3 sec and it declined in about 3 sec.3. Much higher increases in [K](e) were found during repetitive stimulation of an afferent input. The highest increase (by 3 mM) was at 100 Hz, but even at 1 Hz a significant increase of 0.25 mM was observed. Equilibration of accumulated K(+) was slow with a time constant of about 6 sec, which is much longer than could be expected for the same process in free solution.4. A characteristic distribution of increased [K](e) was found in the spinal cord in response to 100 Hz afferent stimulation. The highest increase of 3 mM was found in and around the intermediate nucleus, but at depths between 0.9-1.8 mm the [K](e) increase exceeded 1 mM.5. In the ventral horns afferent stimulation (100 Hz) increased [K](e) by 0.25 mM, while the same stimulation of the ventral root resulted in a [K](e) increase of less than 0.05 mM.6. The consequences of K(e) (+) accumulation after neuronal discharge are discussed in respect to its possible role in the depolarization of primary afferent terminals.     

Properties of sarcolemmal delayed rectification in glycerol-treated fibers of frog sartorius muscle.

Total current-voltage relations were analyzed on nine glycerol-treated surface fibers of frog sartorius muscles in tetrodotoxin-containing isotonic normal Ringer solution. The results indicate that delayed rectification occurs in the sarcolemma on large depolarization and that delayed rectification is only partially inactivated during 1 sec of depolarization and not converted into anomalous rectification. The time to peak, the time course of inactivation and the potassium activation potential determined in glycerol-treated fibers were comparable to those analyzed previously in intact fibers. The value of the conductance increase during delayed rectification in glycerol-treated fibers appeared to be smaller than that in intact fibers.     

The effect of potassium and chloride ions on the volume and membrane potential of single barnacle muscle cells

1. In single barnacle skeletal muscle cells cell diameter has been measured as a function of external osmolality, and cell diameter and membrane potential have been measured during changes of external K and Cl concentrations ([K](o) and [Cl](o)) like those described in frog muscle by Hodgkin & Horowicz (1959). The diameter was monitored microscopically with a precision of 0.2-0.4% (S.D.). [K](o) was varied from 1 to 18 mM, a range of concentrations which does not cause contracture.2. At pH 8.0 the Cl permeability was so low that net KCl and water movements were absent. Such net movements were present at pH 4.5, corresponding to a change in the ratio (Cl conductance/K conductance) from approximately 1/12 at pH 8.0 to 1/2 at pH 4.5.3. Characteristically long time constants were observed for membrane potential responses to a change in [K](o) and/or [Cl](o), even at constant [K](o).[Cl](o). This phenomenon is attributed to a delayed equilibration by diffusion within the system of sarcolemmal invaginations and T-tubules. The delay in the response was increased by introducing polyvinylpyrrolidone (PVP) into this system, presumably because PVP raises intratubular viscosity.4. At pH 4.5 anomalous rectification for net movements of K was demonstrated by measurements of cell diameter and of membrane potential.     

The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres

1. The intracellular Na activity of sheep heart Purkinje fibres has been measured using recessed-tip Na(+)-sensitive glass micro-electrodes.2. The internal Na activity was 7.2 +/- 2.0 mM (mean +/- S.D., n = 32) at the normal external Na concentration, [Na](o), in these experiments of 140 mM (equivalent to an external Na activity of 105 mM). The equilibrium potential for Na across the fibre membrane was therefore approximately + 70 mV.3. When the [K](o) was altered the internal Na activity changed, reaching a new level within about 20 min. Increasing the [K](o) from 4 to 25 mM decreased the internal Na by approximately 30%, while decreasing the [K](o) from 4 to 1 mM increased internal Na by 20%.4. The removal of external K produced an easily reversible increase in the internal Na with an initial rate equivalent to a concentration change of 0.24 +/- 0.07 m-mole/min (mean +/- S.D., n = 8).5. Ouabain produced increases in the internal Na activity that were only very slowly reversible. The threshold concentration for producing an increase was approximately 10(-7)M.6. When [Na](o) was reduced the internal Na activity fell rapidly with a single exponential time course (time constant 3.3 +/- 0.8 min, mean +/- S.D., n = 16) to a new, relatively stable level. The recovery of internal Na on return to the normal [Na](o) did not have a simple time course. It was normally complete within 10-30 min.7. The relationship of the stabilized level of the internal Na activity to the [Na](o) was approximately linear over the range 140-14 mM-[Na](o). When [Na](o) was reduced from 140 to 14 mM the internal Na activity fell by 72 +/- 5% (mean +/- S.D., n = 21).8. When the [Na](o) was reduced, the decrease in the internal Na activity was partially inhibited by Mn or by removal external Ca.9. When the [Ca](o) was altered over the range 0.2-16 mM the internal Na activity was reduced by approximately 50% for a tenfold increase in the [Ca](o).10. The relationship between internal Na and contractility is discussed.     

Accessibility of T-tubule vacuoles to extracellular dextran and DNA: mechanism and potential application of vacuolation

In addition to its function in excitation–contraction coupling, the ability of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. The mechanism of reversible vacuolation has been investigated by confocal microscopy using fluorescein dextran to probe the accessibility of T-tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60–100mm glycerol for 30 minutes and then returning them to glycerol-free medium. Devacuolation was subsequently induced by the reentry of glycerol. During their formation from T-tubules, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. However, it is apparent from the present work that, although the tubules of the treated fibres are constricted, the vacuoles maintain an open connection with the external medium for smaller macromolecules. In the light of these experiments, it is proposed that vacuolation is caused by water moving into T-tubules from the cytoplasm faster thanit can exit to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules has also been investigated. DNA penetrated into the vacuoles from the extracellular medium less well than dextran, but many vacuoles containing fluorescent DNA were observed in the superficial layers of vacuolated fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by direct injection.This revised version was published online in September 2005 with corrections to the Cover Date.     

Membrane calcium current in ventricular myocardial fibres

1. A slow inward current in ventricular preparations of the dog heart can be measured by the voltage clamp method without interference from the initial rapid sodium current if the sodium system is inactivated by conditioning depolarization.2. The slow inward current is very sensitive to variation in [Ca](o). It occurs above the equilibrium potential of I(Na) immediately after changing the bathing fluid to a sodium-free solution and persists under this condition for a long time without much alteration, while I(Na) is rapidly abolished. Tetrodotoxin and [Mg](o) have no effect on this current component. These results strongly support the view that the slow inward current in cardiac tissue is carried by calcium ions.3. The threshold for initiation of the calcium current is around -35 mV in Tyrode solution and is shifted to more negative potentials by either increasing [Ca](o) or reducing [Na](o).4. Calcium sensitive inward current tails associated with repolarization are assumed to represent a proportional measure of calcium conductance activated during the preceding depolarization. Calcium conductance declines rapidly with time in the inside negative potential range and slowly at positive potentials. The time constants for this ;inactivation' process vary between 40 and 700 msec in the potential range -35 to +50 mV.5. By using instantaneous current-voltage relations the reversal potential of calcium current was estimated to be about +60 mV in normal Tyrode solution. As shown in the Appendix, however, the calcium equilibrium potential cannot be considered to be constant.6. The importance of the calcium current for the plateau of the cardiac action potential is discussed.     

Accumulation of caesium and rubidium in vivo by red and white muscles of the rat

1. Rats were given drinking water containing either 20 mM-CsCl or 20 mM-RbCl for a period of 2 weeks. Samples of blood were then taken from the rats under anaesthetic. They were immediately centrifuged and the plasma taken for analysis. Soleus muscles, diaphragm, extensor digitorum longus, white gastrocnemius and vastus lateralis muscles were then taken from the dead animals and these and the plasma were analysed for potassium, and for caesium or rubidium by means of the flame photometer.2. The concentrations of potassium and rubidium or caesium in the fibre water of these various muscles and in the samples of plasma water were then calculated.3. It was found that the red muscles including soleus and diaphragm generally tended to accumulate caesium and rubidium to a greater extent than did the white muscles such as the white gastrocnemius and vastus lateralis.4. When the concentration ratio [K](i)/[K](o) was divided into the ratio [Rb](i)/[Rb](o) for the different muscles, values of about 1.3 were obtained for the red muscles compared with values about 1.14 for white muscles.5. When in the case of the caesium-treated rats the ratio [K](i)/[K](o) was divided into the ratio [Cs](i)/[Cs](o) values ranged from 1.94 +/- 0.12 for the red soleus to 1.08 +/- 0.09 for the white gastrocnemius.6. When these values in the caesium-treated animals were plotted against the percentage of red fibres in the five muscle types (as obtained from the data of Sreter & Woo, 1963) the graph indicated that the white fibres had similar ionic gradients for Cs(+) and K(+) and that affinity for Cs(+) was confined to the red fibres.7. The membrane potential measured in soleus and extensor muscles immersed in plasma from the same animal was not significantly different from E(K) but was much less than E(Cs).8. These results are interpreted in terms of permeability differences between the slow red fibres and white twitch fibres.     

Local activation of frog muscle fibres with linearly rising currents

1. Single fast muscle fibres isolated from the semitendinosus muscles of the frog were locally activated by applying linearly rising current pulses to a pipette whose tip (diameter, 10-40 mu) was in contact with the fibre surface.2. In normal or choline-Ringer solution with 1.8 mM-Ca, the threshold depolarization for producing a just perceptible local contraction was nearly constant over a wide range of the duration of linearly rising currents (0.5-20 sec or more).3. If [Ca](o) was reduced to 0.25-0.1 mM, the threshold depolarization increased steeply with the increase in the duration of linearly rising currents, indicating a marked increase in the rate of an accommodation process in excitation-contraction coupling mechanism.4. In the low-Ca media, the threshold depolarization was observed to rise exponentially during the application of a linearly rising current of 3-10 sec duration, and to return exponentially to its initial value within 3-10 sec after the removal of the current.5. The marked accommodation in the low-Ca media was partially inhibited by the addition of Mg (5-10 mM), and completely eliminated in the presence of caffeine (1 mM), suggesting that the accommodation process may occur in the link between membrane depolarization and release of Ca from sarcoplasmic reticulum.6. Microscopic observation of local contractions in the low-Ca media indicated that the accommodative rise of threshold depolarization was most marked at the superficial layer of the fibre.7. From these results, it is suggested that the removal of bound Ca or some other kind of charged particle from the transverse tubular system or from its vicinity might be the cause of accommodation in excitation-contraction coupling.     

Reversible vacuolation of the transverse tubules of frog skeletal muscle: a confocal fluorescence microscopy study

Summary A confocal microscope was used to investigate the reversible vacuolation of frog skeletal muscle fibres produced by the efflux and entry of glycerol (80–100 mm). The formation, development and disappearance of vacuoles was observed in the fibres by staining simultaneously with two fluorescent membrane probes, RH414 and DiOC₆(3). The styryl dye, RH414, stains only the plasmalemma and the membranes of the transverse tubules. In normal and glycerol-loaded fibres, RH414 revealed regular, narrow dotted bands located at the position of the Z-lines. Glycerol removal produced, within 2–10 min, many empty round vacuoles (0.4–1.5 m in diameter) that were apparently anchored to the stained bands. Later on, individual vacuoles tended to enlarge and align into longitudinal chains of vacuoles. Neighbouring vacuoles that contacted each other fused to form large vacuoles up to several sarcomeres in length. Neither the T-tubules, nor the vacuoles, were stained by DiOC₆(3). However, glycerol efflux was also accompanied by a redistribution of sarcoplasmic reticulum membranes and by changes in mitochondria that were revealed on staining the same fibres with the carbocyanine dye, DiOC₆(3). The alterations in staining patterns revealed by RH414 and DiOC₆(3) were completely reversible. Within 5–10 min after a second application of glycerol, the pattern of staining returned to normal with the exception of very bright, spots stained with RH414, which appeared in place of many but not all of the vacuoles, and probably correspond to the irregular nets of T-tubules observed under the electron microscope in such fibres. They are considered to be defects in regeneration of the T-system after vacuolation. The vacuolation/devacuolation cycle could be repeated several times following glycerol efflux and entry. The development and disappearance of vacuoles then mainly involved conversion of bright spots to large vacuoles and vice versa. Some possible mechanisms of vacuole formation and disappearance are discussed, and it is suggested that vacuolation of the T-system may be important in relation to regulating the volume of skeletal muscle cells.     

Differential effects of glycerol treatment on membrane capacity and excitation-contraction coupling in toad sartorius fibres

Changes in membrane capacity and excitation-contraction coupling caused by glycerol movements have been investigated in toad sartorius fibres using a standard glycerol-Ringer solution containing 400 mM glycerol.1. The rates of glycerol movement, in and out of fibres, were determined by measuring diameter changes in single fibres. Glycerol equilibrated across the surface membrane within 20-25 min after changes in extracellular glycerol concentration.2. The reduction in membrane capacity, which occurs when glycerol-loaded fibres are returned to Ringer solution, was slower than, and not dependent on, changes in fibre volume.3. To obtain the maximum reduction in membrane capacity, it was necessary to expose fibres to glycerol-Ringer for 50-60 min and then to return them to Ringer for at least 60 min. If preparations were not kept in Ringer for the full 50-60 min, the reduction in membrane capacity could be partially or completely reversed by returning the fibres to glycerol-Ringer.4. When fibres were exposed to glycerol-Ringer there was an initial transient contracture: twitches and K contractures were rapidly inhibited, and then slowly recovered over the next 40-50 min. In some preparations, eventual potentiation of twitches was seen.5. When returned to Ringer solution after 60 min in glycerol-Ringer, preparations lost twitches and K contractures within 5-10 min. The time course of this effect was very similar to the time course of the recovery of the normal volume after the initial swelling.6. The briefer the exposure to the glycerol-Ringer, the slower the loss of twitches and K contractures on return to Ringer. In contrast to the loss of membrane capacity, the lesion of excitation-contraction coupling was essentially irreversible. Exposure times as brief as 5 min were eventually effective in producing paralysed fibres which, however, still responded to caffeine.7. The differences in the glycerol load-times required to produce decoupling of excitation and contraction, and disconnexion of the transverse tubules, show that the two effects are separable and that the first does not depend on the second.8. It is proposed that the two lesions obtained in glycerol-treated fibres may be related to distension of the transverse tubular system. It is shown in an Appendix that the outward movement of glycerol from sarcoplasm to transverse tubules would be expected to produce some early swelling of the tubules and this is related to the loss of contraction. Furthermore, much greater swelling would occur if a slow-loading compartment (presumed to be the sarcoplasmic reticulum) dumped glycerol into the transverse tubules: it is thought that this is related to the disconnexion of the transverse tubules.