Amphibian Skeletal Muscle Research Articles

Detubulation abolishes membrane potential stabilization in amphibian skeletal muscle

A recently reported stabilization ('splinting') of the resting membrane potential ( Em) observed in amphibian skeletal muscle fibres despite extracellular hyperosmotic challenge has been attributed to high resting ratios of membrane Cl- to K+ permeability ( P Cl/ P K) combined with elevations of their intracellular Cl- concentrations, [Cl-]i, above electrochemical equilibrium by diuretic-sensitive cation-Cl-, Na-Cl (NCC) and/or Na-K-2Cl (NKCC), co-transporter activity. The present experiments localized this co-transporter activity by investigating the effects of established detubulation procedures on Em splinting. They exposed fibres to introduction and subsequent withdrawal of 400 mM extracellular glycerol, high divalent cation concentrations, and cooling. An abolition of tubular access of extracellularly added lissamine rhodamine fluorescence, visualized by confocal microscopy, and of the action potential afterdepolarization together confirmed successful transverse (T-) tubular detachment. Fibre volumes, V , of such detubulated fibres, determined using recently introduced confocal microscope-scanning methods, retained the simple dependence upon 1/[extracellular osmolarity], without significant evidence of the regulatory volume increases described in other cell types, previously established in intact fibres. However detubulation abolished the Em splinting shown by intact fibres. Em thus varied with extracellular osmolarity in detubulated fibres studied in standard, Cl(-)-containing, Ringer solutions and conformed to simple predictions from such changes in assuming that intracellular ion content was conserved and membrane potential change DeltaEm was principally determined by the K+ Nernst potential. Furthermore, cation--Cl- co-transport block brought about by [Cl-]o or [Na+]o deprivation, or inclusion of bumetanide (10 microM) and chlorothiazide (10 microM) in the extracellular fluid gave similar results. When taken together with previous reports of significant Cl- conductances in the surface membrane, these findings suggest a model that contrastingly suggests a T-tubular location for cation--Cl- co-transporter activity or its regulation.

Detubulation experiments localise delayed rectifier currents to the surface membrane of amphibian skeletal muscle fibres.

Ionic currents in intact and detubulated frog sartorius muscle fibres were compared at room temperature using a loose-patch voltage clamp configuration in four experimental groups. The test fibres (i) were detubulated by a previously established osmotic shock protocol that involved the introduction and withdrawal of extracellular glycerol followed by exposure to Ca2+/Mg2+-Ringer solution and cooling. The control fibres were spared osmotic shock and (ii) simply studied in normal Ringer solution, (iii) exposed to 30 min of steady cooling to 9-10 degrees C before electrophysiological study or (iv) exposed to and studied in glycerol-Ringer solution. The presence or absence of detubulation was confirmed for all the experimental groups through assessing for the abolition or otherwise of the delayed after-depolarisation normally associated with action potential propagation into the transverse (T) tubules. All fibre groups showed similar resting potentials (-80 to -90 mV) thus ensuring consistent baseline voltages from which the voltage clamp steps were imposed. The intact muscle fibres in the three control groups (ii)-(iv) spared osmotic shock showed both inward Na+ and delayed rectifier outward (K+) currents. In contrast, patches from detubulated muscle fibres in the test group (i) showed only delayed outward currents, consistent with contrasting contributions to Na+ and K+ currents from regions of membrane affected or spared by the detubulation procedure. Nevertheless, the voltage dependence, maximum steady state amplitudes and timecourses of the delayed outward currents were conserved through all the experimental groups. These findings suggest that the surface as opposed to the tubular membrane contributes the greater part of the delayed rectifier current in amphibian skeletal muscle.

Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions.

This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (E(m)) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of E(m) despite cell shrinkage, which was attributable to elevation of intracellular [Cl(-)] above electrochemical equilibrium through Na(+)-Cl(-) and Na(+)-K(+)-2Cl(-) cotransporter action following exposures to extracellular hypertonicity. Fibre volumes (V) determined by confocal microscope x z - scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl(-)-free, normal and Na(+)-free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady-state resting potentials (E(m)). Fibres in isotonic Cl(-)-free, normal and Na(+)-free Ringer solutions showed similar E(m) values consistent with previously reported permeability ratios P(Na)/P(K)(0.03-0.05) and P(Cl)/P(K) ( approximately 2.0) and intracellular [Na(+)], [K(+)] and [Cl(-)]. Increased extracellular osmolarities produced hyperpolarizing shifts in E(m) in fibres studied in Cl(-)-free Ringer solution consistent with the Goldman-Hodgkin-Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such E(m) shifts, suggesting a Cl(-)-dependent stabilization of membrane potential. This stabilization of E(m) was abolished by withdrawing extracellular Na(+) or by the combined presence of the Na(+)-Cl(-) cotransporter (NCC) inhibitor chlorothiazide (10 microM) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) inhibitor bumetanide (10 microM), or the Na(+)-K(+)-ATPase inhibitor ouabain (1 or 10 microM) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a hyperpolarization after a time delay, as expected for passive Cl(-) equilibration. These findings suggest a model that implicates the NCC and/or NKCC in fluxes that maintain [Cl(-)](i) above its electrochemical equilibrium. Such splinting of [Cl(-)](i) in combination with the high P(Cl)/P(K) of skeletal muscle stabilizes E(m) despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for regulatory volume increases (RVIs). In situations where P(Cl)/P(K) is low, the same co-transporters would instead permit RVIs but at the expense of a capacity to stabilize E(m).

Static and Dynamic X-Ray Diffraction Recordings from Living Mammalian and Amphibian Skeletal Muscles

Static and time-resolved two-dimensional x-ray diffraction patterns, recorded from the living mouse diaphragm muscle, were compared with those from living frog sartorius muscle. The resting pattern of mouse muscle was similar to that of frog muscle, and consisted of actin- and myosin-based reflections with spacings basically identical to those of frog. As a notable exception, the sampling pattern of the myosin layer lines (MLL's) indicated that the mouse myofilaments were not organized into a superlattice as in frog. The intensity changes of reflections upon activation were also similar. The MLL's of both muscles were markedly weakened. Stereospecific (rigorlike) actomyosin species were not significantly populated in either muscle, as was evidenced by the 6th actin layer line (ALL), which was substantially enhanced but without a shift in its peak position or a concomitant rise of lower order ALL's. On close examination of the mouse pattern, however, a few lower order ALL's were found to rise, slightly but definitely, at the position expected for stereospecific binding. Their quick rise after the onset of stimulation indicates that this stereospecific complex is generated in the process of normal contraction. However, their rise is still too small to account for the marked enhancement of the 6th ALL, which is better explained by a myosin-induced structural change of actin. Since the forces of the two muscles are comparable regardless of the amount of stereospecific complex, it would be natural to consider that most of the force of skeletal muscle is supported by nonstereospecific actomyosin species.

Unitary Ca2+ current through mammalian cardiac and amphibian skeletal muscle ryanodine receptor Channels under near-physiological ionic conditions.

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1–30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (∼55%) in mammalian and amphibian channels. Two amplitudes, differing by ∼35%, were found in amphibian channel studies, probably corresponding to α and β RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (∼40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.

Comparison between the Predictions of Diffusion-Reaction Models and Localized Ca 2+ Transients in Amphibian Skeletal Muscle Fibers

We developed a three-dimensional cylindrical diffusion-reaction model of a single amphibian myofibril in which Ca 2+ release occurred only at the Z-line. The model incorporated diffusion of Ca 2+, Mg 2+, and all relevant buffer species, as well as the kinetic binding reactions between the buffers and appropriate ions. Model data was blurred according to a Gaussian approximation of the point spread function of the microscope and directly compared with experimental data obtained using the confocal spot methodology. The flux parameters were adjusted until the simulated Z-line transient matched the experimental one. This model could not simultaneously predict key parameters of the experimental M- and Z-line transients, even when model parameters were adjusted to unreasonably extreme values. Even though the model was accurate in predicting the Z-line transient under conditions of high [EGTA], it predicted a significantly narrower Ca 2+ domain than observed experimentally. We modified the model to incorporate a broader band of release centered at the Z-line. This extended release model was superior both in simultaneously predicting critical features of the Z- and M-line transients as well as the domain profile under conditions of high [EGTA]. We conclude that a model of release occurring exclusively at the Z-line cannot explain our experimental data and suggest that Ca 2+ may be released from a broader region of the sarcoplasmic reticulum than just the T-tubule-sarcoplasmic reticulum junction.

The effect of extracellular tonicity on the anatomy of triad complexes in amphibian skeletal muscle.

Ultrastructural features of tubular-sarcoplasmic (T-SR) triad junctions and measures of cell volume following graded increases of extracellular tonicity were compared under physiological conditions recently shown to produce spontaneous release of intracellularly stored Ca2+ in fully polarized amphibian skeletal muscle fibres. The fibres were fixed using solutions of equivalent tonicities prior to processing for electron microscopy. The resulting anatomical sections demonstrated a partially reversible cell shrinkage corresponding to substantial increases in intracellular solute or ionic strength graded with extracellular tonicity. Serial thin sections through triad structures confirmed the presence of geometrically close but anatomically isolated transverse (T-) tubular and sarcoplasmic reticular (SR) membranes contrary to earlier suggestions for the development of luminal continuities between these structures in hypertonic solutions. They also quantitatively demonstrated accompanying decreases in T-SR distances, increased numbers of sections that showed closely apposed T and SR membranes, tubular luminal swelling and reductions in luminal volume of the junctional SR, all correlated with the imposed increases in extracellular osmolarity. Fully polarized fibres correspondingly showed elementary Ca(2+)-release events ('sparks', in 100 mM-sucrose-Ringer solution), sustained Ca2+ elevations and propagated Ca2+ waves (> or = 350-500 mM sucrose) following exposure to physiological Ringer solutions of successively greater tonicities. These were absent in hypotonic, isotonic or less strongly hypertonic (approximately 50 mM sucrose-Ringer) solutions. Yet exposure to hypotonic solutions also disrupted T-SR junctional anatomy. It increased the tubular diameters and T-SR distances and reduced their area of potential contact. The spontaneous release of intracellularly stored Ca2+ thus appears more closely to correlate with the expected changes in intracellular solute strength or a reduction in absolute T-SR distance rather than disruption of an optimal anatomical relationship between T and SR membranes taking place with either increases or decreases in extracellular tonicity.

Differential effects of sarcoplasmic reticular Ca(2+)-ATPase inhibition on charge movements and calcium transients in intact amphibian skeletal muscle fibres.

A hypothesis in which intramembrane charge reflects a voltage sensing process allosterically coupled to transitions in ryanodine receptor (RyR)-Ca(2+) release channels as opposed to one driven by release of intracellularly stored Ca(2+) would predict that such charging phenomena should persist in skeletal muscle fibres unable to release stored Ca(2+). Charge movement components were accordingly investigated in intact voltage-clamped amphibian fibres treated with known sarcoplasmic reticular (SR) Ca(2+)-ATPase inhibitors. Cyclopiazonic acid (CPA) pretreatment abolished Ca(2+) transients in fluo-3-loaded fibres following even prolonged applications of caffeine (10 mM) or K(+) (122 mM). Both CPA and thapsigargin (TG) transformed charge movements that included delayed (q(gamma)) "hump" components into simpler decays. However, steady-state charge-voltage characteristics were conserved to values (maximum charge, Q(max) approximately equal to 20-25 nC microF(-1); transition voltage, V* approximately equal to -40 to-50 mV; steepness factor, k approximately equal to 6-9 mV; holding voltage -90 mV) indicating persistent q(gamma) charge. The features of charge inactivation similarly suggested persistent q(beta) and q(gamma) charge contributions in CPA-treated fibres. Perchlorate (8.0 mM) restored the delayed kinetics shown by "on" q(gamma) charge movements, prolonged their "off" decays, conserved both Q(max) and k, yet failed to restore the capacity of such CPA-treated fibres for Ca(2+) release. Introduction of perchlorate (8.0 mM) or caffeine (0.2 mM) to tetracaine (2.0 mM)-treated fibres, also known to restore q(gamma) charge, similarly failed to restore Ca(2+) transients. Steady-state intramembrane q(gamma) charge thus persists with modified kinetics that can be restored to its normally complex waveform by perchlorate, even in intact muscle fibres unable to release Ca(2+). It is thus unlikely that q(gamma) charge movement is a consequence of SR Ca(2+) release rather than changes in tubular membrane potential.

Separation of detubulation and vacuolation phenomena in amphibian skeletal muscle.

Sartorius muscle fibres from cold-adapted Rana temporaria were exposed to variants of an established detubulation procedure (Koutsis et al. (1995) J Muscle Res Cell Motil 16, 519-528) to test the extent to which detubulation and tubular vacuolation phenomena could be separated using different conditions of osmotic shock. A control procedure was optimised to a 28-min exposure to 400 mM glycerol-Ringer. This was followed by a recovery step involving its replacement by a Ca2+/Mg(2+)-Ringer solution and steady cooling over 30 min from room temperature (approximately 18 degress C) to approximately 10 degress C, followed by the restoration of the normal Ringer solution. This procedure successfully abolished the action potential after-depolarisation component, reflecting a loss of tubular conduction ('detubulation') in 74.3 +/- 5.9% of the fibres studied. Omitting the cooling during the recovery step sharply reduced the incidence of detubulation. So did omitting either the high-[Ca2+] and/or [Mg2+] in the recovery solutions in test procedures, but to significantly different extents (P < 5%). Yet trapping of fluorescent Sulfhorhodamine B dye in 'closed' vacuoles persisted albeit with reduced proportions of fibre volume occupied by vacuoles. Furthermore, the variations in recovery conditions produced similar levels of vacuolation despite smaller vacuole sizes in the cooled fibres (P < 0.05). These findings demonstrate that fibre vacuolation and detubulation are phenomena that are potentially separable through varying the conditions of osmotic shock, with detubulation requiring significantly more stringent conditions than vacuolation.

Normal conduction of surface action potentials in detubulated amphibian skeletal muscle fibres.

1. The influence of the transverse (T) tubules on surface action potential conduction was investigated by comparing electrophysiological and confocal microscopic assessments of tubular changes in osmotically shocked and control fibres from frog sartorius muscle. 2. The membrane-impermeant fluorescent dye, di-8-ANEPPs spread readily from the bathing extracellular solution into the tubular membranes in control, intact fibres. Prior exposure of muscles to a hypertonic glycerol-Ringer solution, its replacement by an isotonic Ca(2+)-Mg(2+) Ringer solution and cooling sharply reduced such access. In contrast, dye application in the course of this osmotic shock procedure stained the large tubular vacuoles hitherto associated with successful muscle detubulation. 3. Conduction velocities in intact, control fibres (1.91 +/- 0.048 m s(-1), mean +/- S.E.M., n = 32 fibres) agreed with earlier values reported at room temperature (18-21 degrees C) and were unaffected by prior episodes of steady cooling to 8-10 degrees C (1.91 +/- 0.043 m s(-1), n = 30). 4. Cooling to 11.5 degrees C reduced these velocities (1.47 +/- 0.081 m s(-1), n = 25) but action potential waveforms still included early overshoots and the delayed after-depolarizations associated with tubular electrical activity. 5. In contrast, action potentials from cooled, superficial fibres in osmotically shocked muscles lacked after-depolarization phases implying tubular detachment. Their mean conduction velocities (1.62 +/- 0.169 m s(-1), n = 25) were not significantly altered from values obtained in untreated controls or in intact fibres in muscle similarly treated with glycerol, in direct contrast to earlier results. 6. Cooling produced similar reductions in maximum rates of voltage change dV/dt in action potentials from all fibre groups with lower rates of change shown by detubulated fibres. 7. Use of an antibody to a conserved epitope of the alpha-subunit of voltage-gated sodium channels suggested a concentration of sodium channels close to the mouths of the T tubules. 8. These electrophysiological and anatomical findings are consistent with a partial independence of electrical events in the transverse tubules from those responsible for the rapid conduction of surface regenerative activity. 9. The findings are discussed in terms of a partial separation of the electrical activity propagated over the surface membrane, from the initiation of propagated activity within the T tubules, by the triggering of the sodium channels clustered selectively around the mouths of the T tubules.

Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release.

Caffeine (4 mM) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl--free (SO(4)2-) medium] substantially increased, and both Na+-free medium and Ni2+ (5 mM) reduced, the caffeine-induced depolarization. In voltage-clamped (-60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 +/- 0.48 microA cm(-2). This caffeine-induced current was reduced by 60% in Na+-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni2+, suggesting that the activation of the Na+-Ca2+ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K+. Net K+ efflux increased from 3.5 +/- 0.2 (control) to 22.1 +/- 2.3 pmol s(-1) cm(-2) (caffeine). It is shown that in the presence of the drug, [K+] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K+ efflux was not reduced by 5 mM Ni2+. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni2+. The Ni2+-sensitive fraction of the caffeine-induced current, assumed to represent the Na+-Ca2+ exchanger current, had an estimated reversal potential close to 12 mV ([Na+]o = 115 mM; [Ca2+]o = 1 mM). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na+-Ca2+ exchanger in its forward mode, and (b) the rise of the external [K+] in restricted spaces like the T tubules.

Charge movements in intact amphibian skeletal muscle fibres in the presence of cardiac glycosides.

1. Intramembrane charge movements were examined in intact voltage-clamped amphibian muscle fibres following treatment with cardiac glycosides in the hypertonic gluconate-containing solutions hitherto reported to emphasise the features of q(gamma) at the expense of q(beta) charge. 2. The application of chlormadinone acetate (CMA) at concentrations known selectively to block Na(+)-K(+)-ATPase conserved the steady-state voltage dependence of intramembrane charge, contributions from delayed (q(gamma)) charging transients, and their inactivation characteristics brought about by shifts in holding potential. 3. The addition of either ouabain (125, 250 or 500 nM) or digoxin (5 nM) at concentrations previously reported additionally to influence excitation-contraction coupling similarly conserved the steady-state charge-voltage relationships, Q(V), in fully polarised fibres to give values of maximum charge, Q(max), transition voltage, V*, and steepness factor, k, that were consistent with a persistent q component as reported on earlier occasions (Q(max) approximately = 25-27 nC F-1, V* approximately = -45 to -50 mV, k approximately = 7-9 mV). 4. In both cases shifts in holding potential from -90 to -50 mV produced a partial inactivation that separated steeply and more gradually voltage-dependent charge components in agreement with previous characterisations. 5. However, charge movements that were observed in the presence of either digoxin or ouabain were monotonic decays in which delayed (q(gamma)) transients could not be distinguished from the early charging records. These features persisted despite the further addition of chlormadinone acetate over a 10-fold concentration range (5-50 microM) known to displace ouabain from the Na(+)-K(+)-ATPase. 6. Ouabain (500 nM) restored the steady-state charge movement that was previously abolished by the addition of 2.0 mM tetracaine in common with previous results of using ryanodine receptor (RyR)-specific agents. 7. Perchlorate (8.0 mM) restored the delayed 'on' relaxations and increased the prominence of the 'off' decays produced by q(gamma) charge following treatment with cardiac glycosides. This was accompanied by a negative (approximately 10-15 mV) shift in the steady-state charge-voltage relationship but an otherwise conserved maximum charge, Q(max), and steepness factor, k, in parallel with previously reported effects of perchlorate following treatments with RyR-specific agents. 8. The features of cardiac glycoside action thus parallel those of other agents that act on RyR-Ca(2+) release channels yet influence the kinetics but spare the steady-state properties of intramembrane charge.

Role of Calcium Currents in E-C Coupling and Evolution of the T-System and SR in Developing Cultured Embryonic Amphibian Skeletal Muscle Cells

Abstract Adult frog phasic skeletal muscle cells have slow inward calcium current (ICa) (Stanfield, 1977) carried through L-type voltage dependent calcium channels. It has been suggested that ICa may play a role in E-C coupling (Cota &amp; Stefani, 1981, 1989). However, phasic skeletal muscle cells contract for several minutes after the extracellular Ca2+ concentration ([Ca2+]o) is lowered to &amp;lt;10-8 M (Armstrong et al, 1972). Therefore, extracellular Ca2+ (Ca2+o) is not essential for contraction in these fibers. It has been also shown, by blocking Ica that Ica is not essential for triggering contraction (Gonzalez-Serratos et al., 1982). These results have led to the conclusion that Ica has no obvious role in E-C coupling in adult amphibian phasic skeletal muscle. The question arises then as to what is the biological role Ica in phasic skeletal muscle? We have observed that embryonic skeletal muscle cells are capable of contracting during the first day of development in culture (Cordoba-Rodriguez, et al., 1996), before the T-system and the sarcoplasmic reticulum (SR)may have fully developed (Flucher, et al., 1993).

Sodium/calcium exchange in amphibian skeletal muscle fibers and isolated transverse tubules

The Na(+)/Ca(2+) exchanger participates in Ca(2+) homeostasis in a variety of cells and has a key role in cardiac muscle physiology. We studied in this work the exchanger of amphibian skeletal muscle, using both isolated inside-out transverse tubule vesicles and single muscle fibers. In vesicles, increasing extravesicular (intracellular) Na(+) concentration cooperatively stimulated Ca(2+) efflux (reverse mode), with the Hill number equal to 2.8. In contrast to the stimulation of the cardiac exchanger, increasing extravesicular (cytoplasmic) Ca(2+) concentration ([Ca(2+)]) inhibited this reverse activity with an IC(50) of 91 nM. Exchanger-mediated currents were measured at 15 degrees C in single fibers voltage clamped at -90 mV. Photolysis of a cytoplasmic caged Ca(2+) compound activated an inward current (forward mode) of 23 +/- 10 nA (n = 3), with an average current density of 0.6 muA/muF. External Na(+) withdrawal generated an outward current (reverse mode) with an average current density of 0.36 +/- 0.17 muA/muF (n = 6) but produced a minimal increase in cytosolic [Ca(2+)]. These results suggest that, in skeletal muscle, the main function of the exchanger is to remove Ca(2+) from the cells after stimulation.

Loop diuretics inhibit detubulation and vacuolation in amphibian muscle fibres exposed to osmotic shock.

The effect of loop diuretics at concentrations known to influence cellular water entry coupled to Na-K-Cl co-transport, upon the vacuolation and detubulation following osmotic shock, was investigated in amphibian skeletal muscles. These were exposed to a glycerol-Ringer solution (18 min), an isotonic Ca2+/Mg2+ Ringer solution and cooling. Adding bumetanide (1.0 and 2.0 microM) to these solutions sharply reduced the incidence of detubulation, assessed by abolition or otherwise of action potential after-depolarisations, from 93.9 +/- 4.7% (n = 6) to 5.0 +/- 1.1% (n = 4: mean +/- SEM: 2.0 microM bumetanide). It dramatically reduced the number and fraction of muscle volume occupied by tubular vacuoles, measured using confocal microscopy, from 60.3 +/- 4.3% (n = 10) to 9.0 +/- 1.1% (n = 35). The incidence of large horseradish peroxidase-lined tubular vacuoles, viewed using electronmicroscopy, similarly was reduced with 2 microM bumetanide in the glycerol-Ringer solution. Bumetanide acted through cellular volume adjustments early in the detubulation protocol. Thus, it exerted its maximum effect when added to the glycerol-Ringer, rather than the Ca2+/Mg2+ Ringer solution. Furthermore, whereas fibre diameters measured using scanning electron microscopy returned to normal during glycerol treatment relative to those of control fibres left in isotonic Ringer, addition of 2.0 microM bumetanide in the glycerol Ringer left markedly smaller fibre diameters. Finally equipotent concentrations of the chemically distinct loop diuretics. furosemide and ethacrynic acid similarly influenced detubulation. These findings implicate Na-K-Cl co-transport in the water entry into muscle fibres that would be expected following introduction of extracellular glycerol. This might then enable the subsequent Na-K-ATPase dependent water extrusion that produces the tubular distension (vacuolation) and detachment (detubulation) following glycerol withdrawal, phenomena also observed in muscular dystrophy.

SH Oxidation Stimulates Calcium Release Channels (Ryanodine Receptors) From Excitable Cells

The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors) of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal) modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub microM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed.

Contribution of Ryanodine Receptor Type 3 to Ca 2+ Sparks in Embryonic Mouse Skeletal Muscle

The kinetic behavior of Ca 2+ sparks in knockout mice lacking a specific ryanodine receptor (RyR) isoform should provide molecular information on function and assembly of clusters of RyRs. We examined resting Ca 2+ sparks in RyR type 3-null intercostal myotubes from embryonic day 18 (E18) mice and compared them to Ca 2+ sparks in wild-type (wt) mice of the same age and to Ca 2+ sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly smaller, briefer, and had a faster time to peak than sparks from wt cells (280 events) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (ΔF/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt embryonic sparks were considerably more variable in size and kinetics than sparks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca 2+ spark activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt embryonic and adult cells but not in RyR type 3-null cells. These results confirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR type 3-null cells is entirely consistent with observations by other investigators. There are three conclusions from this study: i) RyR type-1 engages in Ca 2+ spark activity in the absence of other RyR isoforms in RyR type 3-null myotubes; ii) Ca 2+ sparks with parameters similar to those reported in adult amphibian skeletal muscle can be detected, albeit at a low frequency, in adult mammalian skeletal muscle cells; and iii) a major contributor to the unusually large Ca 2+ sparks observed in normal (wt) embryonic muscle is RyR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR type 1 and RyR type 3 channels co-contribute to Ca 2+ release during the same spark and that Ca 2+ sparks undergo a maturation process which involves a decrease in RyR type 3.

Measurement of sarcoplasmic reticulum Ca2+content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro-m-cresol (4-CmC, 0.2– 5 mM) elevated [Ca2+]ireversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]iwas 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+content. Tetanic stimulations (100 Hz) increased [Ca2+]ifrom a resting level of 105 ± 47 nM (n= 10) to 1370 ± 220 nM (n= 6). Application of 2 mM 4-CmC produced a contracture that was 54 ± 16% (n= 6) of the tetanic force and elevated [Ca2+]ito a peak of 3520 ± 540 nM (n= 8). Both force and [Ca2+]ilevels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca2+-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 ± 10% (n= 4). The magnitude of the peak of the 4-CmC-induced Ca2+transient was not significantly changed by removal of extracellular Ca2+nor by inhibiting the SR Ca2+pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+content in the same fibre with 4-CmC without loss of normal muscle function.

Cardiac glycosides inhibit detubulation in amphibian skeletal muscle fibres exposed to osmotic shock.

It has recently been suggested that the 'vacuolation' of the transverse tubular system that follows the imposition of an osmotic shock is a component process in the eventual 'detubulation' of amphibian skeletal muscle. However, such a hypothesis requires net fluid transfers from the intracellular space into the lumina of the transverse tubules against the prevailing transmembrane osmotic gradients. The present experiments tested the effects of cardiac glycosides on the consequences of established osmotic protocols known reliably to achieve high levels of both detubulation and vacuolation in Rana temporaria sartorius muscle. Tubular isolation (detubulation) was assessed through electrophysiological observations of the abolition or otherwise of the after-depolarisation components of muscle action potentials. Vacuolation was assessed by stereological estimation of the volume fraction of muscle that was occupied by fluorescence-labelled vacuoles observed using confocal microscopy. Introduction of ouabain in the osmotic shock solutions sharply reduced such measures of vacuolation from 48.5 +/- 3.6% (mean +/- SEM; n = 70) to 12.1 +/- 2.7% (n = 190) of the total fibre volume. This was accompanied by sharp reductions in the incidence of detubulation (detubulation index reduced from 96.3 +/- 2.6% to 0.0 +/- 0.0%). The presence of ouabain was critical at the osmotic shock stage in the procedures at which the hypertonic glycerol-containing solutions were replaced by isotonic Ca(2+)-Mg(2+)-Ringer solutions. Finally, the alternative cardiac glycosides, strophanthidine and digoxin, exerted similar effects. These findings support a scheme in which the osmotic shock initiates a metabolically dependent fluid expulsion. This distends the transverse tubules into vacuoles that in turn lead to fibre detubulation.

Fast calcium removal during single twitches in amphibian skeletal muscle fibres.

Fluorescence signals from the calcium sensitive dyes Fluo-3 or Rhod-2 were obtained simultaneously with isometric tension in single fibres isolated from the anterior tibialis muscle of Leptodactylus insularis (20-22 degrees C). Fluo-3 fluorescence signals were transformed into [Ca2+]i transients as previously described. Most of the decay phase of single twitch transient is well fitted by a single exponential (tau of about 10 ms), followed by a slower declining component lasting tens of milliseconds. During short periods, 10 to 20 s, of low frequency stimulation, between 0.2 and 5 Hz, the basal [Ca2+]i increased slowly from 0.1 to about 0.4 microM, with only minor changes in the exponentially decaying phase. In fibres poisoned with thapsigargin or cyclopiazonic acid (1-2 microM) the tau of decay of fluorescence or Ca2+ transients of single twitches was very similar to that observed in non-poisoned fibres. Nevertheless, in poisoned fibres challenged with repetitive stimulation. the tau of Ca2+ transients decay increased from about 10 ms to >40 ms, while the basal [Ca2+]i increased from 0.1 to 2 microM. Short rest periods (about 5 min) could reverse these effects, indicating that they were not a direct consequence of SR Ca 2+ -ATPase inhibition. The correlation coefficient between tau of decay and basal [Ca2+]i was >0.8 (P<0.0001). Qualitatively similar results were obtained measuring Rhod-2 fluorescence signals. A lumped, two-compartment model could account for these results. Loading the fibres with EGTA-AM, diminished the effects of prolonged stimulation observed in poisoned fibres. Moreover, we show that the Na+ - Ca2+ exchange mechanism does not participate appreciably in fast Ca2+ removal.