SUMMARY1. 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.2. We tested this hypothesis by recording tension in isolated muscle fibres stimulated tetanically (up to 60 shocks/sec). In low [Na+] solutions, output tension was initially similar to that in normal Ringer, but then fell smoothly to a substantially lower value. 3. The activity of individual myofibrils was recorded directly with cinemicrographs during isotonic contractions while the fibres were stimulated at high frequencies. In low [Na+]o wavy myofibrils appeared in the centre of the fibre and spread towards the periphery, indicating failure of activation. Wavy myofibrils never appeared in normal Ringer.4. Intracellular action potentials recorded during the tetanic stimulation indicated that the inactivated myofibrils present in low [Na+] solutions cannot be explained by the changes in size and duration of the action potential. 5. Our results strongly suggest the existence of a regenerative Na+ conductance in the tubular membrane during the inward spread of an excitatory process.
Tetrodotoxin (TTX) binding was measured in muscles which were either in normal condition or which had been "detubulated" by glycerol-induced osmotic shock. In both cases the binding of TTX was found to saturate at high TTX concentrations. Maximum binding in normal fibers was 35 pmol/g wet weight, and that figure was reduced to 16 pmol/g after glycerol treatment. The dissociation constant for binding to the surface membrane was 3 nM, which is the range of values obtained by electrophysiological measurements of the effect of TTX on the maximum rate of rise of the action potential. The results suggest that the dissociation constant in the transverse tubules may be higher than that in the surface. Control experiments indicate that the effects of glycerol treatment are limited to the accessibility of the receptors to the toxin and result in no alteration of the affinity of the binding site. TTX receptors in the transverse tubules may be recovered after glycerol treatment by homogenization of the fibers. The measurements suggest that the density of sodium channels in surface membrane is about 175/muM2 and that in the transverse tubular membrane is 41-52/mum2.
The binding of the cardiosteroid 3H-ouabain to frog skeletal muscle was determined by studying the kinetics of its uptake and release. The amount of ouabain bound as a function of drug concentration in the external medium follows a hyperbolic relationship with a maximum binding (Bmax) of the order of 2500 molecules per square micrometer of surface membrane and an affinity constant (K) of 2.2 X 10(-7)M. The data do not suggest a drug-receptor (Na pump site) relation other than one-to-one. Ouabain molecules are released from whole muscle into ouabain-free media very slowly. The release is a single exponential function of time (tau approximately equal to 25 hr). When re-binding is prevented by the presence of unlabeled ouabain in the external medium, the loss of labeled ouabain is increased (tau approximately equal to 15 hr). Increasing [K+]O from 2.5 to 10 mM slows the time course of binding without any significant change in binding capacity of the muscle fibers. Experiments on detubulated muscles indicate that the density of pump sites is considerably higher in the surface than in the T-tubular membrane. These findings agree with the report by Narahara et al. [Narahara, H.T., Vogrin, V.G., Green, J.D., Kent, R.A., Gould, M.K. (1979) Biochim. Biophys. Acta 552:247] on the distribution of (Na+ + K+)- ATPase among different cell membrane fractions from frog skeletal muscle.
The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na+ transport (Venosa, R.A., 1978, Biochim. Biophys. Acta 510:378-383), promoted changes in (i) active K+ influx, (ii) passive Na+ and K+ fluxes, and (iii) the number of 3H-ouabain binding sites. The results indicate that a reduction of external osmotic pressure (pi) to one-half of its normal value (pi = 0.5) produced the following effects: (i) an increase in active K+ influx on the order of 160%, (ii) a 20% reduction in Na+ influx and K+ permeability (PK), and (iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na+ pump is not caused by an increased leak of either Na+ (inward) or K+ (outward). It is unlikely that the stimulation of active Na+ extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na+ transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance pi = 2 to pi = 1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na+ and K+ fluxes were not significantly altered by hypertonicity (pi = 2).
SUMMARY1. Paired frog sartorius muscles were exposed to Ringer solutions labelled with 22Na+ for about 20 min. At the end of this exposure one of them was stimulated supramaximally one hundred to two hundred times. Immediately after the stimulation both members of the pair were washed in a series of tubes filled with a Na+-free medium containing 3 x 10-5 M strophanthidin.2. Under the above conditions the intracellular component of the efflux was exponential with an average time constant (r) of 388 min, that is, approximately four times longer than in the presence of normal Ringer. On the other hand the mean r for the washout of the interfibre space was 3-2 min. 3. From the extrapolation to time zero of the intracellular component of the washout curve the initial intracellular radioactivity of both muscles was obtained and the resting and extra Na+ influx were calculated.4. The mean surface membrane area/muscle weight ratio was found to be 552 cm2.
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