Protein kinases A and C stimulate the Na+ active transport in frog skeletal muscle without an appreciable change in the number of sarcolemmal Na+ pumps.

AIM: The activation of both protein kinases A (PKA) and protein kinases C (PKC) in some cell types increases and in others reduces active Na+ efflux. These effects have been ascribed to either a change in the rate of ionic translocation by a fixed number of Na+ pumps or, a change in the number of plasma membrane pumps. The purpose of the present experiments was to study the effect of activating PKA and PKC on the Na+ extrusion by the Na+ pump in frog skeletal muscle. METHODS: Na+ (22Na+) fluxes and ouabain (3H-ouabain) binding were measured in frog sartorius muscles. RESULTS: Both activation of PKA and PKC increased the active Na+ extrusion by a factor of two; these effects were not additive. Ouabain binding experiments indicated that the pump stimulation by activation of these kinases is not associated with any significant increase in the number of plasma membrane pumps. Stimulation of the active Na+ efflux by protein kinase activation (no change in the number of sarcolemmal pumps) and by hypotonicity (increase in the number of pumps) could be elicited in the same preparation and they were additive. CONCLUSION: It is concluded that in frog skeletal muscle fibres, (1) activation of both PKA and PKC stimulate the Na+ pump by increasing its rate of ionic translocation; and (2) two modes of Na+ active transport (with and without an increase in the number of pumps) are operative, and can be at work simultaneously, a phenomenon to be reckoned with.

Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton.

Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (pi) from its normal value (pi = 1) to one-half (pi = 0.5), but also in muscles that are returned to pi = 1 after equilibration in pi = 2 medium. The equilibration in pi = 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+-K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 microM). Under those conditions, the pi = 1 pi = 0.5 transfer produced a 43 % increase in pump sites, while the pi = 2 pi = 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 microM) and intermediate filaments by acrylamide (4 mM) did not alter the hypotonic effect. Likewise, genistein (100 microM), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 microM) and latrunculin B (10 microM) reduced the increase in Na+ efflux induced by pi = 1 pi = 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by pi = 1 pi = 0.5 and pi = 2 pi = 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport.

Sodium influx during action potential in innervated and denervated rat skeletal muscles.

Resting Na(+) influx (J(i)(Na)) was measured in innervated and denervated (1-6 days) rat extensor digitorum longus muscle in the absence and presence of 2 micromol/L tetrodotoxin (TTX). The mean value of Na(+) permeability (P(Na)) in innervated muscles was 49.6 +/- 2.6 pm.s(-1). At the second day postdenervation, it decreased by about 45%. This was followed, between the second and fourth days, by a sharp rise, which by the sixth day reached a steady value approximately 2.5 times greater than that of innervated muscles. This, most likely, generated the 30% increase in internal [Na(+)] concentration ([Na(+)](I)) observed at this time. Tetrodotoxin reduced P(Na) of both innervated and denervated muscles by about 25%. In 6-day denervated muscles, virtually all the TTX effect on P(Na) represents the blockage of TTX-resistant Na(+) channels. Denervation produced a depolarization of about 20 mV by the sixth day. The extra J(i)(Na) per action potential (AP) decreased monotonically with time after denervation from 20.0 +/- 3.8 in innervated to 11.1 +/- 1.0 nmol.g(-1).AP(-1) in 6-day denervated muscles. The overshoot of the AP decreased from 15 +/- 1 in innervated to 7 +/- 1 mV in 6-day denervated muscles. Likewise, the maximum rate of rise (+dV/dt), an expression of the inward Na(+) current, fell from 305 +/- 14 in innervated to 188 +/- 18 V.s(-1) in 6-day denervated muscles. The estimated 6-day denervated/innervated ratio of peak Na(+) conductance (g(Na)) was 0.67. The changes in AP parameters promoted by denervation were substantially reduced when both innervated and denervated fibers were hyperpolarized to -90 mV. These results suggest that the depolarization, mainly due to the increase in P(Na) /P(K) ratio, increases Na(+) inactivation and consequently reduces peak g(Na), in spite of the absolute increment in resting TTX-sensitive P(Na). This, in addition to the moderate reduction in the inward driving force on Na(+), decreases the inward Na(+) current and the extra J(i)(Na) per AP.

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.

Effect of caffeine on K+ efflux in frog skeletal muscle.

The exposure of frog skeletal muscle to caffeine (3-4 mM) generates an increase of the K+ (42K+) efflux rate coefficient (kK,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca2+ ([Ca2+]i), because the effect is mimicked by ionomycin (1.25 microM), a Ca2+ ionophore. Second, the inhibition of caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) by 40 microM tetracaine significantly reduced the increase in kK,o (DeltakK,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca2+-dependent K+ channels (BKCa channels) reduced DeltakK,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca2+-dependent K+ channels (SKCa channels), did not affect DeltakK,o. Fifth, tolbutamide (800 microM), an inhibitor of KATP channels, reduced DeltakK,o by about 23%. Sixth, Ba2+, a blocker of most K+ channels, did not preclude the caffeine-induced DeltakK,o. Seventh, omitting Na+ from the external medium reduced DeltakK,o by about 40%. Eight, amiloride (5 mM) decreased DeltakK,o by 65%. It is concluded that the caffeine-induced rise of [Ca2+]i increases K+ efflux, through the activation of: (1) two channels (BKCa and KATP) and (2) an external Na+-dependent amiloride-sensitive process.

Characteristics of Na(+)-Ca2+ exchange in frog skeletal muscle.

1. Fluxes studies were carried out to investigate the Na(+)-dependent outward movement of Ca2+ in intact frog sartorius muscle from Leptodactylus ocellatus, a preparation for which published data on the subject are sparse. 2. Under normal resting conditions the Na(+)-Ca2+ exchange was not readily detectable. 3. When muscles were exposed to 4 mM caffeine, the rate of fractional loss of Ca2+ (kCa,o) increased by about 50%. Most of this increase exhibits characteristics typical of the Na(+)-Ca2+ antiport working in the forward mode found in other cells. 4. The increase in kCa,o promoted by caffeine was decreased by: (a) 72% in the absence of external Na+ (Nao+); (b) 73% in Na(+)-loaded muscles ([Na+]i = 98 mM); (c) 70% when fibres were depolarized to -27 mV ([K+]o = 50 mM); and (d) 80% in the presence of 5 mM amiloride. 5. Ni2+ (5 mM), an inhibitor of the Na(+)-Ca2+ exchanger current, unexpectedly increased the caffeine-promoted rise in kCa,o. This effect of Ni2+ was associated with a concomitant caffeine-stimulated Ni2+ influx. In the absence of caffeine Ni2+ did not affect kCa,o. 6. It was concluded that: (a) under resting conditions the sarcolemmal Ca2+ pump suffices to handle the cytosolic calcium concentration ([Ca2+]i); (b) Na(+)-Ca2+ activity becomes apparent when [Ca2+]i is substantially increased by caffeine-induced Ca2+ release from the sarcoplasmic reticulum; and (c) the blocking effect of Ni2+ on the current generated by a Na(+)-Ca2+ exchange with a coupling ratio > 2 might actually represent a shift of the antiport mode toward an electroneutral 1 Ni(2+)-1Ca2+ exchange.

Frog striated muscle is permeable to hydroxide and buffer anions.

Hydroxide, bicarbonate and buffer anion permeabilities in semitendinosus muscle fibers of Rana pipiens were measured. In all experiments, the fibers were initially equilibrated in isotonic, high K2SO4 solutions at pHo = 7.2 buffered with phosphate. Two different methods were used to estimate permeabilities: (i) membrane potential changes were recorded in response to changes in external ion concentrations, and (ii) intracellular pH changes were recorded in response to changes in external concentrations of ions that alter intracellular pH. Constant field equations were used to calculate relative or absolute permeabilities. In the first method, to increase the size of the membrane potential change produced by a sudden change in anion entry, external K+ was replaced by Cs+ prior to changes of the anion under study. At constant external Cs+ activity, a hyperpolarization results from increasing external pH from 7.2 to 10.0 or higher, using either CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) or CHES (2-[N-cyclohexylamino]-ethanesulfonic acid) as buffer. For each buffer, the protonated form is a zwitterion of zero net charge and the nonprotonated form is an anion. Using reported values of H+ permeability, calculations show that the reduction in [H+]o cannot account for the hyperpolarizations produced by alkaline solutions. Membrane hyperpolarization increases with increasing total external buffer concentration at constant external pH, and with increasing external pH at constant external buffer anion concentration. Taken together, these observations indicate that both OH- and buffer anions permeate the surface membrane. The following relative permeabilities were obtained at pHo = 10.0 +/- 0.3: (POH/PK) = 890 +/- 150, (PCAPS/PK) = 12 +/- 2, (PCHES/PK) = 5.3 +/- 0.9, and (PNO3/PK) = 4.7 +/- 0.5. PNO3/PK was independent of pHo up to 10.75. At pHo = 9.6, (PHCO3/PK) = 0.49 +/- 0.03; at pHo = 8.9, (PCl/PK) = 18 +/- 2 and at pHo = 7.1, (PHEPES/PK) = 20 +/- 2. In the second method, on increasing external pH from 7.2 to 10.0, using 2.5 mM CAPS (total buffer concentration), the internal pH increases linearly with time over the next 10 min. This alkalinization is due to the entry of OH- and the absorption of internal H+ by entering CAPS- anion. The rate of CAPS- entry was determined in experiments in which the external CAPS concentration was increased at constant external pH. Such increases invariably produced an increase in the rate of internal alkalinization, which was reversed when the CAPS concentration was reduced to its initial value.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha-melanocyte-stimulating hormone stimulates sodium excretion in the salt gland of the duck.

Salt glands of ducks were induced to secrete sodium through the ingestion of salt water. In salt-adapted animals the administration of melanocyte-stimulating hormone (MSH) produced a rise in the sodium excreted by the salt gland, an effect which was not mimicked by adrenocorticotropin. Studies in vitro using incubations of gland slices and radioactive sodium ion showed that MSH increased sodium efflux, indicating that it acted directly upon the gland. We have previously observed that MSH has no effect on the pigmentary system of the duck. It is proposed that in the evolutionary process this hormone has acquired new target tissues in these birds.

Ionic movements mediated by monensin in frog skeletal muscle.

Monensin-mediated ionic movements were studied in frog skeletal muscle. The ionophore, which forms electrically neutral complexes with monovalent cations, induced dose dependent fluxes of Na+, K+ and H+ in and out of the fibers. Monensin concentrations ([MON]) ranged from 2 to 40 microM. In the presence of normal Ringer's solution the following maximum ionic exchanges were generated by monensin (in pmol cm-2 s-1): (1) Nai+/Nao+ 112, (2) Nai+/Ho+ 30.7, (3) Ki+/Nao+ 14.2 (4) Hi+/Nao+ 49. The maximum net fluxes produced by these exchanges (i.e. for [MON] = infinity) are (in pmol cm-2 s-1): Na+ (inward) 32.5, K+ (outward) 14.2, H+ (outward) 18.3. The last one appears to be largely offset by a passive (monensin-independent) H+ influx down an inwardly directed electrochemical gradient promoted by pH reduction of the T-tubular lumen content as a consequence of the monensin-mediated net H+ efflux. Maximum unidirectional cationic fluxes mediated by monensin amounted to 206 pmol cm-2 s-1 and had the following composition: influx: 85% Na+ and 15% H+; efflux: 69% Na+, 7% K+, 24% H+.

Inward movement of Ca2+ in K(+)-depolarized rat vascular smooth muscle.

The kinetics of the Ca2+ (45Ca2+) uptake in segments of rat aorta and its relationship with tension development in the presence of K+ concentrations ([K+]o) ranging from 3.5 to 100 mM was investigated. It was found that: 1) The curve relating 45Ca2+ uptake and time was virtually linear during the first 3 min. Therefore, the influx calculated as uptake in 3 min/3 min was not significantly different from that calculated as the slope of the uptake curve. The actual uptake of the cation during the loading period would be underestimated by about 30 percent if the loss of Ca2+ from that compartment during a 2 h clearing of the extracellular space in La3+ medium, is ignored. 3) 45Ca2+ activity remaining in the tissue after a 3 min labelling period followed by a 2 h washout can reliably be corrected to calculate Ca2+ influx. 4) The K(+)-induced tension development showed no significant correlation with the total exchangeable Ca2+ while it appeared as a steep function of Ca2+ influx between 15 and 80 mM [K+]o. 5) In [K+]o = 100 mM Ca2+ influx was 5 to 6 times larger than in normal [K+]o (5.3 mM). The estimated Ca2+ permeability (PCa) increased linearly as a function of [K+]o (5.3 to 100 mM). In [K+]o = 100 mM, PCa was estimated to be about 17-fold that in [K+]o = 5.3 mM. Similarly, the increased in Ca2+ conductance (GCa) for the same change in [K+]o was estimated to be 14-fold.

Hypo-osmotic stimulation of active Na+ transport in frog muscle: apparent upregulation of Na+ pumps.

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).

Role of sodium and potassium permeabilities in the depolarization of denervated rat muscle fibres.

1. Na+ and K+ flux measurements and membrane potential (Vm) determinations were performed on normal and denervated rat extensor digitorum longus (e.d.l.) muscles. 2. The mean Vm in normal muscle fibres was -74.6 mV. During the first week after denervation Vm fell about 20 mV following an S-shaped time course. 3. In that period the Na+ permeability (PNa) increased and the K+ permeability (PK) decreased, so that by the sixth day post-denervation, the PNa/PK ratio was increased by a factor of 2.7. 4. The decrease in PK preceded the increase in PNa. 5. No major contribution to the fall of Vm by a reduced activity of an electrogenic Na+ pump could be detected. 6. A good agreement was found between the experimental values of the depolarization and those calculated using the constant-field equation assuming Cl- is at equilibrium and no significant change of the intracellular K+ concentration ([K+]i) during the first week after denervation. 7. It is concluded that the depolarization promoted by denervation in e.d.l. rat muscle fibres can be fully explained in terms of changes in PNa and PK.

Increased sensitivity to nifedipine of smooth muscle from hypertensive rats.

The sensitivity of smooth muscle from aortas of spontaneously hypertensive rats (SHR), renal hypertensive rats: two kidney-one clip and one kidney-one clip (2K-1C, 1K-1C) and DOCA salt hypertensive rats to the relaxant effect of nifedipine (NIF) was studied. A parallel leftward shift of the concentration-relaxation curve was detected in the K-precontracted aortic smooth muscle from hypertensive rats. This increased sensitivity seems to be related to the degree of hypertension and independent of the experimental method used to produce the high blood pressure level. No change in sensitivity was detected either in SHR or in renal hypertensive rats when nitroglycerin was used as a vasodilator.

Potassium movements in denervated frog sartorius muscle.

The movement of 42K+ across the sarcolemma and the resting membrane potential (VM) of normal and denervated frog sartorius muscle were studied under several experimental conditions in preparations initially equilibrated in 100 mM K+ and 219 mM Cl-. The results can be summarized as follows. In the absence of any driving force on K+, i.e., when the difference between VM and the K+ equilibrium potential (EK) is zero (VM - EK = 0), the K+ conductance (gK) was 368 microseconds . cm-2 in control and 282 microseconds. in denervated muscle. The reduced gK of denervated muscles results from the addition of the opposite changes in the conductances of a Rb+-sensitive inward rectifying pathway (gIR), which decreases, and a Rb+-insensitive linear channel (gL), which increases. Thus in control muscles gK (368 microseconds . cm-2) equals gIR (359 microseconds . cm-2) plus gL (9 microseconds . cm-2), while in denervated muscles gK (282 microseconds . cm-2) equals gIR (198 microsecond . cm-2) plus gL (84 microseconds . cm-2). Denervation significantly reduces the inward rectifying properties of the resting K+ permeability system. In the presence of outward driving forces on K+ (VM - EK greater than 0) of 35-50 mV, the Rb+-sensitive inward rectifier channel appears to close completely in both control and denervated muscles. In the latter, however, the effect was not as well maintained as in the former, suggesting that its closing mechanism might be altered by denervation. No changes were observed during the first 2 wk after denervation.

Denervated frog skeletal muscle. Some electrical and mechanical properties.

The effects of denervation on several mechanical and electrical parameters of frog sartorius muscle have been investigated. In denervated muscles, there is no change in the resting potential and a relatively small change in the action potential. The first alteration in the action potential is a reduction of about 30% in the maximum rate of repolarization in muscles that have been denervated for 40 days or longer. Later, the overshoot and maximum rate of depolarization also decline. No tetrodotoxin resistant action potentials could be detected. Fibrillatory potentials were observed infrequently and in most cases in depolarized fibers. Twitch tension is significantly reduced by denervation while the tetanus tension is practically unaffected by denervation. The experiments suggest that the decline in twitch tension produced by denervation reflect a defect in some step of the excitation contraction coupling sequence. On the other hand, post-tetanic potentiation of the twitch is much larger in denervated than in control muscles. This potentiation in denervated muscles is paralleled by an increased action potential duration which returns to its pretetanic duration with a time course indistinguishable from that of the twitch potentiation.

Caffeine contractures in denervated frog muscle.

Caffeine contracture tension, effect of caffeine on the resting membrane potential, and caffeine influx in normal and denervated frog sartorius muscle have been investigated. Peak caffeine contracture tension is increased after denervation at all caffeine concentrations. The percentage increases in tension are highest for lower caffeine concentrations. The caffeine concentration required for half maximum tension is decreased from about 3.6 mM in control muscles to 2.6 mM in denervated muscles. Caffeine at 3.5 mM produces a depolarization of about 6 mV in control muscles and 16mV in denervated muscles. The large contracture tensions observed in denervated muscles are not due to the greater depolarization produced by the drug in denervated muscles since innervated muscles depolarized to the same level by external K+ do not enhance caffeine contracture tension. Both control and denervated muscles are highly permeable to caffeine. The increases in sarcoplasmic reticulum development ( Moscatello et al. 1965) and calcium content ( Picken and Kirby 1976) promoted by denervation may explain the larger tension elicited by caffeine in denervated muscles.

Healing-over in rat crystalline lens.

1. 42K efflux has been studied in normal and injured rat crystalline lenses to test their ability to heal over. Following injury, a sizeable, but transient, increase in fractional loss of 42K takes place. 2. The lens healing-over is Ca-dependent and cannot be accounted for by membrane resealing. This is excluded by the fact that Procion Yellow gains access to damaged fibres even 2 hr after injury (when the healing-over is completed). 3. The occlusion of the junctional channels is the basic mechanism of healing-over. This is supported by the observation that Procion Yellow does not diffuse from damaged to intact fibres and by the Ca dependency of the phenomenon.

Density and apparent location of the sodium pump in frog sartorius muscle.

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.

Stimulation of the Na+ pump by hypotonic solutions in skeletal muscle.

The fractional loss of 22 Na+ from frog sartorius muscle is increased when the tonicity of the external solution is reduced. The effect, which is larger the lower the osmolarity, exhibits the following characteristics: (1) quick onset and reversibility, (2) is not reduced in the absence of external Na+, (3) is completely abolished by strophanthidin (3. 10-5 M), (4) is neither the result of membrane depolarization nor K+ accumulation in the extracellular space.

Density and distribution of tetrodotoxin receptors in normal and detubulated frog sartorius muscle.

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.