Experimental verification of an expected relation between time of incubation and magnitude of the fast and slow fractions of the sodium efflux from amphibian eggs.

The Na+ efflux curves of single ovarian eggs are separable into two fractions. The magnitude of the slow fraction increases slowly with time of exposure of the eggs to labeled Na+, long after the fast fraction has reached equilibrium. The data agree with the theory that the fast fraction is rate-limited by surface permeation and that the slow fraction is rate-limited by desorption from intracellular adsorption sites.     

How does reduced external K+ concentration affect the rate of Na+ efflux? Evidence against the K-Na coupled pump but in support of the association-induction hypothesis.

Recent frog-muscle studies produced the following findings: 1. Contrary to the theory of K+--Na+ coupled pump, reduction of external K+ concentration to near zero did not significantly reduce the rate of efflus of the fraction of cell Na+ conventionally regarded as rate-limited by membrane permeability. 2. Reduction of external K+ concentration profoundly reduced the rate of the efflux of this fraction only if the muscles were exposed to the low K+ while being loaded with radioactive Na+. 3. The data indicate that the fraction of Na+ efflux which in normal cells at room temperature has a half-time exchange (t1/2) of 20-40 min is not rate-limited by membrane permeability but by desorption from cellular adsorption sites. Surface-limited Na+ exchange between free Na+ in the cell and the external environment is represented by a faster fraction with a t1/2 of 2 to 4 min. 4. The data further indicate that the slow-down of the rate of efflux of the (slow) fraction arises from a cooperative shift of those beta- and gamma-carboxyl groups from adsorbing K+ to adsorbing Na+ when external K+ concentration is reduced below a critical level. The enhanced adsorption energy of the newly adsorbed Na+ raises the activation energy, hence a slower rate of exchange is seen as a slow-down in the "efflux curves." It is therefore only when free labeled Na+ is present in the cell water and thus available to the newly emerging Na+ adsorption sites that the effect of low external K+ can be visualized in a labeled-Na+ efflux study. Application of low K+ Ringer's solution after free labeled Na+ in and out of the cells has been washed away only causes enhanced adsorption of non-labeled Na+, which is not detected in isotope efflux study.     

Na v1.4 and Na v1.5 are modulated differently during muscle immobilization and contractile phenotype conversion

Muscle immobilization leads to modification in its fast/slow contractile phenotype. Since the properties of voltage-gated sodium channels (Na(v)) are different between "fast" and "slow" muscles, we studied the effects of immobilization on the contractile properties and the Na(v) of rat peroneus longus (PL). The distal tendon of PL was cut and fixed to the adjacent bone at neutral muscle length. After 4 or 8 wk of immobilization, the contractile and the Na(v) properties were studied and compared with muscles from control animals (Student's t-test). After 4 wk of immobilization, PL showed a faster phenotype with a rightward shift of the force-frequency curve and a decrease in both the Burke's index of fatigability and the tetanus-to-twitch ratio. These parameters showed opposite changes between 4 and 8 wk of immobilization. The maximal sodium current in 4-wk immobilized fibers was higher compared with that of control fibers (11.5 ± 1.2 vs. 7.8 ± 0.8 nA, P = 0.008), with partial recovery to the control values in 8-wk immobilized fibers (8.6 ± 0.7 nA, P = 0.48). In the presence of tetrodotoxin, the maximal residual sodium current decreased continuously throughout immobilization. Using the Western blot analysis, Na(v)1.4 expression showed a transient increase in 4-wk muscle, whereas Na(v)1.5 expression decreased during immobilization. Our results indicate that a muscle immobilized at optimal functional length with the preservation of neural inputs exhibits a transient fast phenotype conversion. Na(v)1.4 expression and current are related to the contractile phenotype variation.     

Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: delta psi-dependent 22Na+ efflux from whole cells.

The Na+/H+ antiporter of Bacillus alcalophilus was studied by measuring 22Na+ efflux from starved, cyanide-inhibited cells which were energized by means of a valinomycin-induced potassium diffusion potential, positive out (delta psi). In the absence of a delta psi, 22Na+ efflux at pH 9.0 was slow and appreciably inhibited by N-ethylmaleimide. Upon imposition of a delta psi, a very rapid rate of 22Na+ efflux occurred. This rapid rate of 22Na+ efflux was competitively inhibited by Li+ and varied directly with the magnitude of the delta psi. Kinetic experiments with B. alcalophilus and alkalophilic Bacillus firmus RAB indicated that the delta psi caused a pronounced increase in the Vmax for 22Na+ efflux. The Km values for Na+ were unaffected by the delta psi. Upon imposition of a delta psi at pH 7.0, a retardation of the slow 22Na+ efflux rate at pH 7.0 was caused by the delta psi. This showed that inactivity of the Na+/H+ antiporter at pH 7.0 was not secondary to a low delta psi generated by respiration at this pH. Indeed, 22Na+ efflux activity appeared to be inhibited by a relatively high internal proton concentration. By contrast, at a constant internal pH, there was little variation in the activity at external pH values from 7.0 to 9.0; at an external pH of 10.0, the rate of 22Na+ efflux declined. This decline at typical pH values for growth may be due to an insufficiency of protons when a diffusion potential rather than respiration is the driving force. Non-alkalophilic mutant strains of B. alcalophilus and B. firmus RAB exhibited a slow rate of 22Na+ efflux which was not enhanced by a delta psi at either pH 7.0 or 9.0.     

Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle.

Fast (extensor digitorum longus) and slow (soleus) rat skeletal muscles served as the source for isolation and biochemical comparison of two distinct surface membrane fractions with properties of the sarcolemma and transverse tubular system. Enriched sarcolemmal membrane from soleus demonstrated a lighter density after sucrose density centrifugation. Sialic acid content was 1.5-fold higher in soleus (62 nmol/mg) than extensor (40 nmol/mg). The specific activity of (Na+ + K+ + Mg2+)-ATPase was similar (1.40 and 1.65 micronmol Pi/mg per 5 min) with the soleus enzyme displaying a (1) greater resistance to inhibition by ouabain, and (2) broader ionic ratio (Na+/K+) requirement than extensor enzyme. The polypeptide and phospholipid composition showed no major differences between the two muscle types. The second surface membrane fraction, tentatively identified as transverse tubule, differed in membrane composition. The major polypeptide of extensor was of 95 000 molecular weight whereas for soleus a Mr=28 000 species was dominant. Total phospholipid content of soleus was 1.5-fold greater than extensor due mostly to increased levels of phosphatidylcholine and phosphatidylethanolamine. Endogenous membrane protein kinase for the 28 000 molecular weight polypeptide was found exclusively in this membrane. The reaction conditions were identical for extensor and soleus since both required divalent cations (Ca2+ and Mg2+) and neither was affected by cyclic AMP. Soleus showed a 2-fold higher capacity for phosphate incorporation than extensor. These studies show that surface membrane fractions derived from fast and slow muscles differ in terms of functional and compositional properties. These differences are specific not only for the surface membrane but for the muscle type and may relate to the known physiological differences observed between fast and slow mammalian muscle.     

Multiple fractions of sodium exchange in human lymphocytes

Human lymphocytes contain a large, saturable fraction of K⁺ that exchanges slowly with K⁺ in the external medium, and a small non-saturable fraction that exchanges rapidly. We determined whether or not Na⁺ exchanges in a similar manner with external Na⁺. Cells were pre-equilibrated to ensure absence of net ion movements. Efflux was studied by loading with ²²Na and transferring without washing to a non-labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na⁺ exchange, with half-times of 2, 14, and 120 minutes. At normal external K⁺, most cell Na⁺ exchanges rapidly, while at lower external K⁺ the Na⁺ that replaces cell K⁺ exchanges slowly. Parallel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous ⁴²K and ²²Na fluxes and by a quantitative analysis of the combined K⁺ and Na⁺ content and flux data over a range of external K⁺ and Na⁺ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclea exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K⁺ and Na⁺ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate-limited by adsorption onto and desorption from cellular macromolecules. The data support the association-induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.     

A study on the ouabain-sensitive (Na+ + K+)-ATPase activities of skeletal muscle and brain membrane fractions from genetically dystrophic mice (author's transl)]

The ouabain-sensitive (Na+ + K+)-ATPase activities of membrane fractions from hind-leg muscle and brain of normal and genetically dystrophic mice (C57BL/6J-dy strain) were studied, and the following results were obtained. 1) The ouabain-sensitive (Na+ + K+)-ATPase activity of frozen muscle sarcolemmal fraction from normal mice was several times higher than that of fresh one. 2) The ouabain-sensitive (Na+ + K+)-ATPase activity of frozen muscle sarcolemmal fraction from dystrophic mice was almost equal to that from normal one. But the muscle membrane yield from dystrophic mice was considerably low compared with the yield from normal one. 3) With brain membrane fractions, no differences were observed between normal and dystrophic mice in the ouabain-sensitive (Na+ + K+)-ATPase activity as well as in the yield of membrane fractions.     

Fast Na+ channels in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).     

Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability

The composition of Na+ currents in dorsal root ganglia (DRG) neurons depends on their neuronal phenotype and innervation target. Two TTX-resistant (TTX-R) Na+ currents [voltage-gated Na channels (Nav)] have been described in small DRG neurons; one with slow inactivation kinetics (Nav1.8) and the other with persistent kinetics (Nav1.9), and their modulation has been implicated in inflammatory pain. This has not been studied in neurons projecting to the colon. This study examined the relative importance of these currents in inflammation-induced changes in a mouse model of inflammatory bowel disease. Colonic sensory neurons were retrogradely labeled, and colitis was induced by instillation of trinitrobenzenesulfonic acid (TNBS) into the lumen of the distal colon. Seven to ten days later, immunohistochemical properties were characterized in controls, and whole cell recordings were obtained from small (<40 pF) labeled DRG neurons from control and TNBS animals. Most neurons exhibited both fast TTX-sensitive (TTX-S)- and slow TTX-R-inactivating Na+ currents, but persistent TTX-R currents were uncommon (<15%). Most labeled neurons were CGRP (79%), tyrosine kinase A (trkA) (84%) immunoreactive, but only a small minority bind IB4 (14%). TNBS-colitis caused ulceration, thickening of the colon and significantly increased neuronal excitability. The slow TTX-R-inactivating Na current density (Nav1.8) was significantly increased, but other Na currents were unaffected. Most small mouse colonic sensory neurons are CGRP, trkA immunoreactive, but not isolectin B4 reactive and exhibit fast TTX-S, slow TTX-R, but not persistent TTX-R Na+ currents. Colitis-induced hyperexcitability is associated with increased slow TTX-R (Nav1.8) Na+ current. Together, these findings suggest that colitis alters trkA-positive neurons to preferentially increase slow TTX-R Na+ (Nav1.8) currents.     

Effects of detergents on Na+ + K+-dependent ATPase activity in plasma-membrane fractions prepared from frog muscles. Studies of insulin action on Na+ and K+ transport.

The increase in Na+/K+ transport activity in skeletal muscles exposed to insulin was analysed. Plasma-membrane fractions were prepared from frog (Rana catesbeiana) skeletal muscles, and examination of the Na,K-ATPase (Na+ + K+-dependent ATPase) activity showed that it was insensitive to ouabain. In contrast, plasma-membrane fractions prepared from ouabain-pretreated muscles, by the same procedures, showed extremely low Na,K-ATPase activity. On adding saponin to the membrane suspension, the Na,K-ATPase activity increased, according to the detergent concentration. The maximum activity was about twice the control value, at 0.33 mg of saponin/mg of protein. Thus saponin makes vesicle membranes leaky, allowing ouabain in assay solutions to reach receptors on the inner surface of vesicles. Addition of insulin to saponin-treated membrane suspensions had no effect on the Na,K-ATPase activity, whereas the maximum activity of Na,K-ATPase in whole muscles was stimulated by exposure to insulin. The results show that the stimulation of Na+/K+ transport by insulin is not directly due to insulin binding to receptors on the cell surface, but rather support the view that the increase in the Na,K-ATPase induced by insulin requires an alteration of intracellular events.     

Subcellular distribution and immunocytochemical localization of Na,K-ATPase subunit isoforms in human skeletal muscle

Summary

The expression of Na,K-ATPase isoforms was investigated in human skeletal muscle membranes isolated by subcellular fractionation. The α1, α2, α3 and β1 subunits were detectable in membranes prepared from the human soleus muscle. The α1 subunit was largely detected in a fraction enriched with plasma membranes (PM), its abundance in an Intracellular membrane fraction (IM) accounted for only 4% of that in the PM fraction. No α1 subunits were detected in membranes of sarcoplasmic reticulum (SR) origin. The PM and IM fractions were enriched with α2 subunits which were less abundant in the SR-enriched fraction. The abundance of α2 molecules within the IM fraction was about 75% of that in the PM fraction when the total protein content for the two fractions was taken into account. Immuno-cytochemical studies confirmed the localization of the α1 subunit to the muscle cell surface. The α2 subunit was also found to be present in the cell surface but the observation that α2 immuno-fluorescence was diffusely dispersed throughout the muscle fibre indicated that it was also present intracellularly, consistent with its biochemical localization in the PM and IM membrane fractions. The α3 subunit was detected largely in the PM fraction but the lack of good antibodies to this isoform precluded an analysis of its immunocytochemical localization. The β1 subunit was enriched in the PM fraction but was also detected to a modest extent in the IM. A polyclonal anti-β2 antibody, which reacted positively with both human brain microsomes and rat skeletal muscle membranes, revealed that human skeletal muscles contained no immunoreactive β2 subunits. Our results indicate that the human soleus expresses the α1 and α2 (and possibly the α3) subunits of the Na,K-ATPase and that the activity of these isoforms must be supported by the β1 subunit in this muscle.     

Endothelial Na+-D-glucose cotransporter: no role in insulin-mediated glucose uptake.

A recent report indicates that the Na+-D-glucose cotransporter SGLT1 is present in capillaries of skeletal muscle and is required for insulin-mediated glucose uptake in myocytes. This result is based on the complete inhibition of insulin-mediated muscle glucose uptake by phlorizin, an inhibitor of SGLT1. Using the pump-perfused rat hind limb, we measured glucose uptake, lactate efflux, and radioactive 2-deoxyglucose uptake into individual muscles with saline (control), phlorizin, insulin, and insulin plus phlorizin, as well as with saline and insulin using normal and low Na+ perfusion buffer. Insulin-mediated glucose uptake was not inhibited after correction for phlorizin interference in the glucose assay. Lactate efflux and 2-deoxyglucose uptake by individual muscles were unaffected by phlorizin. Low Na+ buffer did not affect insulin-mediated glucose uptake, lactate efflux, or 2-deoxyglucose uptake. We conclude that endothelial SGLT1 exerts no barrier for glucose delivery to myocytes.     

Some properties of transport ATPases in functionally different muscles]

The properties of Ca-transporting system in sarcoplasmic reticulum membranes in fast and slow frog muscles as well as some properties of sarcolemma Na, K-ATPase of the same object were investigated. The rate of Ca2+ uptake, Ca-ATPase activity and Ca/ATP ratio for the reticulum of fast muscle demonstrated higher values than those for the reticulum of slow muscle. The rate of Ca2+ accumulation by the fragments of the rectus reticulum and Ca/ATP ratio were found to decrease under the influence of acetylcholine (0.05-5 mM). The transport system of the sartorius reticulum was found to be less sensitive to acetylcholine. The peak activity of Na, K-ATPase in femoral muscles of the frog occurred at 80 mM NaCl and 60 mM KCl, whereas in the rectus abdominal muscle it equalled 100 mM NaCl and 40 mM KCl. Thus, Na, K-ATPase activity in the slow muscle was predominantly higher than that in the mixed (femoral) muscles. If the sarcolemma preparations of the muscles of both types the inhibitory effect of acetylcholine on Na; K-ATPase was registered. The enzyme of slow muscles exhibited higher sensibility to acetylcholine.     

Insulin stimulates the translocation of Na+/K(+)-dependent ATPase molecules from intracellular stores to the plasma membrane in frog skeletal muscle.

The mechanism of the stimulation of Na+/K+ transport by insulin in frog skeletal muscle was studied. The ouabain-binding capacity in detergent-treated plasma membranes of insulin-exposed muscles was increased 1.9-fold compared with that of controls. Na+/K(+)-ATPase activity was found in an intracellular 'light fraction' (fraction II) prepared by using anion-exchange chromatography. Marker enzyme activities for plasma and Golgi membranes were not detected in this fraction. The specific activity of Na+/K(+)-ATPase in fraction II from insulin-exposed muscles was 58% of that in an identical fraction from control muscles. No significant difference in the protein yield of the plasma membrane preparation was observed between these two groups. In parallel with the decrease in the Na+/K(+)-ATPase activity in fraction II from insulin-exposed muscles, the ouabain-binding capacity in this fraction was also decreased. The addition of saponin to fraction II increased both Na+/K(+)-ATPase activity and ouabain binding, indicating that some of the Na+/K(+)-ATPase is located in sealed vesicles. These findings support the view that insulin stimulates the translocation of Na+/K(+)-ATPase molecules from fraction II to the plasma membrane.     

Breakdown of Na+/K+-exchanging ATPase phosphoenzymes formed from ATP and from inorganic phosphate during Na+-ATPase activity.

The reactivity towards Na+ and K+ of Na+/K+-ATPase phosphoenzymes formed from ATP and Pi during Na+-ATPase turnover and that obtained from Pi in the absence of ATP, Na+ and K+ was studied. The phosphoenzyme formed from Pi in the absence of cycling and with no Na+ or K+ in the medium showed a biphasic time-dependent breakdown. The fast component, 96% of the total EP, had a decay rate of about 4 s(-1) in K+-free 130 mm Na+, and was 40% inhibited by 20 mm K+. The slow component, about 0.14 s(-1), was K+ insensitive. Values for the time-dependent breakdown of the phosphoenzymes obtained from ATP and from Pi during Na+-ATPase activity were indistinguishable from each other. In K+-free medium containing 130 mm Na+, the decays followed a single exponential with a rate constant of 0.45 s(-1). The addition of 20 mm K+ markedly increased the decays and made them biphasic. The fast components had a rate of approximately 220 s-1 and accounted for 92-93% of the total phosphoenzyme. The slow components decayed at a rate of about 47-53 s(-1). A second group of experiments examined the reactivity towards Na+ of the E2P forms obtained with ATP and Pi when the enzyme was cycling. In both cases, the rate of dephosphorylation was a biphasic function of [Na+]: inhibition at low [Na+], with a minimum at about 5 mm Na+, followed by recovery at higher [Na+]. Although qualitatively similar, the phosphoenzyme formed from Pi showed slightly less inhibition and more pronounced recovery. These results indicate that forward and backward phosphorylation during Na+-ATPase turnover share the same intermediates.     

Effects of temperature and Na0+ on the relaxation of phasic and tonic tension of guinea-pig ureter muscle

Effects of temperature and Na0+ on the relaxation of guinea-pig ureter smooth muscle were studied. Relaxation of phasic contraction was found to be highly temperature-dependent, practically independent of Na0+ and Ca02+, and resistant to vanadate. The relaxation of the tonic tension of both high-K and low-Na contracture was less temperature-dependent and affected by Na0+. The relaxation of tonic tension produced by introduction of Na0+ was about 3-5 times faster than that produced by Ca-free solution. La3+ ions were found to block the relaxation of the tonic component of the Na+-free contracture initiated by removal of Ca02+. Three systems of regulation of cell calcium are suggested to be operative in the ureter muscle: a fast one which is highly temperature-dependent and responsible for the relaxation of the phasic contraction (probably the sarcoplasmic reticulum), and two slow membrane-linked carriers, one of which is dependent on Na0+ (probably Na-Ca exchange) and another one which is independent of Na0+ and inhibited by La3+ (probably Ca-pump).     

Effects of internal Na and external K concentrations on Na/K coupling of Na,K-pump in frog skeletal muscle

Summary To clarify the dependency of the Na/K coupling of the Na,K-pump on internal Na and external K concentrations in skeletal muscle, the ouabain-induced change in membrane potential, the ouabain-induced change in Na efflux and the membrane resistance were measured at various internal Na and external K concentrations in bullfrog sartorius muscle.Upon raising the internal Na concentration from 6 mmol/kg muscle water to 20 mmol/kg muscle water, the magnitude of the ouabain-induced change in membrane potential increased about eightfold and the magnitude of the ouabain-induced change in Na efflux increased about fivefold while the membrane resistance was not significantly changed. As the external K concentration increased from 1 to 10mm, the magnitude of the ouabain-induced change in membrane potential decreased (1/5.5 fold), while the magnitude of the ouabain-induced change in Na efflux increased (about 1.5-fold). The membrane resistance decreased upon raising the external K concentration from 1 to 10mm (1/2-fold). These observations imply that the values of the Na/K coupling of the Na,K-pump increases upon raising the internal Na concentration and decreases upon raising the external K concentration.     

The effect of metabolic inhibition on ion contents and sodium exchange in human lymphocytes

Lymphocytes depleted of ATP by incubation in iodoacetate (IAA) and nitrogen (N2) lost K and gained Na. Isotopic Na exchange showed a fast fraction and a slower exponential fraction, the latter conventionally assumed to reflect surface membrane properties. The gain of cell Na was not accounted for by a decrease in 22Na efflux in either the slow or the fast fraction. After 3-5 hours, Na efflux increased. These results led us to question the concept that normal cell ion levels are maintained by an ATPase pump and could not be explained by exchange diffusion, co-transport, countertransport, or other inherently dissipative mechanisms. The data are, on the other hand, consistent with the concept that cell ion contents are determined by their relative exclusion from cell water coupled with selective adsorption onto fixed macromolecular anionic sites within the cell. In this view, the IAA,N2-induced rise in cell Na is due to the occupancy of adsorption sites losing K, while the increased isotopic exchange is due to a decreased activation energy for ion-site interaction.     

Kinetic investigation of K+ efflux during glycerol treatment of muscle

The kinetics of K+ efflux was investigated in the membranes of frog sartorius muscle after disintegration by glycerol treatment. Data of the K+ concentration in the muscle as a function of time of the glycerol treatment fitted well the sum of two exponential fractions (with the correlation coefficient of more than 0.98). The half-lives of the two fractions of the K+ efflux were 1 and 75 hours respectively. On the basis of the value of its half life the efflux of the faster fraction was suggested to correspond to the free diffusion. At low temperature the magnitude of the faster fraction increased in a Na+-containing milieu. This could be due to K+-Na+ ion exchange. From the rate of loss of the slower fraction of K+ one finds that movement of K+ in cells without membranes is significantly slower than free diffusion. Presumably, part of the bound potassium exists in intra- or intermolecular "ion-bridges" of muscle proteins.     

The effect of the internal Na concentration on the electrogenicity of the insulin-stimulated Na,K-pump in frog skeletal muscles

Insulin induced a hyperpolarization of the membrane by stimulating the Na,K-pump in frog skeletal muscles. The Na,K-pump activity was dependent on the internal Na concentration. As the internal Na concentration was raised from 5 mmol/kg muscle water to 18 mmol/kg muscle water, the magnitude of the insulin-induced increase in the ouabain-sensitive Na efflux (an index of the Na,K-pump activity) rose by 5-fold and the magnitude of the insulin-induced hyperpolarization rose by 8.5-fold. On the other hand, the specific membrane resistance was not significantly changed by a rise in the internal Na concentration. The Na/K coupling of the Na,K-pump was calculated at low, normal or high internal Na concentration by using the values of the insulin-induced changes in the ouabain-sensitive Na efflux and the membrane potential. As a result of the calculation, it was suggested that in frog skeletal muscles the Na/K coupling would increase with a rise of the internal Na concentration.