Effects of Sodium Azide on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes from frog''s striated muscle were measured in the presence of 0 to 5 mM sodium azide. With azide concentrations of 2 and 5 mM the Na efflux was markedly stimulated; the Na efflux with 5 mM azide was about 300 per cent greater than normal. A similar increase was present when all but the 5.0 mM sodium added with azide was replaced by choline. 10^-5 M strophanthidin abolished the azide effect on Na²⁴ efflux. Concentrations of azide of 1.0 mM or less had no effect on Na efflux. The Na influx, on the other hand, was only increased by 41 per cent in the presence of 5 mM NaN₃. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of azide. The hypothesis is advanced that the active transport of Na is controlled by the transmembrane potential and that the stimulation of Na efflux is produced as a consequence of the membrane depolarization caused by the azide.     

Sodium fluxes in internally dialyzed squid axons

The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed squid axons were examined. Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion. These compounds were: AcP, PEP, G-3-P, ADP, AMP, GTP, CTP, PA, and UTP.¹ the compound d-ATP supported 25–50% of the normal sodium extrusion, while ATP supported 80–100%. The relation between internal ATP and sodium efflux was nonlinear, rising most steeply in the range 1 to 10 µM and more gradually in the range 10 to 10,000 µM. There was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 µM. The relation between internal sodium and sodium efflux was linear in the range 2 to 240 mM. The presence of external strophanthidin (10 µM) changed the sodium efflux to about 8–12 pmoles/cm² sec regardless of the initial level of efflux; this changed level was not altered by subsequent dialysis with large concentrations of ATP. Sodium influx was reduced about 50 % by removal of either ATP or Na and about 70 % by removing both ATP and Na from inside the axon.     

Sodium Movements in Perfused Squid Giant Axons : Passive fluxes

Sodium movements in internally perfused giant axons from the squid Dosidicus gigas were studied with varying internal sodium concentrations and with fluoride as the internal anion. It was found that as the internal concentration of sodium was increased from 2 to 200 mM the resting sodium efflux increased from 0.09 to 34.0 pmoles/cm²sec and the average resting sodium influx increased from 42.9 to 64.5 pmoles/cm²sec but this last change was not statistically significant. When perfusing with a mixture of 500 mM K glutamate and 100 mM Na glutamate the resting efflux was 10 ± 3 pmoles/cm²sec and 41 ± 10 pmoles/cm²sec for sodium influx. Increasing the internal sodium concentration also increased both the extra influx and the extra efflux of sodium due to impulse propagation. At any given internal sodium concentration the net extra influx was about 5 pmoles/cm²impulse. This finding supports the notion that the inward current generated in a propagated action potential can be completely accounted for by movements of sodium.     

Choline permeability in cardiac muscle cells of the cat

Permeability of the cardiac cell membrane to choline ions was estimated by measuring radioactive choline influx and efflux in cat ventricular muscle. Maximum values for choline influx in 3.5 and 137 mM choline were respectively 0.56 and 9 pmoles/cm²·sec. In 3.5 mM choline the intracellular choline concentration was raised more than five times above the extracellular concentration after 2 hr of incubation. In 137 mM choline, choline influx corresponded to the combined loss of intracellular Na and K ions. Paper chromatography of muscle extracts indicated that choline was not metabolized to any important degree. The accumulation of intracellular choline rules out the existence of an efficient active pumping mechanism. By measuring simultaneously choline and sucrose exchange, choline efflux was analyzed in an extracellular phase, followed by two intracellular phases: a rapid and a slow one. Efflux corresponding to the rapid phase was estimated at 16–45 pmoles/cm²·sec in 137 mM choline and at 1.3–3.5 pmoles/cm²·sec in 3.5 mM choline; efflux in 3.5 mM choline was proportional to the intracellular choline concentration. The absolute figures for unidirectional efflux were much larger than the net influx values. The data are compared to Na and Li exchange in heart cells. Possible mechanisms for explaining the choline behavior in heart muscle are discussed.     

Structure-activity relationships of several cardiotonic steroids with respect to inhibition of ion transport in frog muscle

Cesium uptake by sodium-loaded frog sartorius muscles was inhibited 100% by 10^-6 M ouabain and 10^-6 M cymarin. The doses for 50% inhibition of cesium uptake by five cardiotonic aglycones were 1.5 x 10^-6 M for strophanthidin, 2 x 10^-7 M for telocinobufagin, 1.6 x 10^-6 for digitoxigenin, 2.4 x 10^-6 M for periplogenin, and 6.3 x 10^-6 M for uzarigenin. Because of the limited solubility of sarmentogenin the maximum concentration studied was 2 x 10^-6 M which inhibited cesium uptake about 36%. Inhibition of cesium uptake by cymarin was not reversed during a 3.5 hr incubation in fresh solution while the muscles treated with ouabain and strophanthidin recovered partly during this time. Cymarin was a more potent inhibitor of sodium efflux than strophanthidin and periplogenin was less potent. Increased cesium ion concentration in the external solution decreased the strophanthidin inhibition of cesium uptake but 25 mM cesium did not overcome the inhibition by 10^-8-10^-6 M strophanthidin. Increased potassium ion concentration in the external solution decreased but did not completely overcome inhibition of sodium efflux by strophanthidin. It is concluded that potassium or cesium ions do not compete with these drugs for a particular site on the ion transport complex. The same structural features of the drugs are necessary for inhibition of ion transport in frog muscle as are required for inhibition of ion transport in other tissues, inhibition of sodium-potassium-stimulated ATPases, and toxicity to animals.     

Strophanthidin-sensitive components of potassium and sodium movements in skeletal muscle as influenced by the internal sodium concentration

"Low sodium" muscles were prepared which contained around 5 mmoles/kg fiber of intracellular sodium. "High sodium" muscles containing between 15 and 30 mmoles/kg fiber of intracellular sodium were also prepared. In low sodium muscles application of 10^-5 M strophanthidin reduced potassium influx by about 5%. Potassium efflux was unaffected by strophanthidin under these conditions. In high sodium muscles, 10^-5 M strophanthidin reduced potassium influx by 45% and increased potassium efflux by 70%, on the average. In low sodium muscles sodium efflux was reduced by 25% during application of 10^-5 M strophanthidin while in high sodium muscles similarly treated, sodium efflux was reduced by about 60%. Low sodium muscles showed a large reduction in sodium efflux when sodium ions in the Ringer solution were replaced by lithium ions. The average reduction in sodium efflux was 4.5-fold. Of the amount of sodium efflux remaining in lithium. Ringer''s solution, 40% could be inhibited by application of 10^-5 M strophanthidin. The total sodium efflux from low sodium muscles exposed to Ringer''s solution in which lithium had been substituted for sodium ions for a period of 1 hr can be fractionated as 78% Na-for-Na interchange, 10% strophanthidin-sensitive sodium pump, and 12% residual sodium efflux. It is concluded that large strophanthidin-sensitive components of sodium and potassium flux can be expected only at elevated sodium concentrations within the muscle cells.     

The Dual Effect of Lithium Ions on Sodium Efflux in Skeletal Muscle

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3°C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer''s solution containing 60 mM LiCl and 50 mM NaCl at 20°C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10^-5 M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer''s solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10^-5 M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer''s solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.     

Potassium fluxes in dialyzed squid axons

Measurements have been made of K influx in squid giant axons under internal solute control by dialysis. With [ATP]_i = 1 µM, [Na]_i = 0, K influx was 6 ± 0.6 pmole/cm² sec; an increase to [ATP]_i = 4 mM gave an influx of 8 ± 0.5 pmole/cm² sec, while [ATP]_i 4, [Na]_i 80 gave a K influx of 19 ± 0.7 pmole/cm² sec (all measurements at ∼16°C). Strophanthidin (10 µM) in seawater quantitatively abolished the ATP-dependent increase in K influx. The concentration dependence of ATP-dependent K influx on [ATP]_i, [Na]_i, and [K]_o was measured; an [ATP]_i of 30 µM gave a K influx about half that at physiological concentrations (2–3 mM). About 7 mM [Na]_i yielded half the K influx found at 80 mM [Na]_i. The ATP-dependent K influx responded linearly to [K]_o from 1–20 mM and was independent of whether Na, Li, or choline was the principal cation of seawater. Substances tested as possible energy sources for the K pump were acetyl phosphate, phosphoarginine, PEP, and d-ATP. None was effective except d-ATP and this substance gave 70% of the maximal flux only when phosphoarginine or PEP was also present.     

Some factors influencing sodium extrusion by internally dialyzed squid axons

Squid giant axons were internally dialyzed by a technique previously described. In an axon exposed to cyanide seawater for 1 hr and dialyzed with an ATP-free medium, the Na efflux had a mean value of 1.3 pmole/cm²sec when [Na]_i was 88 mM, in quantitative agreement with flux ratio calculations for a purely passive Na movement. When ATP at a concentration of 5–10 mM was supplied to the axoplasm by dialysis, Na efflux rose almost 30-fold, while if phosphoarginine, 10 mM, was supplied instead of ATP, the Na efflux rose only about 15-fold. The substitution of Li for Na in the seawater outside did not affect the Na efflux from an axon supplied with ATP, while a change to K-free Na seawater reduced the Na efflux to about one-half. When special means were used to free an axon of virtually all ADP, the response of the Na efflux to dialysis with phosphoarginine (PA) at 10 mM was very small (an increment of ca. 3 pmole/cm²sec) and it can be concluded that more than 96% of the Na efflux from an axon is fueled by ATP rather than PA. Measurements of [ATP] in the fluid flowing out of the dialysis tube when the [ATP] supplied was 5 mM made it possible to have a continuous measurement of ATP consumption by the axon. This averaged 43 pmole/cm²sec. The ATP content of axons was also measured and averaged 4.4 mM. Estimates were made of the activities of the following enzymes in axoplasm: ATPase, adenylate kinase, and arginine phosphokinase. Values are scaled to 13°C.     

Cation regulation in the smooth muscle of frog stomach

Cation composition of frog smooth muscle cells was investigated. Fresh stomach muscle rings resembled skeletal muscle, but marked Na gain and K loss followed immersion. Mean Na (49.8–79.7 mM/kg tissue) and K (61.8–80.1 mM/kg tissue) varied between batches, but were stable for long periods in vitro. Exchange of 6–30 mM Na/kg tissue with ²²Na was extremely slow and distinct. Extracellular water was estimated from sucrose-¹⁴C uptake. Calculated exchangeable intracellular Na was 9 mM/kg cell water, and varied little. Thus steady-state transmembrane cation gradients appeared to be steep. K-free solution had only slight effects. Ouabain (10^-4 M) caused marked Na gain and reciprocal K loss; at 30°C, Na and K varied linearly with time over a wide range of contents, indicating constant net fluxes. Net fluxes decreased with temperature decrease. ²²Na exchange in ouabain-treated tissue at 20–30°C was rapid and difficult to analyze. The best minimum estimates of unidirectional Na fluxes at 30°C were 10–12 times the constant net flux; constant pump efflux may explain these findings. The rapidity of Na exchange may not reflect very high permeability, but it does require a high rate of transport work.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

Cat Heart Muscle in Vitro : VI. Potassium exchange in papillary muscles

The exchange of cell K with K⁴², J _K, has been measured in cat right ventricular papillary muscle under conditions of a steady state with respect to intracellular K concentration. Within the limits of the measurement, all of cell K exchanged at a single rate. Cells from small cats are smaller and have larger surface/volume ratios than cells from large cats. The larger surface/volume ratio results in larger flux values. J _K increases in an approximately linear manner as the external K concentration is increased twentyfold, from 2.5 to 50 mM, at constant intracellular K concentration. The permeability for K ions, P _K, calculated from the influx and membrane potential, remains very nearly constant over this range of external K concentrations. J _K is not affected by replacement of O₂ by N₂, or by stimulated contractions at 60 per minute, but K influx decreases markedly in 10^-5 M and 10^-8 M ouabain.     

Sodium extrusion by internally dialyzed squid axons

A method has been developed which allows a length of electrically excitable squid axon to be internally dialyzed against a continuously flowing solution of defined composition. Tests showed that diffusional exchange of small molecules in the axoplasm surrounding the dialysis tube occurred with a half-time of 2–5 min, and that protein does not cross the wall of the dialysis tube. The composition of the dialysis medium was (mM): K isethionate 151, K aspartate 151, taurine 275, MgCI₂ 4–10, NaCl 80, KCN 2, EDTA 0.1, ATP 5–10, and phosphoarginine 0–10. The following measurements were made: resting Na influx 57 pmole/cm²sec (n = 8); resting potassium efflux 59 pmole/ cm²sec (n = 4); stimulated Na efflux 3.1 pmole/cm²imp (n = 9); stimulated K efflux 2.9 pmole/cm²imp (n = 3); resting Na efflux 48 pmole/cm²sec (n = 18); Q ₁₀ Na efflux 2.2 (n = 5). Removal of ATP and phosphoarginine from the dialysis medium (n = 4) or external application of strophanthidin (n = 1) reversibly reduced Na efflux to 10–13 pmole/cm²sec. A general conclusion from the study is that dialyzed squid axons have relatively normal passive permeability properties and that a substantial fraction of the Na efflux is under metabolic control although the Na extrusion mechanism may not be working perfectly.     

Amino Acid and Sugar Transport in Rabbit Ileum

L-Alanine and 3-O-methyl-D-glucose accumulation by mucosal strips from rabbit ileum has been investigated with particular emphasis on the interaction between Na and these transport processes. L-Alanine is rapidly accumulated by mucosal tissue and intracellular concentrations of approximately 50 mM are reached within 30 min when extracellular L-alanine concentration is 5 mM. Evidence is presented that intracellular alanine exists in an unbound, osmotically active form and that accumulation is an active transport process. In the absence of extracellular Na, the final ratio of intracellular to extracellular L-alanine does not differ significantly from unity and the rate of net uptake is markedly inhibited. Amino acid accumulation is also inhibited by 5 x 10^-5 M ouabain. 3-O-methyl-D-glucose accumulation by this preparation is similarly affected by ouabain and by incubation in a Na-free medium. The effects of amino acid accumulation, of ouabain, and of incubation in a Na-free medium on cell water content and intracellular Na and K concentrations have also been investigated. These results are discussed with reference to the two hypotheses which have been suggested to explain the interaction between Na and intestinal nonelectrolyte transport.     

Nature of the Schwann cell electrical potential. Effects of the external ionic concentrations and a cardiac glycoside

The effects on the Schwann cell electrical potential of external ionic concentrations and of K-strophanthoside were investigated. Increasing (K)_o depolarized the cell. The potential is related to the logarithm of (K)_o in a quasi-linear fashion. The linear portion of the curve has a slope of 45 mv/ten-fold change in (K)_o. Diminutions of (Na)_o and (Cl)_o produced only small variations in the potential. Calcium and magnesium can be replaced by 44 mM calcium without altering the potential. Increase of (Ca)_o to 88 mM produced about 10 mv hyperpolarization. The cell was hyperpolarized by 11 mv and 4 mv within 1 min after applying K-strophanthoside at concentrations of 10^-3 and 10^-5 M, respectively. No variations of cellular potassium, sodium, or chloride were observed 3 min after applying the glycoside. The hyperpolarization caused by 10^-3 M K-strophanthoside was not observed when (K)_o was diminished to 1 or 0.1 mM or was increased to 30 mM. At a (K)_o of 30 mM, 10^-2 M strophanthoside was required to produce the hyperpolarizing effect. In high calcium, the cell was further hyperpolarized by the glycoside. The initial hyperpolarization caused by the glycoside was followed by a gradual depolarization and a decrease of the cellular potassium concentration. The results indicate that the Schwann cell potential of about -40 mv is due to ionic diffusion, mainly of potassium, and to a cardiac glycoside-sensitive ion transport process.     

Ion Fluxes and Short-Circuit Current in Internally Perfused Cells of Valonia ventricosa

Ion transport in the giant celled marine alga, Valonia ventricosa, was studied during internal perfusion and short-circuiting of the vacuole potential. The perfusing and bathing solutions were similar to natural Valonia sap and contained the following concentrations of major ions: Na 51, K 618, and Cl 652 mM. The average short-circuit current (I _sc) was 97 pEq/cm² sec (inward positive current), and the average open-circuit potential difference (PD) was 74 mv (vacuole positive to external solution). Perfused and short-circuited cells showed a small net influx of Na (2.0 pEq/cm² sec) and large net influxes of K (80 pEq/cm² sec) and Cl (50 pEq/cm² sec). Unidirectional K influx was proportional to I _sc, but more than one-half of the I _sc remained unaccounted for. Both the I _sc and PD were partly light-dependent, declining rapidly during the first 1–2 min of darkness. Ouabain (5 x 10^-4 M) had little effect on the influx of Na or K and had no effect on I _inf or PD. Fluid was absorbed at a rate of about 93 pliter/cm² sec. Reversing the direction of fluid movement by adding mannitol to the outside solution had little effect on ion movements. The ionic and electrical properties of normal and perfused cells of Valonia are compared.     

Sodium and potassium absorption by bean stem tissue

The effect of various periods of pretreatment in CaSO₄ solutions (aging) on the absorption of Na and K by bean stem slices was investigated. Freshly sliced tissue absorbed Na over the entire range of concentrations studied (0.02-50 mm). Potassium absorption by fresh tissue was nil at concentrations below 0.5 mm but at higher concentrations was similar to that of Na. When tissue was aged by aerating slices for 20 hr in 0.5 mm CaSO₄, K absorption was substantial over the entire range (0.01-50 mm), with evidence of a dual mechanism of absorption, whereas Na absorption was nil at concentrations below 0.2 mm. The formation of K-absorbing capacity with aging, and the loss of Na-absorbing capacity at low concentrations, were temperature-dependent and did not result from significant changes in rates of efflux of either ion. The absorption of Na by fresh tissue and K by aged tissue was sensitive to antimetabolites, with K uptake the more sensitive. Benzyladenine, an analog of kinetin, suppressed the formation of K-absorbing capability in aged tissue but did not prevent the loss of Naabsorbing capacity. Possible mechanisms for this alteration in ion-specificity of transport mechanisms are discussed.     

The Action of Serotonin on Calcium-45 Efflux from the Anterior Byssal Retractor Muscle of Mytilus edulis

⁴⁵Ca efflux was studied in resting anterior byssal retractor muscle. The data are described by a three-compartment system. The most rapidly exchanging compartment, with an average time constant of 7 min, contains about 0.9 mM Ca/liter muscle, and probably represents extracellular space. A second compartment, with a time constant of 83 ± 5 min, contains 1.2 mM Ca/liter, and may represent a membrane calcium store. The presence of a third, or more, compartments, probably representing sarcoplasmic reticulum and contractile proteins, is indicated by the fact that the final time constant is 10 times the 83 min time constant of the second compartment. Serotonin (5HT), on initial application, increases ⁴⁵Ca efflux from this third compartment(s). This effect has a typical dose-response relationship with a maximum response appearing at 10^-7 M5HT. In addition, removal of 5HT causes a secondary increase in ⁴⁵Ca efflux which has a maximum at a 5HT concentration of 10^-7 M and declines at both higher and lower doses.     

Concentrative accumulation of choline by human erythrocytes

Influx and efflux of choline in human erythrocytes were studied using ¹⁴C-choline. When incubated at 37°C with physiological concentrations of choline erythrocytes concentrate choline; the steady-state ratio is 2.08 ± 0.23 when the external choline is 2.5 µM and falls to 0.94 ± 0.13 as the external concentration is raised to 50 µM. During the steady state the influx of choline is consistent with a carrier system with an apparent Michaelis constant of 30 x 10^-6 and a maximum flux of 1.1 µmoles per liter cells per min. For the influx into cells preequilibrated with a choline-free buffer the apparent Michaelis constant is about 6.5 x 10^-6 M and the maximum flux is 0.22 µmole per liter cells per min. At intracellular concentrations below 50 µmole per liter cells the efflux in the steady state approximates first order kinetics; however, it is not flux through a leak because it is inhibited by hemicholinium. Influx and efflux show a pronounced exchange flux phenomenon. The ability to concentrate choline is lost when external sodium is replaced by lithium or potassium. However, the uphill movement of choline is probably not coupled directly to the Na⁺ electrochemical gradient.     

Quinine and caffeine effects on 45Ca movements in frog sartorius muscle

1 mM caffeine, which produces only twitch potentiation and not contracture in frog sartorius muscle, increases both the uptake and release of ⁴⁵Ca in this muscle by about 50 %, thus acting like higher, contracture-producing concentrations but less intensely. Quinine increases the rate of release of ⁴⁵Ca from frog sartorius but not from the Achilles tendon. The thresholds for the quinine effect on ⁴⁵Ca release and contracture tension are about 0.1 and 0.5 mM, respectively, at pH 7.1. Quinine (2 mM) also doubles the uptake of ⁴⁵Ca by normally polarized muscle. However, there are variable effects of quinine upon ⁴⁵Ca uptake in potassium-depolarized muscle. Quinine (2 mM), increases the Ca, Na, and water content of muscle while decreasing the K content. Both caffeine (1 mM) and quinine (2 mM) act to release ⁴⁵Ca from muscles that have been washed in Ringer''s solution from which Ca was omitted and to which EDTA (5 mM) was added. These results, correlated with those of others, indicate that a basic effect of caffeine and quinine on muscle is to directly release activator Ca²⁺ from the sarcoplasmic reticulum in proportion to the drug concentration. The drugs may also enhance the depolarization-induced Ca release caused by extra K⁺ or an action potential. In respect to the myoplasmic Ca²⁺ released by direct action of the drugs, a relatively high concentration is required to activate even only threshold contracture, but a much lower concentration, added to that released during excitation-contraction coupling, is associated with the condition causing considerable twitch potentiation.