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.     

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.     

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.     

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.     

NOTES ON ULTRASTRUCTURE AND SOME PROPERTIES OF TRANSPORT WITHIN THE LIVING MITOTIC SPINDLE

The sites of lead phosphate precipitation in mouse bladder smooth muscle incubated with adenosine triphosphate and lead nitrate were studied by electron microscopy. The media constituents and incubating conditions were independently varied so that we could determine optimal conditions for histochemical demonstration of ATPase activity in agranular endoplasmic reticulum. Specimens of glutaraldehyde-fixed bladder muscle, frozen, cut into 10–40-µ sections, and incubated for 1 hr at 25°C in medium containing 0.025 M ATP, 0.0025 M lead nitrate, 0.05 M magnesium chloride, and 0.09 M sodium acetate buffer at pH 6.2, exhibited microcrystalline deposits in agranular endoplasmic reticulum and pinocytotic vesicles. Lead salt deposition was also noted in terminal cisternae of sarcoplasmic reticulum in skeletal muscle similarly treated, suggesting that the organelle systems in the two types of muscle cells subserve a common function.     

Sodium and potassium fluxes in isolated barnacle muscle fibers

Sodium and potassium influxes and outfluxes have been studied in single isolated muscle fibers from the giant barnacle both by microinjection and by external loading. The sodium influxes and outfluxes were 49 and 39 pmoles /cm²-sec (temperature = 15–16°C) respectively. The potassium influxes and outfluxes were 28 and 60 pmoles/cm²-sec (temperature = 13–16°C) respectively. Replacement of external sodium by lithium reduced sodium outflux by 67% but had no effect on potassium outflux. Removal of external potassum reduced the sodium outflux by 51% but had no effect on potassium outflux. External strophanthidin (10–30 µM) reduced sodium outflux by 80–90% and increased potassium outflux by 40% in normal fibers. The time constant for sodium exchange increased linearly with internal sodium concentration, as did the fraction of sodium outflux insensitive to a maximally inhibitory concentration of external strophanthidin in the range of 10 tO 80 mM internal sodium. The strophanthidin-sensitive component of sodium outflux could be related to the internal sodium concentration by the following empirical formula: See PDF for Equation     

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.     

Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

Calcium efflux from frog twich muscle fibers

An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of ⁴⁵Ca from twitch muscle fibers. The efflux can be described by three time constants 18 ± 2 min, 300 ± 40 min, and 882 ± 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 ± 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 ± 0.04 pM Ca/cm². sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.     

Sodium flux in the smooth muscle of frog stomach

Sodium efflux from rings of frog stomach muscle was measured at 5° and 15°C in three different steady states. After incubation in normal, K-free, or ouabain (10^-4 M) solutions, intracellular cations stabilized at markedly differing levels. At 5°C, inhibition of Na extrusion was shown in the rate coefficients for ²²Na efflux, which were slightly smaller in K-free than in normal solutions, and much smaller in ouabain. Due to the intracellular Na concentration differences, total Na efflux was similar in K-free and ouabain solutions, and only ⅕ as large in normal solution. At 15°C, normal total Na flux was only 1/7;–1/10 inhibitors, and may be underestimated. The total flux differences may involve dependence of the Na pump and Na permeation on internal Na concentration. The Q ₁₀ of the steady-state fluxes was 3.7 in ouabain, 2.8 in K-free solution, and 1.9 in normal solution. The high temperature dependence of influx as well as efflux suggests transport mechanisms other than simple diffusion. Sodium turnover in the cell water was 46–66 mM/hr in inhibitors at 15°C, and a high rate of Na extrusion in normal muscle is suggested. However, cell volume:surface ratio is only 1.6 µ and all estimates of Na flux were under 3 pmoles/cm² per sec, indicating low Na permeability.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Increased chloride conductance as the proximate cause of hydrogen ion concentration effects in Aplysia neurons

A fall in extracellular pH increased membrane conductance of the giant cell in the abdominal ganglion of Aplysia californica. Chloride conductance was trebled whereas potassium conductance was increased by 50%. Half the giant cells were hyperpolarized (2–8 mv) and half were depolarized (3–10 mv) by lowering the pH. The hyperpolarizing response always became a depolarizing response in half-chloride solutions. When internal chloride was increased electrophoretically, the hyperpolarization was either decreased or changed to depolarization. The depolarizing response was reduced or became a hyperpolarizing response after soaking the cell in 10.0 mM chloride, artificial seawater solution for 1 hr. Depolarization was unaffected when either external sodium, calcium, or magnesium was omitted. A glass micropipette having an organic liquid chloride ion exchanger in its tip was used to measure intracellular chloride activity in 14 giant cells; 7 had values of 27.7 ± 1.8 mM (SEM) and 7 others 40.7 ± 1.5 mM. Three of the first group were hyperpolarized when pH was lowered and three of the second group were depolarized. In all six cells, these changes of membrane potential were in the direction of the chloride equilibrium potential. Intracellular potassium activity was measured by means of a potassium ion exchanger microelectrode.     

Studies on streaming. I. Inhibition of protoplasmic streaming and cytokinesis of Chaos chaos by adenosine triphosphate and reversal by magnesium and calcium ions

In Chaos chaos streaming, motility, and cytokinesis were inhibited nearly 100% for several hours by 2.5–5 mM sodium adenosine triphosphate (ATP)¹ added to culture fluid. All three effects were completely prevented by the addition of equimolar Mg⁺⁺ or Ca⁺⁺ ions but not Na⁺ to the ATP/culture fluid solution. The effects of ATP were not reproduced by EDTA, EGTA, colchicine, or AMP. Sodium pyrophosphate produced about 50% inhibition at 5 mM. Studies with ¹⁴C-ATP showed that 5 x 10^-5 to 5 x 10^-4 mmole of ATP was firmly associated with each milliliter of packed cells after an hour''s incubation at 24°C. Labeling studies also showed that prevention of the ATP effects by Mg⁺⁺ ions was not due to a decrease in the amount of ATP associated with the cells.     

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.     

Caffeine and excitation-contraction coupling in the guinea pig taenia coli

The effects of caffeine (0.2–10 mM) on the electrical and mechanical activities of guinea pig taenia coli were investigated with the double sucrose-gap method. Caffeine evoked a small tension with a latency of 20–30 sec, then phasic contraction developed and finally relaxation. The initial tension development also appeared in the Na-free solution without any marked changes in the membrane potential and membrane resistance. The phasic contraction disappeared in the Na-free solution. The relaxation in the presence of caffeine was accompanied by depolarization block of the spike generation. The minimum concentration of Ca ion needed to evoke the tension development by the caffeine was 10^-7 M. Caffeine also potentiated the twitch tension below a concentration of 5 mM either in the Na-free solution or at low temperature (5°C). NO₃ ^- and Br^- showed a similar response to caffeine on the potentiation of the twitch tension at low temperature.     

Kinetic analyses of calcium movements in cell cultures. 3. Effects of calcium and parathyroid hormone in kidney cells

Calcium compartments and fluxes were measured by kinetic analyses in kidney cell suspensions in a three-compartment closed system. The fast phase influx and compartment size increase linearly with the medium calcium and the half-time of exchange is only 1.3 min which suggests that the fast component is extracellular. The slow phase compartment rises linearly from 0.1 to 0.5 mmole calcium/kg cell water when the medium calcium is raised from 0.02 to 2.5 mM. The slow phase calcium influx exhibits the pattern of saturation kinetics with a V _max of 0.065 µµmole cm^-2 sec^-1 and a K_m of 0.3 mM indicating that it is a carrier-mediated transport process. PTH has no effect on the fast phase of calcium influx, but increases both calcium influx and the calcium pool size of the slow component. The maximum effect is obtained at medium calcium concentration of 1.3 mM. Below 0.3 mM extracellular calcium, the effects of the hormone cannot be demonstrated. PTH increases the V _max of calcium influx from 0.065 to 0.128 µµmole cm^-2 sec^-1 while the K_m rises from 0.3 to 1.15 mM. These findings suggest that PTH increases the translocation of the calcium-carrier complex across the membrane and not the carrier concentration or its binding affinity for calcium.     

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.     

Effects of acriflavine on the mitochondria and kinetoplast of Crithidia fasciculata. Correlation of fine structure changes with decreased mitochondrial enzyme activity

The effects of acriflavine on the fine structure and function of the mitochondria and the kinetoplast in Crithidia fasciculata have been investigated. A mitochondrial fraction was prepared by differential centrifugation of cells broken by grinding with neutral alumina. Isolated mitochondria or intact cells revealed by spectrophotometric measurements the presence of cytochromes a + a ₃, b, c ₅₅₅ and o. After cells were grown in acriflavine for 3–4 days, the fine structure of the mitochondria and their cytochrome content were affected. Cells grown in 5.0 µM acriflavine had a threefold decrease in cytochrome a + a ₃ and decreased respiratory activity. The mitochondrial preparation from these cells had a fivefold decrease in cytochrome a + a ₃ and a less but significant decrease of other cytochromes present. There was also a decrease in the mitochondrial enzyme activities of NADH, succinic and L-α-glycerophosphate oxidases, and succinic and L-α-glycerophosphate dehydrogenases. Dyskinetoplastic cells could be demonstrated after growth in 1.0 µM acriflavine. At 5 µM, 80–90% of the cells were dyskinetoplastic. The kinetoplastic DNA was condensed, nonfibrillar, and did not incorporate thymidine-³H. The mitochondria in these cells had few cristae and were shorter and more swollen than the controls. Acriflavine may induce the fine structure effects we have observed and may affect the formation of the mitochondria in C. fasciculata.