Intracellular Sodium Ion Research Articles

Electrical and biochemical properties of an enzyme model of the sodium pump.

The electrochemical properties of a widely accepted six-step reaction scheme for the Na+, K+-ATPase have been studied by computer simulation. Rate coefficients were chosen to fit the nonvectorial biochemical data for the isolated enzyme and a current-voltage (I-V) relation consistent with physiological observations was obtained with voltage dependence restricted to one (but not both) of the two translocational steps. The vectorial properties resulting from these choices were consistent with physiological activation of the electrogenic sodium pump by intracellular and extracellular sodium (Na+) and potassium (K+) ions. The model exhibited K+/K+ exchange but little Na+/Na+ exchange unless the energy available from the splitting of adenosine triphosphate (ATP) was reduced, mimicking the behavior seen in squid giant axon. The vectorial ionic activation curves were voltage dependent, resulting in large shifts in apparent Km's with depolarization. At potentials more negative than the equilibrium or reversal potential transport was greatly diminished unless the free energy of ATP splitting was reduced. While the pump reversal potential is at least 100 mV hyperpolarized relative to the resting potential of most cells, the voltage-dependent distribution of intermediate forms of the enzyme allows the possibility of considerable slope conductance of the pump I-V relation in the physiological range of membrane potentials. Some of the vectorial properties of an electrogenic sodium pump appear to be inescapable consequences of the nonvectorial properties of the isolated enzyme. Future application of this approach should allow rigorous quantitative testing of interpretative ideas concerning the mechanism and stoichiometry of the sodium pump.

Relation between intracellular sodium and twitch tension in sheep cardiac Purkinje strands exposed to cardiac glycosides.

Changes in intracellular sodium ion activity (aiNa) produced by several cardiac glycosides were correlated with twitch tension in sheep cardiac Purkinje strands. Simultaneous measurements of aiNa and twitch tension were obtained through the use of Na-sensitive intracellular microelectrodes (ETH 227) in Purkinje preparations stimulated at a frequency of 1 Hz. All concentrations of ouabain, acetylstrophanthidin, and actodigin that were tested caused an increase in aiNa immediately before, or coincident with, a positive inotropic effect. No fall in aiNa was observed at any positive inotropic concentration of digitalis in these beating fibers. In all cases, the onset and washout of the positive inotropic effect were paralleled by the rise and fall in aiNa, respectively. No dissociation between changes in aiNa and twitch tension occurred at any concentration of any of the agents used. The relation between changes in aiNa and twitch tension was linear with 1 mM increase in aiNa producing about a 100% increase in the twitch magnitude. Propranolol did not significantly alter this relationship. The increase in aiNa with digitalis was also associated with a reduction in the maximum depolarization rate of the action potential, presumably as a consequence of a reduction in the transmembrane Na electrochemical gradient. These results indicate that the positive inotropic action of digitalis in sheep Purkinje strands is always associated with a rise in aiNa secondary to inhibition of the Na pump. This increase in aiNa could increase calcium available for contraction via the Na-Ca equilibrium exchange process. In addition, the increase in aiNa reduces Vmax, as a consequence of decreasing the electrochemical gradient for Na.

Changes of intracellular sodium and potassium ion concentrations in frog spinal motoneurons induced by repetitive synaptic stimulation

A post-tetanic membrane hyperpolarization following repetitive neuronal activity is a commonly observed phenomenon in the isolated frog spinal cord as well as in neurons of other nervous tissues. We have now used double-barrelled Na +- and K +-ion-sensitive microelectrodes to measure the intracellular Na +- and K +-concentrations and also the extracellular K +-concentration of lumbar spinal motoneurons during and after repetitive stimulation of a dorsal root. The results show that the posttetanic membrane hyperpolarization occurred at a time when the intracellular [Na +] reached its maximal value, intracellular [K +] had its lowest level and extracellular [K +] was still elevated. The hyperpolarization was blocked by ouabain and reduced by Li +. These data support the previous suggestion that an electrogenic Na +/K + pump mode may be the mechanism underlying the post-tetanic membrane hyperpolarization.

Potassium leakage: not the cause of gossypol induced anti-motility in spermatozoa.

Gossypol induced a dose‐dependent potassium leakage from boar spermatozoa suspended either in Krebs‐Ringer phosphate buffer or in isotonic tris‐HCl buffer. It had, however, practically no effect on the spermatozoal intracellular sodium ion content. As spermatozoal motility arrest was observed at a much lower gossypol concentration, the gossypol anti‐motility effect does not seem to have stemmed from potassium leakage.

Increase in intracellular sodium ion activity during stimulation in mammalian cardiac muscle.

Changes in stimulation rate alter the electrical and mechanical characteristics of myocardial cells. We have investigated the possibility that intracellular sodium activity (aiNa) changes with stimulation and correlates with changes in contraction strength. Two kinds of liquid membrane Na+-selective microelectrodes were used to measure aiNa in guinea pig and sheep ventricular muscle and in sheep Purkinje strands. Stimulation produced a rate- and time-dependent elevation of aiNa. Small increases in aiNa were seen at stimulation rates as slow as 0.2 Hz, and faster rates of stimulation elevated aiNa by over 30%. The changes seen in Purkinje strands and ventricular muscle were similar. Following a period of stimulation, aiNa and Vm returned to their pre-stimulus levels with the same time courses. This is consistent with the suggestion that the post-stimulation hyperpolarization is the result of an increased rate of electrogenic Na+ extrusion. The effects of stimulation on aiNa and tension were compared with those of ouabain. The comparison suggests that rapid stimulation could produce increased contraction strength as the result of a substantial gain in intracellular calcium via a Na-Ca exchange mechanism, but that this is only one of several factors determining the force-frequency relationship.

The role of intracellular sodium ions in the regulation of cardiac contractility

It has been known for a long time that the contractility of cardiac muscle depends on the pattern of stimulation [ 2, 3, 15], but the underlying mechanisms are still a matter of controversy [ 14]. Here I wish to present experimental evidence that both the “positive inotropic effect of activation” (PIEA) [ 2] and the positive inotropic effect of cardiac glycosides may be the result of a change in intracellular sodium activity. This conclusion is based on the direct measurement of the time course of (i) the blockage of the electrogenic pump current by a fast acting cardiac glycoside, dihydro-ouabain, (ii) the resulting change in intracellular sodium activity, (iii) the accompanying change in contractility, and (iv) the decay of the PIEA in guinea-pig ventricular muscle.

Intracellular sodium ion activity and sodium transport in rabbit urinary bladder.

1. Intracellular potentials and the intracellular activities of Na+ and K+ were examined using conventional and ion-selective micro-electrodes. 2. In animals on a normal diet, the intracellular Na+ activity was 8.6 +/- 2.9 mM (mean +/- S.D.) with a mean short-circuit current of 2.8 +/- 0.9 microA/cm2. 3. In animals on a low-Na+ diet, the intracellular Na+ activity was 18.5 +/- 9.9 mM with a short-circuit current of 4.5 +/- 1.3 microA/cm2 (mean +/- S.D.). 4. There was a correlation between short-circuit current and intracellular Na+ activity which could be fitted by a saturating hyperbolic relationship. 5. Treatment of the issue with ouabain and amiloride produced an increase and a decrease, respectively, in the intracellular Na+ activity. 6. Treatment with aldosterone produced a large increase in short-circuit current with a substantial increase in intracellular Na+ activity. 7. Intracellular Na+ activity does not seem to affect apical membrane permeability directly.

Insulin does not act by causing a change in membrane potential or intracellular free sodium and potassium concentration of adipocytes.

The influence of insulin on the intracellular free sodium and potassium ion concentrations ([Na+]i, [K+]i) and resting membrane potential of rat epididymal adipocytes was examined to determine its potential for mediating insulin's action on other cellular processes. Direct intracellular measurements of [Na+]i, [K+]i, and the resting membrane potential were made using ion-selective and conventional microelectrodes. The use of these microelectrodes enabled us to continuously monitor these parameters in the same cell before, during, and after periods of insulin stimulation of up to 20-min duration. The electrical potentials measured in these experiments remained unchanged when the cells were stimulated with insulin (0.01, 0.1, or 1.0 mU/ml). Varying the extracellular glucose concentration had no effect on these results. Our results provide the first direct measurement of [Na+]i and [K+]i in adipose tissue and clearly demonstrate that the response to insulin's association with its receptors on the cell surface and subsequent action on hexose transport and cellular metabolism does not involve a change in membrane potential or intracellular sodium and potassium ions.

The effect of calcium injection on the intracellular sodium and pH of snail neurones.

1. Ion-sensitive glass micro-electrodes were used to measure the intracellular pH (pHi) and the intracellular sodium ion concentration, [Na+]i, in identified Helix aspersa neurones. 2. The injection of small volumes of 0-1 McaCl2, which increased the membrane potential by 10-15 mV for 1-2 min, had little or no effect on [Na+]i. Increases of up to 1 mM in [Na+]i could be reversibly induced by larger injections. 3. Calcium injection caused an immediate decrease in pHi, which appeared to be directly proportional to the amount of calcium injected. Injections causing hyperpolarizations of 10-20 mV which recovered in 2-5 min caused pHi decreases of 0-04-0-15 units. After each of these injections both pHi and the membrane potential recovered exponentially but with different time constants. 4. The injection of calcium at a low rate could decrease pHi without affecting the membrane potential. 5. Neither membrane potential nor pHi were affected by the injection of small volumes of 0-1 M-MgCl2, Injection of CoCl2 produced a large transient decrease in pHi but no significant change in membrane potential. 6. Exposure of the cell to saline equilibrated with 2-5% CO2 greatly reduced the pHi decrease caused by calcium injection but had only small effects on the membrane potential response. 7. It is concluded that most of the injected calcium is exchanged for protons inside the cell.

Induction of mitosis in mature neurons in central nervous system by sustained depolarization.

DNA synthesis and mitosis have been induced in vitro in fully differentiated neurons from the central nervous system by depolarization with a variety of agents that produce a sustained rise in the intracellular sodium ion concentration and a decrease in the potassium ion concentration. Depolarization was followed in less than 1 hour by an increase in RNA synthesis and in 3 hours by initiation of DNA synthesis. Apparently normal nuclear mitosis ensued, but cytokinesis was not completed in most cells; this resulted in the formation of binucleate neurons. The daughter nuclei each contained the same amount of DNA as the diploid preinduction parental neurons; this implies that true mitogenic replication was induced.

Potential-dependent membrane sodium pump current in snail giant neurons

The effect of the membrane potential on the pump current evoked by iontophoretic injection of sodium into the neuron and the effect of the intracellular sodium ion concentration on the potential dependence of the pump current were investigated by the voltage clamp method in isolated and semi-isolated neurons ofHelix pomatia andHelix italiana. The pump current was shown to change its direction in the presence of marked hyperpolarization of the membrane (by more than −80 to −120 mV). An increase in the intracellular sodium ion concentration following injection of excess ions into the neuron increases the potential dependence of the pump current. A possible connection between passive potassium permeability and the activity of the enzymic transport mechanism for the elimination of sodium from the cell is postulated.

Changes of intracellular sodium and potassium ion concentrations in isolated rat superior cervical ganglia induced by depolarizing agents.

1. Na and K contents of isolated rat superior cervical ganglia were measured by flame photometry, and intracellular Na and K concentrations ([Na](i) and [K](i)) calculated using Li and (35)SO(4) to determine extracellular space (e.c.s.).2. Resting concentrations after 1-2 hr incubation at 25 degrees C in normal Krebs solution were: [Na](i), 19.8 +/- 0.9 m-mole (kg cell water)(-1); [K](i), 192.7 +/- 2.8 m-mole (kg cell water)(-1) (mean +/- S.E. of mean of thirty-five ganglia). Correction for losses during e.c.s. measurement gave 22 mM [Na](i) and 207 mM [K](i) as probable fresh concentrations.3. Carbachol (180 muM for 4 min) increased [Na](i) by 47.8 +/- 2.9 m-mole (kg cell water)(-1) and decreased [K](i) by 54.6 +/- 4.3 m-mole (kg cell water)(-1). Maximal exchange with carbachol or nicotine (at approximately 1 mM for 4 min) amounted to 80-100 m-mole (kg cell water)(-1). On washing with Krebs solution containing 2.5 mM hexamethonium recovery of ionic concentrations occurred with a rate constant of 0.3-0.4 min(-1).4. Restitution of ganglionic Na and K after carbachol was inhibited by washing with K-free solution, and slowed by ouabain (0.14 mM), cyanide (2 mM) or cooling (Q(10) 2.7 between 17 and 27 degrees C).5. Equilibrium potentials for Na and K (E(Na), E(K)) at rest were calculated to be +49 and -88 mV. At a membrane potential (E(m)) of -70 mV, the permeability ratio P(Na):P(K) was calculated at 0.04:1 (assuming P(Cl):P(K) < 0.1).

An analysis of the leakages of sodium ions into and potassium ions out of striated muscle cells.

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na](i), and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na](i) and a component that increased linearly with increasing [Na](i). Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na](o), and a steady-state balance of the sodium ion fluxes occurred at [Na](o) = 40 mM. When solutions were K-free and contained 10(-4) M ouabain, net sodium influx varied linearly with [Na](o) and a steady state for the intracellular sodium was observed at [Na](o) = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na](i) but dependent on [Na](o). Replacing external NaCl with MgCl(2) brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na](o) = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl(2). When muscles were enriched with sodium ions, potassium efflux into K-free, Mg(++)-substituted Ringer solution fell to around 0.1 pmol/cm(2).s and was increased 14-fold by addition of ouabain.

The molecular mechanism of cardiac glycoside action

The sequence of events leading to cardiac muscle contraction is: (1) passive diffusion of sodium ions intracellularly and potassium ions extracellularly ( depolarization), (2) release of bound calcium ions from the sarcoplasmic reticulum, (3) formation of a calcium-troponin-tropomyosin complex which frees acceptor sites on actin for interaction with the cross-bridges on myosin, (4) myosin ATPase induces the interaction of actin and myosin thereby causing a contraction, (5) active pumping mechanism—“sodium pump”—for the cellular removal of sodium ions and the cellular reentry of potassium ions by cell membrane ATPase ( repolarization occurs during this process), and (6) active pumping of calcium ions from the actomyosin complex—followed by muscle relaxation. Cardiac glycosides interfere with the sequence of events leading to cardiac muscle contraction by inhibiting cell membrane ATPase (“sodium pump”). This results in: (1) a high intracellular sodium ion concentration, (2) the prevention of the intracellular influx of potassium ions, and (3) an apparent increase in the “free” intracellular calcium ion concentration thereby causing a net increase in myocardial contraction through the interaction of calcium ions and the contractile proteins.

Studies on Ion Distribution in Living Cells: II. Cooperative Interaction between Intracellular Potassium and Sodium Ions

New steady levels of K(+) and Na(+) ion in frog sartorius muscle were reached in 72 hr at 25 degrees C in environments containing 100-mM Na(+)-ion and K(+)-ion concentrations varying from near zero to 10 mM. These steady levels follow a pattern predicted by a cooperative adsorption isotherm presented in 1964. From a total of 13 sets of experiments carried out over a five year period, the average concentration of adsorption sites is 109 mumoles/g of fresh cells. The average intrinsic equilibrium constant for the Na --> K exchange is 135, and the average free energy of nearest neighbor interaction is 0.54 kcal/mole. These values are in good agreement with those obtained by Jones and Karreman from studies on canine carotid arteries.

Sodium ions and the secretion of catecholamines.

1. Perfusion of bovine adrenal glands with a potassium-free Tyrode solution results in an increased spontaneous release of catecholamines and an increased response to stimulation with carbamylcholine.2. Perfusion with a Tyrode solution containing 70 mM-KCl causes a marked secretion of catecholamines and a subsequent inhibition of secretion in response to stimulation with carbamylcholine.3. Perfusion with sodium-free media abolishes or severely reduces the secretory response of the gland to carbamylcholine and to 70 mM-KCl although the basal secretion shows an initial rise.4. Perfusion with sodium-deficient media permits some retention of the secretory response.5. A possible role for intracellular sodium ions in the secretion of catecholamines is discussed.

Ouabain and conduction velocity at different heart frequencies in rabbit auricles.

IN the crista terminalis of isolated rabbit auricles we observed a relationship between the frequency of stimulation and the velocity of conduction, together with a relationship between the frequency of stimulation and the maximal rate of rise of the transmembrane action potential1. An increase in the frequency of stimulation causes an exponential decrease in both the maximal rate of rise and the velocity of conduction. The change in the velocity of conduction is most likely a direct result of the change in the maximal rate of rise2. An augmented intracellular sodium ion concentration caused by a relative insufficiency of the sodium–potassium pump might explain this phenomenon.

Intracellular sodium ion concentration changes in the early amphibian embryo and the influence on nuclear metabolism

During early cleavage the total sodium and potassium concentrations of the R. pipiens embryo remain relatively constant at 70 m M and 100 m M, respectively, per kg dry weight. As the blastocoel forms, intracellular sodium decreases to about 40 m M with no significant change in intracellular potassium. The sodium which leaves the cells can be recovered in the blastocoel fluid. As a result of the redistribution of sodium, the Na K ratio in the blastocoel fluid resembles that of the adult plasma. Thus, prior to gastrulation, the cells lining the blastocoel become exposed to the medium in which differentiated cells normally function, and the intracellular Na K ratio approaches adult cell values. Studies with isolated late blastula stage nuclei indicate that 14C-labelled tryptophan uptake into both the protein and acid-soluble pool is maximal at extranuclear sodium concentrations of about 40 m M but markedly reduced at 70 m M sodium concentrations. One of the factors permitting nucleo-cytoplasmic interactions at the beginning of gastrulation may be a decrease in the cytoplasmic sodium ion concentration.

Membrane lipid depletion in hyperpermeable red blood cells: its role in the genesis of spherocytes in hereditary spherocytosis.

Hereditary spherocytosis (HS) red cells lose membrane lipids excessively during incubation in vitro. Individual phosphatides as well as cholesterol are lost in proportion to their content in membranes, suggesting that fragments of membrane are removed. Supplementation of HS red cells with glucose during incubation has no consistent protective effect, whereas diminishing the excessive sodium flux through these cells by suspending them in either sodium-free or hypertonic media prevents membrane fragmentation. The characteristic excessive increase in osmotic fragility which occurs in incubated HS red cells results both from inordinate accumulation of intracellular sodium ions which produces osmotic swelling, and from depletion of surface material which generates microspherocytosis. Inhibiting both of these processes by incubating HS red cells in sodium-free media completely prevents increases in osmotic fragility despite prolonged incubation. Normal red cells rendered hyperpermeable to cations by exposure either to n-butanol or to inhibitors of membrane sulfhydryl groups, lose membrane lipid upon incubation in a similar fashion to untreated HS red cells; perfectly smooth microspherocytes, akin to those seen in HS, are thereby generated.I conclude that depletion of membrane lipids in HS which leads to microspherocytosis is correlatable with the excessive cation flux and possibly to the stimulated metabolism of acidic phosphatides in these red cells. It is suggested that this relation is derived from the fact that these phosphatides are in some way involved in maintaining the proper alignment of repeating membrane lipoprotein units, and that this function is adversely affected when these molecules are turning over more rapidly in response to increased cation flux.

INTERRELATION OF POTASSIUM AND HYDROGEN ION GRADIENTS IN METABOLIC ALKALOSIS.

To study the interrelationships of potassium and hydrogen ion intra- and extracellularly, as an extension of previous investigations on gastric alkalosis, metabolic alkalosis was produced in dogs by dietary K depletion, NaHCO3 loading, and deoxycorticosterone administration, with and without chloride depletion. Analysis of skeletal muscle and measurement of intracellular pH confirmed the development of cellular K depletion, with increases of intracellular sodium and hydrogen ion associated with extracellular alkalosis and hypokalemia. These changes were not influenced by concomitant dietary chloride restriction. The intracellular K concentration was linearly and inversely related to the intra-extracellular hydrogen ion gradient. The intra-extracellular K gradient was about 10 times greater than the corresponding hydrogen ion gradient. These respective gradients were linearly related, in this proportion, over the range of K depletion studied in this type of metabolic alkalosis as well as that resulting from gastric juice loss. Deviation from this relationship occurred only with the most severe K depletion. These experimental observations demonstrate in vivo that the potassium and hydrogen ion gradients change in the same direction and are nearly constantly related in at least two types of metabolic alkalosis.