Intracellular Sodium Concentration Research Articles

Sodium is elevated in mdx muscles: Ionic interactions in dystrophic cells

Recent evidence suggests that cellular sodium regulation may be abnormal in muscular dystrophy. We have measured intracellular sodium concentration (Na i) in muscles of mdx mice (a model of Duchenne muscular dystrophy) using two techniques. Na i in isolated diaphragm (measured using a microelectrode) was 13.0 ± 0.3 mM and 23.5 ± 0.7 mM (mean ± SE) in the control and mdx mice respectively. Na i in gastrocnemius muscle (calculated from extra- and intracellular volumes using serum and whole-muscle sodium concentrations) was 13 ± 3 mM and 24 ± 2 mM (mean ± SE) in control and mdx, respectively. We argue that this abnormality in mdx tissues could reflect a reduced flux through the Na K ATPase, although a contribution from increased Na leak cannot be ruled out. We also discuss possible consequences of an increased Na i: for example, raised Na i may lead to defective cell volume control in Duchenne dystrophy and the mdx mouse.

Effects of Verapamil, Ouabain, and Ethacrynic Acid on Calcium Retention in Perfused Rat Kidneys

A Sprague‐Dawley rat kidney perfusion technique was used in situ to study the effect of verapamil, ouabain, and ethacrynic acid on renal calcium retention. The technique involves perfusion of the kidneys via the abdominal aorta and then through the left and right renal arteries and dorsal aorta. Verapamil (1mM) in Krebs‐improved Ringer solution increased calcium retention in the kidneys by ~ 117% compared with controls. With Na‐free Krebs‐improved Ringer solution, calcium retention increased by only 92%. However, in Krebs‐improved Ringer solutions containing 59 and 122 mequivalents of Na, calcium retention in the kidney increased by 46 and 43%, respectively, compared with controls. With Krebs‐improved Ringer solution containing 15mM ouabain, calcium retention in the kidney decreased by 29.5%, whereas with 15mM ouabain plus 1mM ethacrynic acid in the perfusate, the effect on calcium retention in the kidney was insignificant compared with controls. These results suggest that two sodium‐dependent calcium‐transporting systems exist at the peritubular side of the kidney tubules: (1) a Na+–Ca+2 countertransport system sensitive to verapamil and (2) a Na+–Ca+2 cotransport system sensitive to the intracellular concentration of sodium.

Bretylium-induced voltage-gated sodium current in human lymphocytes

Using the whole-cell variation of the patch-clamp technique it has been determined that 0.25–3 mM bretylium tosylate (BT) exerts a repolarizing effect on partially depolarized human lymphocytes. The repolarizing effect was ouabain (40 μM)-sensitive, and was inhibited by the removal of external Na + or by the Na +-channel-blocker amiloride (10–44 μM), but K +-channel-blockers 4-aminopyridine (0.1–5 mM) and quinine (100 μM) had no effect. The drug induced a sodium dependent, amiloride-sensitive transient inward current reaching its maximum value approx. 20–30 s after the administration of BT and lasting for 6–10 min. This current was activated by depolarization within 25 ms at around −42 mV, its inactivation took about 2 s and its reversal potential was + 24 ±5 mV. An increase in the intracellular sodium concentration (1.8–3.2 mM) has been observed upon the addition of BT by monitoring the SBFI fluorescence of the dye-loaded cells. It has been shown that whole-cell K + currents are significantly decreased by BT. The existence of voltage and ligand (BT)-gated sodium channels has been postulated in human lymphocytes. These channels are thought to participate in the initiation of membrane repolarization in human lymphocytes, and thereby influence mitogenic or antigen-induced cell-activation processes.

Characteristics of extracellular sodium relaxation in perfused hearts with pathologic interventions.

Sodium spectroscopy and imaging sequences designed to emphasize fast T2 decay or the multiple quantum signal have previously demonstrated a high contrast between normal and pathologic tissue which may be due to changes in intracellular versus extracellular sodium distribution. Since alterations in the amount of signal with fast T2 decay have previously been shown to occur with changes in intracellular sodium content, this study investigated the fast T2 relaxation characteristics of extracellular sodium during pathologic interventions on nonsubmerged perfused rat hearts. T2 data on total sodium content were obtained while global ischemia (stopping all perfusate flow) and extracellular edema (due to long perfusion times) were induced in the heart. The data were fit to a biexponential, with Mf(T2f) the magnitude (time constant) of the fast component of decay. Mf increased significantly in both pathologies (to 319 +/- 26%, n = 3, of baseline for ischemia and to 527 +/- 284%, n = 3, of baseline for edema); the increase with edema was demonstrated to be due to extracellular sodium by intermittently perfusing the heart for a short period with shift reagent. When shift reagent was not used until the conclusion of the edema experiment, Mf increased to 169 +/- 35% of baseline, also due mainly to extracellular sodium. T2f did not exhibit any trends with these experiments, with values ranging from 1.7 to 5.5 ms. We believe that these results indicate that compartmental sodium content will most likely not be quantifiable in pathologic states in the heart with relaxation-based techniques. However, correlations between the pathologic state of the tissue and the sodium NMR signal obtained with pulse sequences or images that emphasize a particular aspect of relaxation may prove to be useful.

Ouabain-induced excitation of colonic smooth muscle due to block of K + conductance by intracellular Na + ions

The mechanism by which ouabain causes excitation of canine colonic circular smooth muscle was investigated. Ouabain-induced depolarization and increase in contractility were related to the concentration of extracellular sodium and prevented by complete substitution of sodium ions with N-methyl-D-glucamine or lithium ions. Absence of external sodium ions did not prevent the depolarization and increase in contractility induced by tetraethylammonium. Exposure of the muscle strips to sodium-free solutions produced a transient hyperpolarization and decrease in the input membrane resistance consistent with the hypothesis that intracellular sodium blocks potassium conductance. The relationship between the membrane potential and the extracellular potassium concentration indicated that the resting membrane potential is mainly determined by the membrane potassium conductance. Our data suggest the following mechanism of action for ouabain: (a) ouabain blocks Na +/K + pump thereby increasing the intracellular sodium concentration; (b) increase in intracellular sodium inhibits membrane potassium conductance, which depolarizes the membrane and prolongs the slow wave plateau, resulting in an increase of the force of contraction. The direct contribution of the sodium pump to the resting membrane potential, if any, can only be minor (< 6 mV).

Sodium pump activation by 5-hydroxytryptamine in human placental veins

The aim of the present study was to determine the influence of the sodium pump on the responses elicited by 5-hydroxy tryptamine (5-HT) in segments of human placental veins. 5-HT (10−9−3 × 10−7M) elicited potent concentration-dependent vasoconstrictor responses, but a fall in tone was observed at higher concentrations. In the presence of 10−7 M ouabain, this fall in tension was abolished. A single concentration of 5-HT (10−6 M) produced a biphasic response, consisting in a fast early contraction followed by a slow relaxation. This relaxant phase was concentration dependently inhibited by ouabain (10−7 and 10−6 M), and abolished by preincubating the vessels in a K+-free solution and reducing bath temperature to 28°C, methods usually employed to inhibit the sodium pump. After adding 7.5 mM K+ or returning the temperature to 37°C, marked relaxation was observed. On the other hand, the relaxant phase with the amine remained unchanged by pretreatment with phenidone, oxyhemoglobin, indomethacin (all at 10−5 M) and endothelium removal. 5-HT (10−7 and 10−6M) elicited increases in ouabain-sensitivc 86Rb+ uptake. These results suggest that: (1) 5-HT activates Na+K+-ATPase, likely by an indirect mechanism that involves an increase of intracellular sodium concentration; and (2) the relaxant phase of 5-HT-evoked vasoactive responses is not mediated by the release of nitric oxide or prostacyclin from the endothelium.

Concentration of sodium and free amino acids in the haemolymph and other fluid compartments in an adephagous and a polyphagous species of beetle

1. 1. The extracellular and intracellular concentrations of sodium and free amino acids were determined in the adephagous beetle Carabus nemoralis and the polyphagous beetle Rhagium inquisitor acclimated to 0 and 25°C. 2. 2. C. nemoralis had substantially higher extracellular sodium concentrations than R. inquisitor, and also substantially lower extracellular concentrations of free amino acids. 3. 3. The possible mechanistic relationship between the transmembrane sodium distribution and the distribution of free amino acids via symport mechanisms is discussed.

Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK1 cells.

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Pollutant-induced depression of the transmembrane sodium gradient in muscles of mussels.

This study deals with the effects of chemical pollutants on the transmembrane potential difference for sodium (delta mu Na) in smooth muscle cells of Mytilus edulis. A method for indirect determination of extracellular space, intracellular ion concentrations and delta mu Na has been developed and is applied in the investigations. The determination is based on concentration data from haemolymph and muscle tissue samples. The precision of the method used was tested by direct measurements of the apparent intracellular concentration of sodium and the membrane potential. On the basis of these tests, the method was evaluated as reasonably good. The method was used to study the sensitivity of the transmembrane delta mu Na in Mytilus edulis to 96 h exposures to various sublethal concentrations of formaldehyde, methanol and mercury. Both formaldehyde and mercury induced a depression of delta mu Na. The observed depressions could be ascribed to a change in both the electrogenic and the chemical components of delta mu Na. A depression of delta mu Na was associated with subsequent clinical injury and death. Methanol did not cause death or any changes in delta mu Na. Because of the observed correlation between depression of delta mu Na and clinical injury, delta mu Na is suggested to have a potential as an indicator of toxicity.

Effects of Temperature on the (Na++ K+)-ATPase and Oxygen Consumption in Hepatocytes of Two Species of Freshwater Fish, Roach (Rutilus rutilus) and Brook Trout (Salvelinus fontinalis)

Effects of temperature on the (Na+ + K+)-ATPase were studied in isolated hepatocytes of two species of freshwater fish acclimated to different temperatures. Binding of 3H+-ouabain to hepatocytes was used to determine the density of (Na+ + K+)-ATPase, and oxygen consumption was measured to estimate the cost of cation pumping. Intracellular sodium concentration was manipulated by incubating hepatocytes at 20°C or 0°-4°C before the measurement of oxygen consumption. In hepatocytes of Rutilus rutilus, the maximal number of ouabain binding sites per 10⁶ cells (Bmax) was 1.86 times higher in the 5° C- acclimated than in the 20° C-acclimated group. The equilibrium dissociation constants (Kd's) in both acclimation groups were not statistically different. In Salvelinus fontinalis,Bmax and Kd values did not change with temperature acclimation. Ouabain-sensitive oxygen consumption (OS) in hepatocytes of R. rutilus was 1.60-1. 73 times higher in the cold- than in the warm-acclimated group. In S. fontinalis, acclimation temperature did not affect OS. On the other hand, cold preincubation of hepatocytes significantly increased OS in both acclimation groups and species. Results are discussed with regard to species-specific differences in the adaptation to temperature change at the cellular level, the main point being that in response to cold-acclimation R. rutilus adjusts the activity of the (Na+ + K+)-ATPase in a compensatory fashion, whereas S. fontinalis modulates passive ion leakage.

Time course of sodium-induced Na(+)-K(+)-ATPase recruitment in rabbit cortical collecting tubule.

In cortical collecting tubules (CCD) of aldosterone-repleted rabbit kidney, an increase in intracellular sodium concentration (Nai) induces the recruitment and/or activation of latent Na(+)-K(+)-ATPase pumps (Blot-Chabaud et al., J. Biol. Chem. 265: 11676-11681, 1990). The present study was addressed to determine the time course of this Nai-dependent pump recruitment and to examine some of the factors possibly involved in this phenomenon. CCD from adrenalectomized rabbits complemented with aldosterone and dexamethasone were incubated at 4 degrees C either in a K(+)-free saline solution (Na(+)-loaded CCD) or in a sucrose solution (control CCD) and then rewarmed for various time periods to allow pump recruitment to occur. The number of pumps in the membrane was determined by specific [3H]ouabain binding; Nai was measured using 22Na. A rise in Nai induced a threefold increase in the number of basolateral pumps, which was fully achieved within 1-2 min. This pump recruitment was reversible within 15 min after restoration of low Nai. It was unaffected by inhibitors of cytoskeleton and Ca2+ ionophore A 23187. The blocker of the Na(+)-H+ antiporter, amiloride, did not prevent it. The protein kinase C activator, phorbol 12-myristate 13-acetate, did not induce it in the absence of Na+. We conclude that Nai is a major determinant of pump recruitment and/or activation, which occurs over a very short period of time. It may constitute a rapid adaptative response to an increase in the cell Na+ load.

Ouabain enhancement of compound 48/80 induced histamine secretion from rat peritoneal mast cells: dependence on extracellular sodium.

Purified populations of rat peritoneal mast cells were used to study the effect of ouabain on compound 48/80-induced histamine secretion and on 86Rb+ uptake. 86Rb+ was used as a tracer for extracellular K+. The calculated value of the ouabain-sensitive uptake of K+ and 86Rb+ was considered a measure of the Na(+)-K+ pump activity of the cells. Ouabain caused an immediate inhibition of the pump activity and a time-dependent increase in histamine secretion in the absence of extracellular calcium. No effect on the secretion was observed in the presence of calcium. The effect of ouabain on the secretion occurs in the presence of sodium but not when sodium was replaced by lithium. Preservation by ouabain of a high intracellular sodium content in sodium-loaded cells was associated with preservation of the secretory response in a calcium-free medium. In the presence of lanthanum in a calcium-free medium, the pump activity was inhibited and the enhancement by ouabain of the secretion of histamine was blocked. A less marked inhibition of the pump was found in a calcium-free medium containing magnesium. The inhibition exerted by magnesium was concentration-dependent (0-5 mM) as was the counteraction of magnesium of the enhancement of ouabain of the secretion of histamine. These observations indicate that the enhancement by ouabain of the secretory response of mast cells preincubated in a calcium-free medium is associated with accumulation of sodium inside the cell. In addition to a decreased rate of sodium-calcium exchange caused by a decreased inward directed sodium gradient, the mechanism by which ouabain enhances the secretory response is likely to involve an increased binding of calcium to membrane binding sites.

Effect of elevated potassium on the ion content of mouse astrocytes and neurons.

Potassium is tightly regulated within the extracellular compartment of the brain. Nonetheless, it can increase 3- to 4-fold during periods of intense seizure activity and 10- to 20-fold under certain pathological conditions such as spreading depression. Within the central nervous system, neurons and astrocytes are both affected by shifts in the extracellular concentration of potassium. Elevated potassium can lead to a redistribution of other ions (e.g., calcium, sodium, chloride, hydrogen, etc.) within the cellular compartment of the brain. Small shifts in the extracellular potassium concentration can markedly affect acid-based homeostasis, energy metabolism, and volume regulation of these two brain cells. Since normal neuronal function is tightly coupled to the ability of the surrounding glial cells to regulate ionic shifts within the brain and since both cell types can be affected by shifts in the extracellular potassium, it is important to characterize their individual response to an elevation of this ion. This review describes the results of side-by-side studies conducted on cortical neurons and astrocytes, which assessed the effect of elevated potassium on their resting membrane potential, intracellular volume, and their intracellular concentration of potassium, sodium, and chloride. The results obtained from these studies suggest that there exists a marked cellular heterogeneity between neurons and astrocytes in their response to an elevation in the extracellular potassium concentration.

Erythrocyte Sodium-Potassium Transport in Cystic Fibrosis

Erythrocytes from 15 patients with cystic fibrosis (CF) aged 8 mo to 22 y (mean age 12.8 y) were analyzed for Na+,K(+)-ATPase activity and sodium, potassium, and ATP concentrations. Sodium concentrations and Na(+)-K+ ratio of erythrocytes were statistically significantly lower in the CF patients [6.6 (SD 1.9) versus 9.2 (SD 1.1) mmol/L (p less than 0.01) and 0.070 (SD 0.023) versus 0.104 (SD 0.016) mmol/L (p less than 0.01), respectively]. The Na+,K(+)-ATPase activity was similar compared with that of reference individuals [536 (SD 100) versus 488 (SD 92) nmol inorganic phosphate/mg protein/h]. Intraerythrocyte sodium concentration and Na(+)-K+ ratio were thus lower in relation to the recorded Na+,K(+)-ATPase activities in controls, indicating a change of the passive transmembrane movements of sodium ions in CF. There was a rise of erythrocyte sodium and Na(+)-K+ ratio despite unchanged Na+,K(+)-ATPase activity after regular infusion of a fat emulsion rich in essential fatty acids, inferring that an altered membrane composition by essential fatty acid deficiency could explain the low intracellular sodium concentration in CF.

Papaverine reduces the sodium permeability of the apical membrane and the potassium permeability of the basolateral membrane in isolated frog skin.

The effect of papaverine, an inhibitor of the phosphodiesterase responsible for breakdown of cAMP, on the transepithelial sodium transport across the isolated frog skin was investigated. Serosal addition of papaverine caused initially an increase in the short-circuit current (SCC), a doubling of the cellular cAMP content and a depolarization of the intracellular potential under SCC conditions (Vscc). The initial increase in the SCC was followed by a pronounced decrease both in the SCC and in the natriferic action of antidiuretic hormone (ADH), but papaverine had no inhibitory effect on the ability of ADH to increase the cellular cAMP content. As SCC declines, no hyperpolarization was observed. The I/V relationship across the apical membrane during the inhibitory phase, revealed that papaverine reduces the sodium permeability of the apical membrane (PNaa) as well as intracellular sodium concentration. These observations and the previously noted effect of papaverine on Vscc indicates that papaverine must have an effect on the cellular Cl or K permeability. The basolateral Na,K,2Cl cotransporter was blocked with bumetanide, which should bring the cellular chloride in equilibrium. Bumetanide had no effect on basal SCC and Vscc. When papaverine was added to skins preincubated with bumetanide, the effect of papaverine on SCC and Vscc was unchanged. Therefore, the depolarization of Vscc, observed during the papaverine-induced inhibition of the SCC, must be due to a reduction in the cellular K permeability. In conclusion, it is suggested that papaverine reduces the sodium permeability of the apical membrane and the potassium permeability of the basolateral membrane of the frog skin epithelium.

Skeletal muscle amino acid and myofibrillar protein mRNA response to thermal injury and infection.

This study investigates alterations in rat skeletal muscle after burn trauma (30% body surface area, full-thickness scald injury) with or without concomitant burn wound infection (immediate postburn inoculation with Pseudomonas aeruginosa), an injury model previously described by the authors.1 The study design allowed evaluation of sequential changes in skeletal muscle membrane integrity after burn or burn-infection injury and whether these occur coordinately with changes in muscle protein mass, specific protein transcription, intracellular amino acid and electrolyte concentrations, and indices of cellular energy status. Four groups of rats were compared: animals subjected to burn injury alone (Burn), burn injury plus wound infection (BI), freely fed, unburned control animals (FF), and unburned animals pair-fed to BI (PF: to control for anorexia in the BI group). Skeletal muscle myofibrillar protein transcription (relative abundance of messenger RNA for actin and myosin heavy chain); indices of muscle mas...

The determination of intracellular sodium concentration in human red blood cells: Nuclear magnetic resonance measurements

The intracellular sodium concentration and intracellular volume of human red blood cells were determined from 23Na and 1H NMR spectra. It is shown that sodium dissolved in the intracellular water has a concentration higher than that previously published. The intracellular sodium concentration measured was 11.4 ± 3.1 m m. A comparison of different NMR methods used to determine sodium concentration is given.

L-Serine Uptake by Trout (Salmo Trutta) Red Blood Cells: the Effect of Isoproterenol

ABSTRACT We studied the uptake of L-serine by trout red blood cells and the effect of a -adrenergic agonist (isoproterenol) on this process. The results obtained indicate that L-serine is taken up by these cells by means of a concentrative process. The uptake seems to be mediated both by a sodium-dependent process and by a sodium-independent process. The sodium-dependent uptake is mediated by a transport system that probably belongs to the ASC system family. Isoproterenol exerts an inhibitory effect on L-serine uptake. This effect is dose-dependent. It is proposed that the inhibitory effect of isoproterenol is mediated by a rise in the intracellular sodium concentration and/or changes in cell volume.

Magnesium supplementation prevents the development of alcohol-induced hypertension.

The effect of chronic alcohol administration on blood pressure was investigated in 7-week-old Wistar rats. Tail-cuff blood pressure was significantly higher in rats who received 15% ethanol in drinking water than in control rats. Intracellular free calcium concentration of lymphocytes was increased, while magnesium concentration of erythrocyte, aorta, and skeletal muscle and erythrocyte ouabain-sensitive 22Na efflux rate constant (Kos) were decreased in alcohol-induced hypertensive rats but not in control rats. Extracellular fluid volume was also increased in alcohol-administered rats. Oral magnesium supplementation (1% MgO in rat chow) attenuated the development of alcohol-induced hypertension accompanied by increased magnesium concentration of erythrocyte, aorta, skeletal muscle, and Kos and decreased intraerythrocyte sodium concentration. Norepinephrine half-life time of the heart and spleen was also increased in magnesium-supplemented rats. Blood pressure significantly correlated positively with intracellular calcium concentration and extracellular fluid volume, negatively with magnesium concentration of erythrocyte, aorta, skeletal muscle, and Kos. These results suggest that increased intracellular calcium, which was partly due to magnesium depletion and suppressed sodium pump activity, and expanded body fluid volume had a possible role in the development of alcohol-induced hypertension. It is also suggested that oral magnesium supplementation had a hypotensive effect on alcohol-induced hypertension possibly through decreased intracellular sodium concentration caused by an activation of sodium pump and decreased sympathetic nervous activity.

Effects of anoxia and inhibitors of energy metabolism on potassium and sodium homeostasis in neurons of gastropod mollusk

The effect of anoxia and inhibitors of energy metabolism on intracellular concentrations of potassium and sodium, membrane potentials, permeability, active and passive ouabain-sensitive transport of potassium (determined with86Rb) was studied in neurons of the freshwater planorbis mollusk (Planorbarius corneus). X-ray microanalysis showed that incubation of isolated ganglia in oxygen-free medium induced no change in intracellular concentrations of potassium and sodium. In the presence of cyanide, absorption of oxygen by the ganglia ceased, but accumulation of86Rb decreased insignificantly. The membrane potential and permeability did not depend on addition of cyanide. Desoxyglucose, an inhibitor of glycolysis, decreased86Rb accumulation more than cyanide did. In the presence of inhibitors of both glycolysis and respiration, which excluded the possibility of mutual compensation of oxidation and glycolytic sources of energy supply,86Rb accumulation decreased to the highest degree. A hypothesis was formulated on the paramount importance of glycolytic ATP for maintaining ion homeostasis of the nerve cells. The problem of functionally facilitated compartmentation of intracellular energy sources is discussed.