Sodium fluxes in human fibroblasts: kinetics of serum-dependent and serum-independent pathways

Sodium influx in serum-deprived human fibroblasts is by way of a pathway which shows saturation kinetics. A plot of initial Na influx versus [Na]0 ([Na]i approximately equal to 10 mM) gives a simple Michaelis-Menten type of curve with a K1/2 = 70.0 +/- 8.1 mM and a Vmax = 14.5 +/- 1.9 mumol/g prot/min. A similar plot of initial Na influx versus [Na]0 in the presence of 10% fetal bovine serum (FBS) gives a nonsaturating curvilinear response which appears to be biphasic. A plot of the serum-dependent Na influx versus [Na]0 (obtained by subtracting the curve in the absence of FBS from the curve in the presence of 10% FBS) shows that there is a linear relationship between serum-induced Na influx and external [Na]. At physiological Na concentrations, in the presence of FBS, the serum-induced Na influx is equal to the amiloride-sensitive Na flux, whereas in the absence of serum amiloride inhibits less than 10% of the Na influx. The effect of intracellular Na on Na flux was tested by preloading cells with Na in a digitoxin-containing medium prior to measurement of Na flux. A plot of steady-state Na exchange flux versus [Na]0 ([Na]i approximately equal to [Na]0) in the absence of serum gives a curve that appears to saturate at approximately 100 mM Na (flux = 100 mumol/g prot/min) and then declines with increasing [Na] (flux = 40 mumol/g prot/min at 150 mM). In contrast to Na influx in control serum-deprived cells, Na flux in Na-loaded cells in dramatically inhibited by the presence of amiloride. Since the peak Na exchange flux of 100 mumol/g prot/min is greatly in excess of the Vmax for Na influx in control serum-deprived cells and the enhanced Na flux is amiloride-sensitive, elevating intracellular Na must somehow activate the amiloride-sensitive Na transport system, which is normally only minimally active in the absence of serum.     

Modulation of erythrocyte Na transport pathway(s) by excess Na intake

Different Na transport pathways were studied in the erythrocytes of 10 normotensive subjects who received 240 meq/day of Na in excess of their usual diet. In most of these subjects the maximal rate (Vmax) of the Na,K pump and the Na,K-cotransport system was markedly decreased on the first day of the diet. In some of these subjects, excess Na intake induced an increase in the apparent affinity for internal Na for the Na,K pump and the Na,K-cotransport system. The decrease in the Na,K pump fluxes was not concomitant to that of the co-transport system and not accompanied with an increase in blood pressure or cation concentration in the plasma. Interestingly, the apparent affinity for internal Li of the Li-Na exchange was markedly increased without alteration of the Vmax. The passive permeability for Na and the cellular Na content were not altered by excess Na intake. Ouabain and bumetanide at low concentrations respectively induced an increase in the apparent affinity for internal Na of the Na,K pump and the Na,K- cotransport system. These results are similar to those observed after excess Na intake. These later agree with the hypothesis that Na homeostasis regulates some endogenous factors with ouabain-like and furosemide-like properties that might contribute to the regulation of cellular Na handling.     

Bacterial Na+ energetics

Novel observations related to the Na+-linked energy transduction in bacterial membranes are considered. It is concluded that besides the well-known systems based on the circulation of protons, there are those based on the circulation of Na+. In some cases, H+ and Na+ cycles co-exist in one and the same membrane. Representatives of the 'sodium world', i.e. cells possessing primary Na+ pumps (delta mu Na generators and consumers) are found in many genera of bacteria. Among the delta mu Na generators, one should mention Na+-NADH-quinone reductase and Na+-terminal oxidase of the respiratory chain, Na+-decarboxylases and Na+-ATPases. For delta mu Na consumers, there are Na+-ATP-synthases, Na+-metabolite symporters and Na+ motors. Sometimes, one and the same enzyme can transport H+ or, alternatively, Na+. For instance, an Na+-ATP-synthase of the F0F1 type translocates H+ when Na+ is absent. Employment of the Na+ cycle, apart from or instead of the H+ cycle, increases the resistance of bacteria to alkaline or protonophore-containing media and, apparently, to some other unfavourable conditions.     

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Although the enzyme (Na+ + K+)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na+ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na+ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na+ transport sensitively. This technique relies on the presence of Na+-Ca2+ exchange and ATP-driven Na+ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na+ uptake is monitored by a subsequent Nai+-dependent Ca2+ uptake reaction (Na+-Ca2+ exchange) using 45Ca2+. We present evidence that the Na+-Ca2+ exchange will be linearly related to the prior active Na+ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na+ isotopes. Applying this method, we measure cardiac ATP-dependent Na+ transport and (Na+ + K+)-ATPase activities in identical ionic media. We find that the (Na+ + K+)-ATPase and the Na+ pump have identical dependencies on both Na+ and ATP. The dependence on [Na+] is sigmoidal, with a Hill coefficient of 2.8. Na+ pumping is half-maximal at [Na+] = 9 mM. The Km for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na+ pumping. This approach should allow other new investigations on ATP-dependent Na+ transport across cardiac sarcolemma.     

Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump

The dependence of Na pump activity on intracellular and extracellular Na+ and K+ was investigated using a suspension of rabbit cortical tubules that contained mostly (86%) proximal tubules. The ouabain- sensitive rate of respiration (QO2) was used to measure the Na pump activity of intact tubules, and the Na,K-ATPase hydrolytic activity was measured using lysed proximal tubule membranes. The dependence (K0.5) of the Na pump on intracellular Na+ was affected by the relative intracellular concentration of K+, ranging from approximately 10 to 15 mM at low K+ and increasing to approximately 30 mM as the intracellular K+ was increased. The Na pump had a K0.5 for extracellular K+ of 1.3 mM in the presence of saturating concentrations of intracellular Na+. Measurements of the Na,K-ATPase activity under comparable conditions rendered similar values for the K0.5 of Na+ and K+. The Na pump activity in the intact tubules saturated as a function of extracellular Na at approximately 80 mM Na, with a K0.5 of 30 mM. Since Na pump activity under these conditions could be further stimulated by increasing Na+ entry with the cationophore nystatin, these values pertain to the Na+ entry step and not to the Na+ dependence of the intracellular Na+ site. When tubules were exposed to different extracellular K+ concentrations and the intracellular Na+ concentration was subsaturating, the Na pump had an apparent K0.5 of 0.4 mM for extracellular K. Under normal physiological conditions, the Na pump is unsaturated with respect to intracellular Na+, and indirect analysis suggests that the proximal cell may have an intracellular Na+ concentration of approximately 35 mM.     

The regulation of the intracellular sodium ion concentration in cultured chick embryo heart cells.

Using digital imaging microscopy with the fluorescent indicator sodium-binding benzofuran isophtalate, we examined the cytosolic Na+ concentration ([Na+]i) in individual chick embryo heart cells. Inhibition of the Na(+)-H+ exchanger using Na(+)-free (Li+ substituted) medium and inhibition of the Na(+)-efflux through the Na(+)-Ca2+ exchanger using Ca(2+)-free medium didn't change the [Na+]i. The opening of voltage-dependent Na+ channels with veratridine (150 micrograms/ml) and inhibition of the Na(+)-K(+)-Cl(-)-cotransporter with bumetanide (10 microM) led to an increase in [Na+]i by 107% and 86%, respectively, suggesting that the Na+ channels and the Na(+)-K(+)-Cl- cotransporter predominantly regulate the [Na+]i in cultured chick embryo heart cells.     

Characterization of the reverse Na/Ca exchange in squid axons and its modulation by Cai and ATP. Cai-dependent Nai/Cao and Nai/Nao exchange modes

We have used dialyzed squid axons to characterize the ouabain- and bumetanide-insensitive Na efflux components and their relation to the operation of the Na/Ca exchange mechanism. In axons dialyzed with solutions containing nearly physiological concentrations of K, Na, and Mg, three components of the Na efflux can be distinguished: Cai-activated, Cao-dependent Na efflux ("reverse" Na/Ca exchange); Cai-activated, Nao-dependent Na efflux; and Cai-independent, ATP-activated, Nao-dependent Na efflux. We have studied the effects of internal alkalinization, Mgi, Cao, and the ATP analogue [gamma-thio]ATP (ATP gamma S) on the different components of the Na efflux. The results show the following: (a) internal alkalinization activates both Cao- and Nao-dependent Na efflux components provided that Cai is present; (b) Mgi inhibits both the Cai-activated, Cao- and Nao-dependent Na efflux components; (c) Cao inhibits the Nao-dependent component by competition for a common site; (d) ATP gamma S activates both Nao- and Cao-dependent Na efflux components only in the presence of Cai; and (e) ATP activates the Nai/Nao and Nai/Cao exchanges, causing a 10-fold increase in the affinity of the reverse Na/Ca exchange toward Cai. In the absence of Cai, ATP stimulates an Nao-dependent Na efflux that is not affected either by internal alkalinization or high Cao. The ATP analogue does not activate the Cai-independent Na/Na exchange system. These experiments demonstrate that the Cai-activated Na/Na exchange is a mode of operation of the Na/Ca exchange mechanism that substantially contributes to Na movement during the activation of the Na/Ca antiporter. The experimental evidence obtained on the Cai-independent Na/Na exchange component shows that this system is not part of the Na/Ca exchange.     

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

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

Intracellular Na+ kinetically interferes with the rotation of the Na(+)-driven flagellar motors of Vibrio alginolyticus

To understand the mechanism of Na+ movement through the force-generating units of the Na(+)-driven flagellar motors of Vibrio alginolyticus, the effect of intracellular Na+ concentration on motor rotation was investigated. Control cells containing about 50 mM Na+ showed good motility even at 10 mM Na+ in the medium, i.e. in the absence of an inwardly directed Na+ gradient. In contrast, Na(+)-loaded cells containing about 400 mM Na+ showed very poor motility at 500 mM Na+ in the medium, i.e. even in the presence of an inwardly directed Na+ gradient. The membrane potential of the cells, which is a major driving force for the motor under these conditions, was not detectably altered, and consistently with this, Na(+)-coupled sucrose transport was only partly reduced in the Na(+)-loaded cells. Motility of the Na(+)-loaded cells was restored by decreasing the intracellular Na+ concentration, and the rate of restoration of motility correlated with the rate of the Na+ decrease. These results indicate that the absolute concentration of the intracellular Na+ is a determinant of the rotation rate of the Na(+)-driven flagellar motors of V. alginolyticus. A simple explanation for this phenomenon is that the force-generating unit of the motor has an intracellular Na(+)-binding site, at which the intracellular Na+ kinetically interferes with the rate of Na+ influx for motor rotation.     

Non-pumped sodium fluxes in human red blood cells. Evidence for facilitated diffusion.

Unidirectional and net Na+ fluxes modified by changes in internal Na+ concentration ([Na+]i) were studied in human red blood cells incubated in K+-free solutions containing 10-minus 4 m ouabain. An increase in [Na+]i brought about (a) a reduction in net Na+ gain, (b) no change in Na+ influx, (c) a reduction in the rate constant for Na+ effux and (d) an increase in Na+ efflux. Similar reductions in net Na+ gain were observed when the changes in [Na+]i were carried out at constant [K+]i. In addition, the rate constant for 42K+ efflux was not affected by changes in [Na+]i. The electrical membrane potential (as determined from the chloride distribution ratio) was also constnat. Furosemide (10-minus 3 M) increased the net Na+ gain in concentration reduced Na+ efflux and increased Na+ influx: the magnitude of these effects was dependent onthe intracellular Na+. The reduction in the net Na+ gain as [Na+]i increased was unaffected by depletion of cellular ATP to values below 10 mumol/1 cells, and this effect was independent of the depletion method used     

Decreased intracellular Na+ concentration is an early event in murine erythroleukemic cell differentiation

A decrease in Na+/K+-pump activity is an early event of Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway. This decreased Na+/K+-pump activity has been proposed to be an essential step in differentiation which would cause a rise in intracellular Na+ concentration and then, by means of Na+/Ca2+ exchange, an increase in intracellular Ca2+. An increase in intracellular Ca2+ has been proposed to be essential for induction of differentiation. A critical prediction of this Na+-Ca2+ hypothesis is the rise in intracellular Na+. To test this prediction we have measured intracellular Na+ using a novel triple isotope method involving 3H2O, [14C]sucrose, and 22Na to measure total water, extracellular fluid, and Na+, respectively. 22Na equilibration occurred in less than 10 min. In uninduced cells, intracellular Na+ was 15.2 +/- 2.2 mM (S.D., n = 22); after induction for 14-16 h with dimethyl sulfoxide, intracellular Na+ decreased significantly (p less than 0.0001) to 8.4 +/- 1.4 mM (n = 21). The time course of the decline in intracellular Na+ paralleled that of the decrease in the Na+/K+-pump activity. These results are in direct contradiction to the Na+-Ca2+ hypothesis and suggest that observed changes in Na+/K+-pump activity can be explained solely on the basis of changes in intracellular Na+. The drop in intracellular Na+ is due to a decrease in Na+ influx. We suggest, however, that the decrease in the Na+ influx is not itself an essential event of differentiation, but may be induced by a change in the flux of another ion coupled to Na+.     

Sodium extrusion by the sea-water-acclimated fiddler crab Uca pugilator: comparison with other marine crustacea and marine teleost fish.

1. Measurements of the blood Na concentration and transepithelial electrical potential (T.E.P.) across Uca pugilator acclimated to sea water indicate that Na is maintained out of electrochemical equilibrium with sea water. The resulting net Na influx as well as the sodium gain due to ingestion of the medium must be balanced by extrarenal Na extrusion. 2. The small T.E.P. (-0.7 mV) and the 'transport numbers' of Na and Cl indicate that the permeability to these ions is equivalent. 3. Removal of external K results in a significant stimulation of unidirectional Na efflux that is dependent upon external Na but is not inhibited by ouabain. 4. Transfer of Uca to K and Na-free sea water results in a 54% decline in unidirectional efflux, which is not due to T.E.P. changes. Readdition of 25mM-K stimulates Na efflux much more than can be accounted for by changes in the T.E.P. Readdition of 25mM-Na to potassium-free sea water does not change the Na efflux. 5. The results indicate that Na extrusion by Uca is via a Na/K exchange mechanism which partially inhibits Na/Na exchange. Cessation of Na/K exchange (in K-free sea water) removes this inhibition and allows rapid Na/Na exchange. It is not known whether Na/K and Na/Na exchange are via the same or parallel carrier systems.     

Relation between intracellular sodium and active sodium transport in rabbit colon: Current-voltage relations of the apical sodium entry mechanism in the presence of varying luminal sodium concentrations

The current-voltage relations of the amiloride-sensitive Na entry pathway across the apical membrane of rabbit descending colon, exposed to a high K serosal solution, were determined in the presence of varying mucosal Na activities, (Na)m, ranging from 6.2 to 99.4 mM. These relations could be closely fit to the "constant field" flux equation yielding estimates of the permeability of the apical membrane to Na, PmNa, and the intracellular Na activity, (Na)c. The following empirical relations emerged: (Na)c increased hyperbolically with increasing (Na)m; PmNa decreased hyperbolically with increasing (Na)m and linearly with increasing (Na)c; spontaneous variations in Na entry rate at constant (Na)m could be attributed entirely to parallel, spontaneous variations in PmNa; the rate of Na entry increased hyperbolically with increasing (Na)m obeying simple Michaelis-Menten kinetics; the relation between (Na)c and "pump rate," however, was sharply sigmoidal and could be fit by the Hill equation assuming strong cooperative interactions between Na and multiple sites on the pump; the Hill coefficient was 2-3 and the value of (Na)c at which the pump-rate is half-maximal was 24 mM. The results provide an internally consistent set of relations among Na entry across the apical membrane, the intracellular Na activity and basolateral pump rate that is also consistent with data previously reported for this and other Na-absorbing epithelia.     

Measurement of intracellular sodium concentration and sodium transport in Escherichia coli by 23Na nuclear magnetic resonance

Escherichia coli is known to actively extrude sodium ions, but little is known concerning the concentration gradient it can develop. We report here simultaneous measurements, by 23Na NMR, of intracellular and extracellular Na+ concentrations of E. coli cells before and after energization. 23Na spectra in the presence of a paramagnetic shift reagent (dysprosium tripolyphosphate) consisted of two resonances, an unshifted one corresponding to intracellular Na+ and a shifted one corresponding to Na+ in the extracellular medium, including the periplasm. Extracellular Na+ was found to be completely visible despite the presence of a broad component in its resonance; intracellular Na+ was only 45% visible. Measurements of Na+ were made under aerobic and glycolytic conditions. Na+ extrusion and maintenance of a stable low intracellular Na+ concentration were found to correlate with the development and maintenance of proton motive force, a result that is consistent with proton-driven Na+/H+ exchange as a means of Na+ transport. In both respiring and glycolyzing cells, at an extracellular Na+ concentration of 100 mM, the intracellular Na+ concentration observed (4 mM) corresponded to an inwardly directed Na+ gradient with a concentration ratio of about 25. The kinetics of Na+ transport suggest that rapid extrusion of Na+ against its electrochemical gradient may be regulated by proton motive force or intracellular pH.     

Relationship of the decreases in protein synthesis and intracellular Na+ during friend murine erythroleukemic cell differentiation

The earliest known ionic event during Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway is a 45% drop in intracellular sodium concentration ([Na+]i) due to a decrease in Na+ influx (Lannigan, D. A., and Knauf, P. A. (1985) J. Biol. Chem. 260, 7322-7324). We have analyzed the mechanism of the decreased Na+ influx. The Na+ influx in uninduced cells was insensitive to dimethylamiloride, bumetanide, and diisothiocyanostilbene disulfonate. The intracellular pH (pHi) did not change up to 15 h after dimethyl sulfoxide induction, at which time Na+ influx has decreased by approximately 40%; thus, the decrease in Na+ influx is not coupled to a change in pHi. A substantial amount of the decrease in Na+ influx seems to result from a drop in amino acid-dependent Na+ transport. This reduction in amino acid-dependent Na+ influx reflects a decrease in net Na+ influx rather than solely in Na+/Na+ exchange and can account for an appreciable portion of the reduction in [Na+]i seen during differentiation. The drop in amino acid-dependent Na+ influx could not be explained by membrane depolarization but was correlated with a decrease in protein synthesis. Inhibition of protein synthesis in uninduced cells by cycloheximide also caused a decrease in Na+ influx. We conclude that during differentiation the reduction in protein synthesis decreases amino acid-dependent Na+ influx which in turn causes a drop in [Na+]i leading to a reduction in the Na+/K+ pump rate.     

整株水平上Na+ 转运体与植物的抗盐性

植物可以利用不同的机制来维持Na+稳态, 从而增强植物的抗盐性。这些机制包括: 限制Na+的内流; 增大Na+的外排; 减少Na+向地上部分的运输; 把进入地上部分的Na+分散到特殊部分(如老叶)或通过泌盐结构排出体外或通过韧皮部的再循环回到根部。本文简要介绍整株水平上Na+转运体与植物抗盐性的研究进展。     

The amiloride-sensitive Na+/H+ exchange system in skeletal muscle cells in culture

Chick skeletal muscle cells in culture have an amiloride-sensitive Na+-transporting system that has the following properties. Na+ uptake is dependent on the extracellular Na+ concentration. The Km value for Na+ is 25 mM and remains constant between pH 7.5 and 8.5. The maximal rate of Na+ transport is higher at alkaline pH. An ionizable group with a pK of 7.6 is essential for the system to be functional. The activity of the amiloride-sensitive Na+ uptake system is controlled by internal Na+ and H+ concentrations. Amiloride inhibition of Na+ uptake is competitively antagonized by increasing Na+ concentration. The dissociation constant for amiloride is 5 microM in Na+-free conditions and is constant between pH 7.5 and 8.5. The Km value for Na+ found from competition experiments is 13 mM. The amiloride-sensitive Na+ influx occurs in parallel with an amiloride-sensitive H+ efflux. This H+ efflux is stimulated by increasing external Na+ concentrations, the Km for Na+ being 15 mM. It is inhibited by amiloride with the same concentration dependence as Na+ influx.     

Nonlabens Dokdonensis视紫红质2中的钠离子结合与pH依赖性的传递

光驱动钠离子泵是近年来发现的全新的光遗传学工具蛋白,在光激发下向膜外主动运输钠离子。然而,其钠离子传递的机制仍不清楚。该家族成员NdR2的可见吸收光谱、荧光光谱和等温滴定量热的结果说明：基态下的NdR2不结合钠离子。进一步的钠离子传递动力学和功能实验发现NdR2在酸性条件下的钠离子传递发生改变。这种pH依赖性的钠离子传递有助于深入理解光驱动钠离子泵中的钠离子的传递机制。     

Fertilization acid release in Urechis eggs. II. The stoichiometry of Na+ uptake and H+ release

We tested the hypothesis that Na+ uptake and H+ release at fertilization of Urechis eggs might occur via a Na+:H+ exchange. Previous studies have shown that (1) Na+ uptake is proportional to the number of entering sperm in seawater with or without lowered Na+ and (2) H+ release is proportional to external pH. Therefore, to determine if Na+ uptake and H+ release are always proportional, we determined the effect of polyspermy on H+ release in natural and low Na+ seawater and the effect of external pH on Na+ uptake and release. Na+ uptake and H+ release do not covary in a manner consistent with a Na+:H+ exchange. H+ release under most conditions was manner consistent with a Na+:H+ exchange. H+ release under most conditions was independent of the number of sperm/egg and in low Na+ seawater was at most 53 +/- 16% of that in natural seawater. In contrast, Na+ uptake in low Na+ seawater can be more than in natural seawater (Jaffe et al., J. Gen. Physiol. 73, 469-492, 1979). In natural seawater Na+ uptake exceeded H+ release; at pH 7 Na+ uptake was 2 pmol/egg, but there was no H+ release. Since Na+ release did not increase at fertilization at pH 7, neither Na+:Na+ nor Na+:H+ exchange could account for the Na+ uptake. An alternate hypothesis is suggested: Na+ uptake is primarily via the channels responsible for the fertilization potential, while H+ release is by another route that is affected by the membrane potential during the fertilization potential.     

Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.