22Na+ fluxes in thymic lymphocytes. I. Na+/Na+ and Na+/H+ exchange through an amiloride-insensitive pathway

The Na+ transport pathways of normal rat thymocytes were investigated. Na+ conductance was found to be lower than K+ conductance, which is consistent with reported values of membrane potential. In contrast, the isotopically measured Na+ permeability was greater than 10-fold higher than that of K+, which indicates that most of the flux is electroneutral. Cotransport with Cl- (or K+ and Cl-) and countertransport with Ca2+ were ruled out by ion substitution experiments and use of inhibitors. Countertransport for Na+ or H+ through the amiloride-sensitive antiport accounts for only 15-20% of the resting influx. In the presence of amiloride, 22Na+ uptake was increased in Na+-loaded cells, which suggests the existence of Na+/Na+ countertransport. Cytoplasmic pH determinations using fluorescent probes indicated that under certain conditions this amiloride-resistant system will also exchange Na+ for H+, as evidenced by an internal Na+- dependent acidification is proportional to internal [Na+] but inversely related to extracellular [Na+]. Moreover, 22Na+ uptake is inhibited by increasing external [H+]. The results support the existence of a substantial amiloride-insensitive, electroneutral cation exchange system capable of transporting Na+ and H+.     

Respiration-driven Na+ pump and Na+ circulation in Vibrio parahaemolyticus.

Sodium circulation in Vibrio parahaemolyticus was investigated. We observed respiration-driven Na+ extrusion from cells by using a Na+ electrode. The Na+ extrusion was insensitive to a proton conductor, carbonyl cyanide m-chlorophenylhydrazone, and sensitive to a respiratory inhibitor, CN-. These results support the idea of the existence of a respiratory Na+ pump in V. parahaemolyticus. The respiration-driven Na+ extrusion was observed only under alkaline conditions.     

Nuclear Magnetic Resonance of Tissue 23Na: I. 23Na Signal and Na+ Activity in Homogenate

The ability to depress the resonance intensity of ²³Na in rat liver tissue was not found in the supernatant fraction. It was exclusively localized in particulate fractions. The intensity and saturation behavior of the ²³Na signal was examined in suspensions containing various amounts of the particulate fraction of rat liver homogenate. The results strongly suggest that the ²³Na signal of tissue reflects quadrupole interactions and does not result from a slow exchange between the free and bound fractions of Na⁺. The activity coefficient of Na⁺ in rat liver homogenate (no medium was added) was 0.59, about 20% less than that in the isotonic saline. Available evidences and discussion indicate that the bound Na⁺ in the homogenate is much less than the so-called “NMR-invisible” fraction of Na⁺.     

Structural and functional organization of Na+ transport in epithelial systems. I. Epithelial Na+ channels

The review summarizes recent data on the structural and functional organization and regulation mechanisms of Na+ transport in epithelial systems. The review is focused on the structure, function, regulation and pathology of epithelial Na+ channels, which are critical for Na+ homeostasis maintenance and blood pressure control.     

Mechanism of epithelial lithium transport. Evidence for basolateral Na:Na and Na:Li exchange

Measurement of transmural sodium fluxes across isolated, ouabain- inhibited turtle colon in the presence of a serosal-to-mucosal sodium gradient shows that in the absence of active transport the amiloride- sensitive cellular path contains at least two routes for the transmural movement of sodium and lithium, one a conductive path and the other a nonconductive, cation-exchange mechanism. The latter transport element can exchange lithium for sodium, and the countertransport of these two cations provides a mechanistic basis for the ability of tight epithelia to actively absorb lithium despite the low affinity of the basolateral Na/K-ATPase for this cation.     

Na,K-ATPase in artificial lipid vesicles. Comparison of Na,K and Na-only pumping mode

Na,K-ATPase from rabbit kidney outer medulla was reconstituted in large unilamellar lipid vesicles by detergent dialysis. Vesicles prepared in the presence or absence of potassium allowed to study two different transport modes: the (physiological) Na,K-mode in buffers containing Na+ and K+ and the Na-only mode in buffers containing Na+ but no K+. The ATP hydrolysis activity was obtained by determination of the liberated inorganic phosphate, Pi, and the inward directed Na+ flux was measured by 22Na-tracer flux. Electrogenic transport properties were studied using the membrane potential sensitive fluorescence-dye oxonol VI. The ratio upsilon(Na,K)/upsilon(Na) of the turnover rates in the Na,K-mode and in the Na-only mode is 6.6 +/- 2.0 under otherwise identical conditions and nonlimiting Na+ concentrations. Strong evidence is found that the Na-only mode exhibits a stoichiometry of 3Na+cyt/2Na+ext/1ATP, i.e. the extracellular (= intravesicular) Na+ has a potassium-like effect. In the Na-only mode one high-affinity binding side for ATP (KM congruent to 50 nM) was found, in the Na,K-mode a high- and low-affinity binding side with equilibrium dissociation constants, KM, of 60 nM and 13 microM, respectively. The sensitivity against the noncompetitively inhibiting ADP (KI = 6 microM) is higher by a factor of 20 in the Na-only mode compared to the Na,K-mode. From the temperature dependence of the pumping activity in both transport modes, activation energies of 160 kJ/mol for the Na,K-mode and 110 kJ/mol for the Na-only mode were determined.     

Furosemide-sensitive Na and K fluxes in human red cells. Net uphill Na extrusion and equilibrium properties

This paper reports experiments designed to find the concentrations of internal and external Na and K at which inward and outward furosemide-sensitive (FS) Na and K fluxes are equal, so that there is no net FS movement of Na and K. The red cell cation content was modified by using the ionophore nystatin, varying cell Na (Nai) from 0 to 34 mM (K substitution, high-K cells) and cell K (Ki) from 0 to 30 mM (Na substitution, high-Na cells). All incubation media contained NaCl (Nao = 130 or 120 nM), and KCl (Ko = 0-30 mM). In high-K cells, incubated in the absence of Ko, there was net extrusion of Na through the FS pathway. The net FS Na extrusion increased when Nai was increased. Low concentrations of Ko (0-6 mM) slightly stimulated, whereas higher concentrations of Ko inhibited, FS Na efflux. Increasing Ko stimulated the FS Na influx (K0.5 = 4 mM). Under conditions similar to those that occur in vivo (Nai = 10, Ki = 130, Nao = 130, Ko = 4 mM, Cli/Clo = 0.7), net extrusion of Na occurs through the FS pathway (180-250 mumol/liter cell X h). The concentration of Ko at which the FS Na influx and efflux and the FS K influx and efflux become equal increased when Nai increased in high-K cells and when Ki was increased in high-Na cells. The net FS Na and K fluxes both approached zero at similar internal and external Na and K concentrations. In high-K cells, under conditions when net Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional flux was found to be 2:3. In high-K cells, the empirical expression (Nai/Nao)2(Ki/Ko)3 remained at constant value (apparent equilibrium constant, Kappeq +/- SEM = 22 +/- 2) for each set of internal and external cation concentrations at which there was no net Na flux. These results indicate that in the physiological region of concentrations of internal and external Na, K, and Cl, the stoichiometry of the FS Na and K fluxes is 2 Na:3 K. In high-Na cells under conditions when net FS Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional fluxes was 3:2 (1).(ABSTRACT TRUNCATED AT 400 WORDS)     

23Na NMR evaluation of human erythrocytes Na+/K+/CL-cotransport. A study in elderly hypertensives.

The behaviour of sodium transport systems across the cell membrane has been poorly investigated in elderly hypertension. Sodium efflux driven by Na+/K+/Cl-cotransport activity was therefore investigated (using a novel NMR-spectroscopy method) in 5 elderly hypertensive males (mean age 78 +/- 5 years) and 5 normotensive controls (mean age 79 +/- 3 years). In order to exclude any change in cotransport activity secondary to metabolic abnormalities, both patients and controls were non-obese and had normal glucose and lipid metabolisms. The Na+/K+/Cl-cotransport evaluation was performed after three months of pharmacological wash-out, under a diet containing 120 mEq of Na+/day. The resulting data showed that Na+ efflux due to outward Na+/K+/Cl-cotransport was higher in hypertensive group than in the normotensive one (0.50 +/- 0.10 mmol Na+/l cells/hr. vs 0.33 +/- 0.03 mmol Na+/l cells/hr., respectively, p < 0.05). Intracellular Na+ content was similar in both groups. At variance with previous data from the literature, our findings indicate that the Na+/K+/Cl-cotransport activity is elevated in elderly hypertensives.     

Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+

Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to - 80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.     

Sodium-calcium exchange current. Dependence on internal Ca and Na and competitive binding of external Na and Ca

Na-Ca exchange current was measured at various concentrations of internal Na [( Na]i) and Ca [( Ca]i) using intracellular perfusion technique and whole-cell voltage clamp in single cardiac ventricular cells of guinea pig. Internal Ca has an activating effect on Nai-Cao exchange beginning at approximately 10 nM and saturating at approximately 50 nM with a half maximum [Ca]i (Km[Ca]i) of 22 nM (Hill coefficient, 3.7). Measurement of Nai-Cao exchange current at various concentration of [Na]i revealed an apparent Km[Na]i of 20.7 +/- 6.9 mM (n = 14) with imax of 3.5 +/- 1.2 microA/microF. For [Ca]i transported by the exchange, a Km[Ca]i of 0.60 +/- 0.24 microM (n = 8) with an imax of 3.0 +/- 0.54 microA/microF was obtained by measuring Nao-Cai exchange current. These values are apparently different from the values for the external binding site which have been reported previously. Whether Na and Ca compete for the external binding site, and if so, how it affects the binding constants was then investigated. Outward Nai-Cao exchange current became larger by reducing [Na]o. The double reciprocal plot of the current magnitude and [Ca]o at different [Na]o revealed a competitive interaction between Na and Ca. In the absence of competitor [Na]o, an apparent Km[Ca]o of 0.14 mM was obtained. When comparing internal and external Km values, the external value is markedly larger than the internal one and thus we conclude that binding sites of the Na-Ca exchange molecule are at least apparently asymmetrical between the inside and outside of the membrane.     

Role and regulation of lung Na,K-ATPase.

The recognition that pulmonary edema is cleared from the alveolar airspace by active Na+ transport has led to studies of the role and regulation of alveolar epithelial Na,K-ATPases. In the lung these heterodimers are predominantly composed of alpha1 and beta1-subunits and are located on the basolateral aspect of alveolar type 2 epithelial cells (AT2). Working with apically positioned epithelial Na+ channels they generate a transepithelial osmotic gradient which causes the movement of fluid out of the alveolar airspace. Accumulating data indicates that in some forms of pulmonary edema alveolar Na,K-ATPases function is reduced suggesting that pulmonary edema may be due, in part, to impairment of edema clearance mechanisms. Other studies suggest that Na,K-ATPase dysfunction or inhibition may contribute to airway reactivity. It is now recognized that lung Na,K-ATPases are positively regulated by glucocorticoids, aldosterone, catecholamines and growth hormones. These findings have led to investigations that show that enhancement of Na,K-ATPase function can accelerate pulmonary edema clearance in vitro, in normal and injured animal lungs in vivo, and in human lung explants. This review focuses on Na,K-ATPase data from lung and lung cell experiments that highlight the importance of Na,K-ATPases in airway reactivity and in maintaining a dry alveolar airspace. Review of data that suggests that there may be a role for therapeutic modulation of alveolar Na,K-ATPases for the purpose of treating patients with respiratory failure are also included.     

Na+ channel and Na+-K+ ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb.

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.     

The structure and function of Na+ channels.

The past year has seen major advances in our understanding of voltage-gated ion channels through a powerful combination of patch-clamp and molecular biological techniques. These approaches have identified regions (in some cases single amino acid residues) that are essential for voltage-dependent activation and inactivation, lining of the pore, and regulation of channel function.     

Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.     

Conductance and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li

The behavior of individual Na channels in the apical membrane of the rat cortical collecting tubule (CCT) was studied at different concentrations of the permeant ions Na and Li. Tubules were opened to expose their luminal surfaces and bathed in K-gluconate medium to minimize tubule-to-tubule variation in cell membrane potential and intracellular Na concentration. The patch-clamp technique was used to resolve currents through individual channels. The patch-clamp pipette was filled with solutions containing variable concentrations of either NaCl or LiCl. In one series of experiments, the concentrations were changed without substitutions. In another series, the ionic strength and Cl concentration were maintained constant by partial substitution of Li with N-methyl-D-glucamine (NMDG). In cell-attached patches, both the single-channel conductance (g) and the single-channel current (i) saturated as functions of the Na or Li activity in the pipette. Without NMDG, the saturation of i was well described by Michaelis-Menten kinetics with an apparent Km of approximately 20 mM activity for Na and approximately 50 mM activity for Li. Km was independent of voltage for both ions. With substitution for Li by NMDG, the apparent Km value for Li transport through the channels increased. The values of the probability of a channel's being open (Po) varied from patch to patch, but no effect of pipette ion activity on Po could be demonstrated. A weak dependence of Po on membrane voltage was observed, with hyperpolarization increasing Po by an average of 2.3%/mV.     

Sites important for Na+ and substrate binding in the Na+/proline transporter of Escherichia coli, a member of the Na+/solute symporter family.

To elucidate the functional importance of transmembrane domain II in the Na(+)/proline transporter (PutP) of Escherichia coli we analyzed the effect of replacing Ser-54 through Gly-58. Substitution of Asp-55 or Met-56 dramatically reduces the apparent affinity for Na(+) and Li(+) in a cation-dependent manner. Conversely, Cys in place of Gly-58 significantly reduces only the apparent proline affinity while substitution of Ser-57 results in a dramatic reduction of the apparent proline and cation affinities. Interestingly, upon increasing the proline concentration the apparent Na(+) affinity of Ser-57 replacement mutants converges toward the wild-type value, indicating a close cooperativity between cation and substrate site(s). This notion is supported by the fact that Na(+)-stimulated site-specific fluorescence labeling of a single Cys at position 57 is completely reversed by the addition of proline. Similar results are obtained upon labeling of a Cys at position 54 or 58. Taken together, these results indicate that Asp-55 and Met-56 are located at or close to the ion-binding site while Ser-54, Ser-57, and Gly-58 may be close to the proline translocation pathway. In addition, the data prod at an involvement of the latter residues in ligand-induced conformational dynamics that are crucial for cation-coupled transport.