The excretion of hydrogen ion by the isolated amphibian skin: Effects of antidiuretic hormone and amiloride

H⁺ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo was studied in order to test for the presence of exchange mechanisms of the type Na⁺/H⁺ and Cl^?/HCO₃^?, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na⁺ transport and the pH-stat technique was utilized to determine the rates of H⁺ extrusion under different experimental conditions. These conditions were either the withdrawal of the ions intervening in the mentioned exchanges (Cl^- or Na⁺, or the addition of drugs with well-known effects on Na⁺ uptake and transport (antidiuretic hormone and amiloride).In the frog skin, H⁺ excretion was detected in solutions containing either Cl^? or SO₄^2?, with identical rates. Again, Na⁺ substitution by Mg²⁺ had no effect on H⁺ excretion rates, neither did the suppression of Na⁺ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase.Experiments in toad skin showed that H⁺ excretion could not be detected when Cl^? was present in the outer medium, but became apparent if an impermeant anion, SO₄^2?, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl^?/HCO₃^?. Secondly, in these preparations H⁺ extrusion increased after stimulation with antidiuretic hormone and decreased when amiloride was used or when Na⁺ was substituted by Mg²⁺, suggesting that at least a fraction of the total H⁺ efflux is linked to Na⁺ influx. In the isolated frog skin this mechanism does not seem to be operative.     

Synthesis of new vasotocin analogues: effects on renal water and ion excretion in rats

Vasopressin and nonmammalian hormone vasotocin are known to increase the water permeability of mammalian collecting ducts, frog skin and the urinary bladder. Neurohypophysial nonapeptides have also been shown to interfere with the regulation of renal ion transport. The subject of this study was a search for vasopressin and vasotocin analogues with selective effects on renal water, sodium and potassium excretion. During this study, we synthesised the following peptides: 13 vasotocin analogues modified at positions 4 (Thr or Arg), 7 (Gly or Leu) and 8 (d ‐Arg, Lys or Glu); 4 vasopressin analogues modified at positions 4 and 8; and 9 peptides shortened or extended at the C‐terminal or with substitutions for Gly‐NH₂. Most of these peptides had mercaptopropionic acid (Mpa) instead of Cys in position 1. The effects of these nonapeptides on renal water, sodium and potassium transport were evaluated in in vivo experiments using Wistar rats. Some nonapeptides possessed antidiuretic, natriuretic and kaliuretic activities ([Mpa¹]‐arginine vasotocin, [Mpa¹, homoArg⁸]‐vasotocin, [Mpa¹, Thr⁴]‐arginine vasotocin and [Mpa¹, Arg⁴]‐arginine vasopressin). Substitutions at positions 4 and 8 increased the selectivity of peptide actions. The antidiuretic [d ‐Arg⁸]‐vasotocin analogues had no effects on sodium excretion. [Mpa¹, Arg⁴]‐arginine vasotocin was antidiuretic and kaliuretic but not natriuretic. [Mpa¹, Glu⁸]‐oxytocin had weak natriuretic activity without any effects on water and potassium transport. In accordance with the data obtained, synthesised vasotocin analogues could be good candidates for pharmaceuticals selectively regulating renal sodium and potassium transport, which is of clinical importance. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and Characterization of C8 Analogs of c-di-GMP

We have synthesized five analogs of c-di-GMP with different substituents at the guanine C8 position, to study their effects on the metal-dependent polymorphism we had previously demonstrated for the parent compound. Of these, only the K⁺ salt of c-di-Br-GMP, 2, forms higher order complexes, predominantly two different syn octamolecular ones. Its Na⁺ salt, as well as both the K⁺ and Na⁺ salts of c-di-thio-GMP, 3, c-di-methylthio-GMP, 4, c-di-phenyl-GMP, 5, and c-di-acetylphenyl-GMP, 6, all form primarily a syn bimolecular structure.     

Effectiveness of potassium sulfate in mitigating salt-induced adverse effects on different physio-biochemical attributes in sunflower (Helianthus annuus L.)

To assess whether foliar application of K+S as potassium sulfate (K₂SO₄) could alleviate the adverse effects of salt on sunflower (Helianthus annuus L. cv. SF-187) plants, a greenhouse experiment was conducted. There were two NaCl levels (0 and 150 mM) applied to the growth medium and six levels of K+S as K₂SO₄ (NS (no spray), WS (spray of water+0.1% Tween 20 solution), 0.5% K+0.21% S, 1.0% K+0.41% S, 1.5% K+0.62% S, and 2.0% K+0.82% S in 0.1% Tween-20 solution) applied two times foliarly to non-stressed and salt-stressed sunflower plants. Salt stress markedly repressed the growth, yield, photosynthetic pigments, water relations and photosynthetic attributes, quantum yield (F_v/F_m), leaf and root K⁺, Mg²⁺, P, Ca²⁺, N as well as K⁺/Na⁺ ratios, while it enhanced the cell membrane permeability, and leaf and root Na⁺ and Cl⁻ concentrations. Foliar application of potassium sulfate significantly improved growth, achene yield, photosynthetic and transpiration rates, stomatal conductance, water use efficiency, leaf turgor and enhanced shoot and leaf K⁺ of the salt-stressed sunflower plants, but it did not improve leaf and root Na⁺, Cl⁻, Mg²⁺, P, Ca²⁺, N as well as K⁺/Na⁺ ratios. The most effective dose of K+S for improving growth and achene yield was found to be 1.5% K+0.62% S and 1% K+0.41% S, respectively. Improvement in growth of sunflower plants due to exogenously applied K₂SO₄ was found to be linked to enhanced photosynthetic capacity, water use efficiency, leaf turgor and relative water content.     

Characterization,quantitation, similarity evaluation and combination with Na+,K+-ATPase of cardiac glycosides from Streblus asper

Streblus asper Lour. (Moraceae) is a medicinal plant in Asian countries including India and Thailand, possessing activities of anti-tumor, anti-allergy, anti-parasitic and anti-bacterial. In this paper, characterization, quantitation and similarity evaluation of cardiac glycosides in different parts of S. asper were investigated by HPLC-Q-TOF-MS and chemometric methods. Then, the inhibition of Na⁺,K⁺-ATPase activity by the compounds isolated from S. asper was measured. Meanwhile, enzyme kinetics and molecular docking were determined to exhibit the combination modes between cardiac glycosides and Na⁺,K⁺-ATPase. As a result, twenty peaks of cardiac glycosides were assigned. Strophanthidin-3-O-α-l-rhamnopyranosyl-(1 → 4)-6-deoxy-β-d-allopyranoside (1), glucostrebloside (2), strebloside (4) and mansonin (8) with a significant activity of inhibiting Na⁺,K⁺-ATPase (IC₅₀ 7.55–13.60 μM) were chosen for the determination of enzyme kinetics, exhibiting anticompetitive inhibitory characteristics towards Na⁺,K⁺-ATPase. Compound 4 could reasonably bind to the active sites of Na⁺,K⁺-ATPase, proved by molecular docking. Furthermore, the contents of the major compounds in four different parts of S. asper were extremely different, analyzed by chemometric methods, similarity analysis and principle compounds analysis. All these findings indicated that the contents of major compounds in different parts of S. asper were extremely different with a significant activity of inhibiting Na⁺,K⁺-ATPase, providing a reference for determination of effective part and administered dosage. The combination modes between cardiac glycosides and Na⁺,K⁺-ATPase were also revealed by enzyme kinetics and molecular docking, which provided a basis for further study of pharmacological activity.     

An unusual conformation from Na+-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na+-binding

The Na⁺,K⁺-ATPase (NKA) and non-gastric H⁺,K⁺- ATPase (ngHKA) share ~65 % sequence identity, and nearly identical catalytic cycles. These pumps alternate between inward-facing (E1) and outward-facing (E2) conformations and differ in their exported substrate (Na⁺ or H⁺) and stoichiometries (3 Na⁺:2 K⁺ or 1 H⁺:1 K⁺). We reported that structures of the NKA-mimetic ngHKA mutant K794S/A797P/W940/R949C (SPWC) with 2 K⁺ occluded in E2-P_i and 3 Na⁺-bound in E1·ATP states were nearly identical to NKA structures in equivalent states. Here we report the cryo-EM structures of K794A and K794S, two poorly-selective ngHKA mutants, under conditions to stabilize the E1·ATP state. Unexpectedly, the structures show a hybrid with both E1- and E2-like structural features. While transmembrane segments TM1-TM3 and TM4's extracellular half adopted an E2-like conformation, the rest of the protein assumed an E1 configuration. Two spherical densities, likely bound Na⁺, were observed at cation-binding sites I and III, without density at site II. This explains the E2-like conformation of TM4's exoplasmic half. In NKA, oxygen atoms derived from the unwound portion of TM4 coordinated Na⁺ at site II. Thus, the lack of Na⁺ at site II of K794A/S prevents the luminal portion of TM4 from taking an E1-like position. The K794A structure also suggests that incomplete coordination of Na⁺ at site III induces the halfway rotation of TM6, which impairs Na⁺-binding at the site II. Thus, our observations provide insight into the molecular mechanism of E2-E1 transition and cooperative Na⁺-binding in the NKA and other related cation pumps.     

Taurine Stimulation of Isolated Hamster Brain Na+, K+-ATPase: Activation Kinetics and Chemical Specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na⁺, K⁺-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na⁺, K⁺-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na⁺, K⁺-ATPase by taurine. Normal whole brain homogenate Na⁺, K⁺-ATPase activity is 5.1 ± 0.4 (4) μmol P_i± h^?1± mg^?1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na⁺, K⁺-ATPase activity of 204.6 ± 5.8 (4) mol P_i± h^?1± mg^?1 Lowry protein. Taurine activates the Na⁺, K⁺-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K_1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na⁺, K⁺-ATPase. Its action is mediated by a membrane-bound protein.     

K+ Excretion: The Other Purpose for Puddling Behavior in Japanese Papilio Butterflies

To elucidate the purpose of butterfly puddling, we measured the amounts of Na⁺, K⁺, Ca²⁺, and Mg²⁺ that were absorbed or excreted during puddling by male Japanese Papilio butterflies through a urine test. All of the butterflies that sipped water with a Na⁺ concentration of 13 mM absorbed Na⁺ and excreted K⁺, although certain butterflies that sipped solutions with high concentrations of Na⁺ excreted Na⁺. According to the Na⁺ concentrations observed in naturally occurring water sources, water with a Na⁺ concentration of up to 10 mM appears to be optimal for the health of male Japanese Papilio butterflies. The molar ratio of K⁺ to Na⁺ observed in leaves was 43.94 and that observed in flower nectars was 10.93. The Na⁺ amount in 100 g of host plant leaves ranged from 2.11 to 16.40 mg, and the amount in 100 g of flower nectar ranged from 1.24 to 108.21 mg. Differences in host plants did not explain the differences in the frequency of puddling observed for different Japanese Papilio species. The amounts of Na⁺, K⁺, Ca²⁺, and Mg²⁺ in the meconium of both male and female butterflies were also measured, and both males and females excreted more K⁺ than the other three ions. Thus, the fluid that was excreted by butterflies at emergence also had a role in the excretion of the excessive K⁺ in their bodies. The quantities of Na⁺ and K⁺ observed in butterfly eggs were approximately 0.50 μg and 4.15 μg, respectively; thus, female butterflies required more K⁺ than male butterflies. Therefore, female butterflies did not puddle to excrete K⁺. In conclusion, the purpose of puddling for male Papilio butterflies is not only to absorb Na⁺ to correct deficiencies but also to excrete excessive K⁺.     

Kinetic analysis of gill (Na⁺,K⁺)-ATPase activity in selected ontogenetic stages of the Amazon River shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): interactions at ATP- and cation-binding sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄ ⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K _M = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺)-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K _0.5 = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄ ⁺ had no modulatory effect. The affinity for Na⁺ (K _0.5 = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄ ⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄ ⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.     

Distinct differences in metal ion specificity of RNA and DNA G-quadruplexes

RNA G-quadruplexes, as their well-studied DNA analogs, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH₄ ⁺, Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺. Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes, and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na⁺, K⁺, Rb⁺, NH₄ ⁺, as well as Sr²⁺ and Ba²⁺ in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K⁺ and Sr²⁺ lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na⁺ ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.     

Effects of vanadate on Na+,K+-ATPase and on the force of contraction in guinea-pig hearts.

Vanadate has been shown to be a potent inhibitor of isolated Na⁺,K⁺-ATPase. Since the inhibition of this enzyme system has been implicated in a mechanism for the positive inotropic action of cardiac glycosides, the cardiac actions of vanadate were examined in connection with its action on Na⁺,K⁺-ATPase. Vanadate inhibited isolated Na⁺,K⁺-ATPase obtained from various tissues. The differences in the vanadate sensitivity due to enzyme source were relatively small. K⁺-stimulated phosphatase activity was more sensitive than Na⁺,K⁺-stimulated ATP hydrolysis. The compounds was more potent than phosphate in supporting [³H] oubain binding in the presence of Mg²⁺, indicating a higher affinity of the enzyme for vanadate. It, however, failed to inhibit oubain sensitive ⁸⁶Rb uptake in electrically stimulated atrial muscle of guinea-pig hearts in concentrations which would inhibit isolated Na⁺,K⁺-ATPase. These latter concentrations of vanadate also failed to produce positive inotropic effects in electrically stimulated left atrial preparations of guinea-pig hearts. Higher concentrations produced marked negative inotropic effects associated with a shortening of the action potential duration. These results indicate that vanadate is a potent inhibitor of isolated Na⁺,K⁺-ATPase, but cannot inhibit the enzyme in intact myocardial cells or produce positive inotropic effects when applied extracellularly. Inhibitory sites on the enzyme are probably located at the internal surface of the cell membrane which are normally inaccessible to vanadate in intact tissue.     

Ouabain receptor interactions in (Na + K)-ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases and SH reagents

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (K_D) of 13 nM was found in the presence of (Mg²⁺ + P_i) and (Na⁺ + Mg²⁺ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of (Mg²⁺ + P_i), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of (Na⁺ + K⁺)-ATPase. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

A Low Affinity Vasopressin V2-Receptor in Inherited Nephrogenic Diabetes Insipidus

Abstract

Congenital nephrogenic diabetes insipidus (NDI) is an X-linked inherited disorder characterized by renal resistance to the antidiuretic hormonal action of vasopressin. This study describes the molecular basis of nephrogenic diabetes insipidus in a dog family. Kidney membranes prepared from NDI-affected male huskies were examined for vasopressin binding and response. Compared to membranes from unaffected canines, those from the kidney inner medulla of NDI-dogs possessed normal V₂-receptor numbers, but with 10–fold lower affinity for [Arg⁸] vasopressin (AVP). Adenylate cyclase stimulation by AVP in contrast to that by forskolin or GTP-analogues was similarly reduced in a dose responsive manner. The NDI-affected dogs showed antidiuretic responses to very high doses of V₂–specific agonists, consistent with their possessing V₂–receptors of lower affinity. Prolonged treatment with V₂–agonists, 1–deamino [D-Arg⁸] VP (dDAVP) and 1–deamino [Va]⁴, Sar⁷] AVP (dVSAVP), rendered the NDI-affected dogs near normal in terms of water intake and urine osmolality.     

Prostaglandin E2 modulates Na+,K+-ATPase activity in rat hippocampus: implications for neurological diseases

Prostaglandin E₂ (PGE₂) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na⁺,K⁺‐ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE₂ decreases Na⁺,K⁺‐ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE₂. Na⁺,K⁺‐ATPase activity was determined by assessing ouabain‐sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE₂ (0.1–10 μM) for 30 min decreased Na⁺,K⁺‐ATPase activity in a concentration‐dependent manner. However, PGE₂ did not alter Na⁺,K⁺‐ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE₂ on Na⁺,K⁺‐ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic α subunit of Na⁺,K⁺‐ATPase. We found that the inhibitory effect of PGE₂ (1 μM) on Na⁺,K⁺‐ATPase activity was receptor‐mediated, as incubation with selective antagonists for EP1 (SC‐19220, 10 μM), EP3 (L‐826266, 1 μM) or EP4 (L‐161982, 1 μM) receptors prevented the PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1–10 μM) increased enzyme activity per se in a concentration‐dependent manner, but did not prevent the inhibitory effect of PGE₂. Incubation with a protein kinase A (PKA) inhibitor (H‐89, 1 μM) and a protein kinase C (PKC) inhibitor (GF‐109203X, 300 nM) also prevented PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. Accordingly, PGE₂ increased phosphorylation of Ser943 at the α subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE₂ decreases Na⁺,K⁺‐ATPase activity in vivo. The results presented here imply Na⁺,K⁺‐ATPase as a target for PGE₂‐mediated signaling, which may underlie PGE₂‐induced increase of brain excitability.     

Relationship of Cation Influxes and Effluxes in Yeast

The Na⁺ efflux from Na⁺-rich yeast cells into a cation-free medium is largely balanced by the excretion of organic anions. In the presence of Rb⁺, K⁺, or high levels of H⁺ (pH 3–4), the Na⁺ efflux is increased and the organic anion excretion is suppressed so that stoichiometric cation exchanges occur. H⁺ participates in the exchanges, moving into or out of the cells depending on the external pH and on the concentration of external Rb⁺(K⁺). The total cation efflux is dependent on the external Rb⁺ concentration in a "saturation" relationship, but the individual cations in the efflux stream are not. The discrimination factor in the efflux pathway between H⁺ and Na⁺ is very large (of the order of 10,000), and between Na⁺ and K⁺ considerable (of the order of 50). For the latter pair, the recycling of K⁺ from the cell wall space is an important factor in the discrimination. In addition, the Na⁺ efflux as a function of Na⁺ content follows a sigmoidal curve so that the discrimination factor is increased at high levels of cellular Na⁺. Although the influx and efflux pathways behave as a tightly coupled system, the mechanism of coupling is not entirely clear. A single system with different cation specificities and kinetic behaviors on the inside and outside faces of the membrane could account for the data.     

The excretion of urate and cationic electrolytes by the kidney of the male domestic fowl (Gallus domesticus)

Summary Four groups of roosters were obtained from combinations of high and low protein diets (33% or 11%), and two drinking solutions (tap water or 1% NaCl solution). Ureteral urine was analyzed for urate, Na⁺ and K⁺, in both the liquid and precipitated fractions of the urine.Birds fed a high protein diet-salt water combination excreted unusually large amounts of urate. In all groups, most of the excreted urate was in the form of a precipitate. This precipitate also contained large amounts of Na⁺ and K⁺ (Table 2).The presence of abundant urinary urate, due to high protein diet and large NaCl intake, aids in the excretion of Na⁺ and K⁺, by reducing their contribution to the osmotic pressure of the urine. However, some of these cations, although present in the urine precipitates, do not appear to be in the form of monobasic urate salts. The significance of urate in the excretion of electrolytes is discussed.We wish to thank Dr. Paul B. Siegel, Department of Poultry Science, V.P.I. and S.U., who generously supplied the roosters used in this study. We also wish to thank Mr. Douglas Bartley for his determination of urine pH values, and Dr. Alan G. Heath for the use of his microelectrode pH apparatus. This work was supported by USPHS-NIH grant, number AM 14991.     

Roles of transmembrane segment M1 of Na+,K+-ATPase and Ca2+-ATPase, the gatekeeper and the pivot

In this review we summarize mutagenesis work on the structure–function relationship of transmembrane segment M1 in the Na⁺,K⁺-ATPase and the sarco(endo)plasmic reticulum Ca²⁺-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca²⁺ interaction/occlusion in Ca²⁺-ATPase and K⁺ interaction/occlusion in Na⁺,K⁺-ATPase. Leu⁶⁵ of the Ca²⁺-ATPase and Leu⁹⁹ of the Na⁺,K⁺-ATPase, located at homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu³⁰⁹/Glu³²⁹. A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the extracytoplasmic gate in both the Ca²⁺-ATPase and the Na⁺,K⁺-ATPase. All numbering of Na⁺,K⁺-ATPase amino acid residues in this article refers to the sequence of the rat α₁-isoform.     

Coordination of AtHKT1;1 and AtSOS1 facilitates Na+ and K+ homeostasis in Arabidopsis thaliana under salt stress

Reducing Na⁺ accumulation and maintaining K⁺ stability in plant is one of the key strategies for improving salt tolerance. AtHKT1;1 and AtSOS1 are not only the salt tolerance determinants themselves, but also mediate K⁺ uptake and transport indirectly. To assess the contribution of AtHKT1;1 and AtSOS1 to Na⁺ homeostasis and K⁺ nutrition in plant, net Na⁺ and K⁺ uptake rate, Na⁺ and K⁺ distributions in Arabidopsis thaliana wild type (WT), hkt1;1 mutant (athkt1;1) and sos1 mutant (atsos1) were investigated. Results showed that under 2.5 mM K⁺ plus 25 or 100 mM NaCl, athkt1;1 shoot concurrently accumulated more Na⁺ and less K⁺ than did WT shoot, suggesting that AtHKT1;1 was critical for controlling Na⁺ and K⁺ distribution in plant; while atsos1 root accumulated more Na⁺ and absorbed lower K⁺ than did WT root, implying that AtSOS1 was determiner of Na⁺ excretion and K⁺ acquisition. Under 0.01 mM K⁺, athkt1;1 absorbed lower Na⁺ than did WT with 100 mM NaCl, suggesting that AtHKT1;1 is involved in Na⁺ uptake in roots; while atsos1 shoot accumulated less Na⁺ than did WT shoot no matter with 25 or 100 mM NaCl, implying that AtSOS1 played a key role in controlling long-distance Na⁺ transport from root to shoot. We present a model in which coordination of AtHKT1;1 and AtSOS1 facilitates Na⁺ and K⁺ homeostasis in A. thaliana under salt stress: under the normal K⁺, the major function of AtHKT1;1 is Na⁺ unloading and AtSOS1 is mainly involved in Na⁺ exclusion, whereas under the low K⁺, AtHKT1;1 may play a dominant role in Na+ uptake and AtSOS1 may be mainly involved in Na⁺ loading into the xylem.     

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons

Binding to Na⁺,K⁺-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na⁺,K⁺-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na⁺,K⁺-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na⁺,K⁺-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein–protein interactions between Na⁺,K⁺-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na⁺,K⁺-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na⁺,K⁺-ATPase.     

The Na+,K+,2Cl−-cotransport system in HeLa cells: Aspects of its physiological regulation

We have previously reported on the biochemical properties of a Na⁺,K⁺,2Cl^?-cotransport in HeLa cells and here we deal with aspects of its physiological regulation. Na⁺,K⁺,2Cl^?-cotransport in HeLa cells was studied by ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺/²²Na⁺ efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on ⁸⁶Rb⁺ flux, mediated by the bumet-anide-sensitive Na⁺, K⁺, 2Cl^?-cotransport system and the ouabain-sensitive Na⁺/K⁺-pump, were investigated. ANP reduced bumetanide-sensitive ⁸⁶Rb⁺ influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide-sensitive ⁸⁶Rb⁺ influx was observed in the presence of 8-bromo-cGMP, while neither isoproterenol as a β-receptor agonist nor 8-bromo-cAMP-could alter bumetanide-sensitive ⁸⁶Rb⁺ influx. Furthermore, efflux of ⁸⁶Rb⁺ and ²²Na⁺ was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim. Biophys. Res. Commun. 168:148–154, 1990), this study indicates that ANP participates in the regulation of the Na⁺, K⁺, 2Cl^?-cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative α-methyl-aminoisobutyric acid (metAlB, 1 and 5 mM, respectively) also reduced Na⁺, K⁺, 2Cl^?-cotransport-mediated ⁸⁶Rb⁺ uptake and diminished the stimulatory effect of hypertonicity on the cotransporter. In addition, the Na⁺/K⁺-pump was markedly stimulated in the presence of amino acids, while neither ANP and 8-Br-cGMP nor isoproterenol and 8-Br-cAMP had a significant effect on the activity of the Na⁺/K⁺-pump.