Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

Characterization of the Na+/H+ exchanger in human epidermoid carcinoma A431 vesicles

A previous report from this laboratory (Rothenberg et al., 1983a) demonstrated the presence of an Na+/H+ exchanger in human epidermoid carcinoma A431 cells. We now characterize surface-derived membrane vesicles from this cell line which contain a functional Na+/H+ exchanger. The Na+/H+ exchanger in A431 vesicles shares a number of characteristics in common with previously described Na+/H+ exchangers including the following: (1) Na+ uptake is stimulated by an outward-directed pH gradient and inhibited by an inward-directed pH gradient. (2) Na+ uptake is inhibited by amiloride and its analogs and their relative effectiveness is similar in vesicles and A431 cells. (3) The Na+/H+ exchanger uses Na+ or Li+ as a substrate but not K+ or Cs+. (4) H+ efflux is stimulated by an inward-directed Na+ gradient and inhibited by the amiloride analog 5-N-dimethylamiloride. The Na+/H+ exchanger in these membrane vesicles is activated allosterically by low intravesicular pH. The apparent pKa of the activating site is 6.4-6.6, characteristic of the NA+/H+ exchanger before activation by mitogens.     

Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles

Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.     

Na+/H+ exchange mechanism in the basolateral membrane of the rat enterocyte

Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.     

Electrogenicity of the Na+-ATPase from the marine microalga Tetraselmis (Platymonas) viridis and associated H+ countertransport

Sodium accumulation by the Na+-ATPase in the plasma membrane (PM) vesicles isolated from the marine alga Tetraselmis (Platymonas) viridis was shown to be accompanied by deltapsi generation across the vesicle membrane (positive inside) and H+ efflux from the vesicle lumen. Na+ accumulation was assayed with 22Na+; deltapsi generation was detected by recording absorption changes of oxonol VI; H+ efflux was monitored as an increase in fluorescence intensity of the pH indicator pyranine loaded into the vesicles. Both ATP-dependent Na+ uptake and H+ ejection were increased by the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CICCP) while deltapsi was collapsed. The lipophilic anion tetraphenylboron ion (TPB-) inhibited H+ ejection from the vesicles and abolished deltapsi. Based on the effects of CICCP and TPB- on H+ ejection and deltapsi generation, the conclusion was drawn that H+ countertransport observed during Na+-ATPase operation is a secondary event energized by the electric potential which is generated in the course of Na+ translocation across the vesicle membrane. Increasing Na+ concentrations stimulated H+ efflux and caused the decrease in the deltapsi observed, thus indicating that Na+ is likely a factor controlling H+ permeability of the vesicle membrane.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Na+-H+ exchange in cardiac sarcolemmal vesicles

The transport of Na+ by a purified sarcolemmal vesicular preparation from canine ventricular tissue was studied as a function of both internal and external pH. The uptake of Na+ into sarcolemmal vesicles increased upon raising the extravesicular pH of the reaction medium. Half-maximal uptake of Na+ was observed at a pHo of about 8.1 and maximal uptake occurred at pH 8.6. The uptake of Na+ by sarcolemma was also dependent upon the intravesicular pH. Na+ uptake into sarcolemmal vesicles was greatly attenuated in the absence of a H+ gradient across the membrane. Transport of Na+ was potently inhibited by amiloride, a known blocker of Na+-H+ exchange. LiCl was also an effective inhibitor of Na+ transport. In the presence of optimal H+ gradients, Na+ uptake was linear for the first 5 seconds of the reaction and exhibited a Vmax of 290 nmol Na+/mg per min and a KNa of 3.5 mM. These experiments strongly indicate the presence of a Na+-H+ exchange system in cardiac sarcolemma. This activity appeared to be relatively specific for this membrane fraction. The identification of Na+-H+ exchange activity in a sarcolemmal vesicular fraction from the heart will permit extensive characterization of the regulation and kinetics of this antiporter in future investigations.     

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+.     

Reconstitution of the mitochondrial non-selective Na+/H+ (K+/H+) antiporter into proteoliposomes

Mitochondria contain two Na+/H+ antiporters, one of which transports K+ as well as Na+. The physiological role of this non-selective Na+/H+ (K+/H+) antiporter is to provide mitochondrial volume homeostasis. The properties of this carrier have been well documented in intact mitochondria, and it has been identified as an 82,000-dalton inner membrane protein. The present studies were designed to solubilize and reconstitute this antiporter in order to permit its isolation and molecular characterization. Proteins from mitoplasts made from rat liver mitochondria were extracted with Triton X-100 in the presence of cardiolipin and reconstituted into phospholipid vesicles. The reconstituted proteoliposomes exhibited electroneutral 86Rb+ transport which was reversibly inhibited by Mg2+ and quinine with K0.5 values of approximately 150 and 300 microM, respectively. Incubation of reconstituted vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of 86Rb+ uptake into proteoliposomes. Incubation of vesicles with [14C]dicyclohexylcarbodiimide resulted in labeling of an 82,000-dalton protein. These properties, which are also characteristic of the native Na+/H+ (K+/H+) antiporter, lead us to conclude that this mitochondrial carrier has been reconstituted into proteoliposomes with its known native properties intact.     

Submicromolar Ag+ increases passive Na+ permeability and inhibits the respiration-supported formation of Na+ gradient in Bacillus FTU vesicles

The effect of Ag+ on Na+ pumping by Na(+)-motive NADH-quinone reductase and terminal oxidase has been studied in Bacillus FTU inside-out vesicles. Very low concentrations of Ag+ (C1/2 = 1 x 10(-8) M or 2 x 10(-12) g ion.mg protein-1) are shown to inhibit the uphill Na+ uptake coupled to the oxidation of NADH by fumarate or of ascorbate + TMPD by oxygen but exert no effect on the H+ uptake by the H(+)-motive respiratory chain. Low Ag+ also induces a specific increase in the Na+ permeability of the vesicles. HQNO, added before and not after Ag+, prevents the Ag(+)-induced permeability increase, with effective HQNO concentrations being similar to those inhibiting the uphill Na(+)-uptake coupled to the NADH-fumarate oxidoreduction. Reduction of terminal oxidase by ascorbate + TMPD in the presence of cyanide sensitizes the Na+ permeability to Ag+. It is suggested that low [Ag+], known as a specific inhibitor of electron transport by the Na(+)-motive NADH-quinone reductase, uncouples the electron and Na+ transports so that the Ag(+)-modified NADH-quinone reductase operates as an Na+ channel rather than an Na+ pump. This effect is discussed in connection with the antibacterial action of Ag+.     

Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

The C-terminally truncated NuoL subunit (ND5 homologue) of the Na+-dependent complex I from Escherichia coli transports Na+

The NADH:quinone oxidoreductase (complex I) from Escherichia coli acts as a primary Na+ pump. Expression of a C-terminally truncated version of the hydrophobic NuoL subunit (ND5 homologue) from E. coli complex I resulted in Na+-dependent growth inhibition of the E. coli host cells. Membrane vesicles containing the truncated NuoL subunit (NuoLN) exhibited 2-4-fold higher Na+ uptake activity than control vesicles without NuoLN. Respiratory proton transport into inverted vesicles containing NuoLN decreased upon addition of Na+, but was not affected by K+, indicating a Na+-dependent increase of proton permeability of membranes in the presence of NuoLN. The His-tagged NuoLN protein was solubilized, enriched by affinity chromatography, and reconstituted into proteoliposomes. Reconstituted His6-NuoLN facilitated the uptake of Na+ into the proteoliposomes along a concentration gradient. This Na+ uptake was prevented by EIPA (5-(N-ethyl-N-isopropyl)-amiloride), which acts as inhibitor against Na+/H+ antiporters.     

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.     

The H(+)-motive and Na(+)-motive respiratory chains in Bacillus FTU subcellular vesicles.

Respiration-dependent pumping of Na+ and H+ into the inside-out subcellular vesicles of alkalotolerant and halotolerant Bacillus FTU grown at alkaline pH was studied. The vesicles were shown to be competent in Na+ and H+ transport coupled to ascorbate oxidation via N,N,N',N'-tetramethyl-p-phenylenediamine or diaminodurene. The uphill Na+ uptake is strongly stimulated by either protonophores or valinomycin, whereas H+ uptake is stimulated by valinomycin and completely inhibited by protonophores. The salt of a penetrating weak base and of the penetrating weak acid, diethylammonium acetate, potentiates the stimulating effect of protonophores on Na+ uptake and abolishes H+ uptake. Na+ transport, supported by ascorbate oxidation, is resistant to 2-heptyl-4-hydroxyquinoline N-oxide, but sensitive to Ag+ and Na+ ionophore, N,N'-dibenzyl-N,N'-diphenyl-1,2-phenylenediacetamide. Micromolar concentrations of cyanide specifically inhibit the H+ uptake but does not affect Na+ uptake. These cyanide concentrations are shown to cause 70% inhibition of respiration, complete reduction of alpha-type cytochromes and partial reduction of c/b-type cytochromes. To inhibit the remaining respiratory activity and Na/ uptake, approximately 100-fold higher cyanide concentrations are necessary. High cyanide concentrations cause some additional increase in absorbance in the region of cytochromes c and/or b. In the presence of a high cyanide concentration, Na+ uptake can be supported by NADH oxidation by fumarate. This Na+ transport is stimulated by protonophores and diethylammonium acetate, being sensitive to very low concentrations of 2-heptyl-4-hydroxyquinoline N-oxide and Ag+. The NADH-fumarate reductase reaction is also found to be competent in H+ uptake, which is inhibited by protonophores and by much higher 2-heptyl-4-hydroxyquinoline N-oxide concentrations, and is resistant to Ag+. It is inferred that Bacillus FTU possesses two respiratory chains: the H(+)-motive and the Na(+)-motive, which strongly differ in their inhibitor sensitivities. Each chain comprises at least two energy-coupling sites which are localized in their initial and terminal segments. It has been indicated that common redox carrier(s) are present in the two chains.     

Generation of Na+ electrochemical potential by the Na+-motive NADH oxidase and Na+/H+ antiport system of a moderately halophilic Vibrio costicola

Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate-dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential.     

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.     

Effect of cisplatin on H+ transport by H+ -ATPase and Na+/H+ exchanger in rat renal brush-border membrane

The effect of the potent anticancer drug cisplatin, cis-diamminedichloroplatinum (II) (CDDP), on H+ -ATPase and Na+/H+ exchanger in rat renal brush-border membrane was examined. To measure H+ transport by vacuolar H+ -ATPase in renal brush-border membrane vesicles, we employed a detergent-dilution procedure, which can reorientate the catalytic domain of H+ -ATPase from an inward-facing configuration to outward-facing one. ATP-driven H+ pump activity decreased markedly in brush-border membrane prepared from rats two days after CDDP administration (5 mg/kg, i.p.). In addition, N-ethylmaleimide and bafilomycin A1 (inhibitors of vacuolar H+ -ATPase)-sensitive ATPase activity also decreased in these rats. The decrease in ATP-driven H+ pump activity was observed even at day 7 after the administration of CDDP. Suppression of ATP-driven H+ pump activity was also observed when brush-border membrane vesicles prepared from normal rats were pretreated with CDDP in vitro. In contrast with H+ -ATPase, the activity of Na+/H+ exchanger, which was determined by measuring acridine orange fluorescence quenching, was not affected by the administration of CDDP. These results provide new insights into CDDP-induced renal tubular dysfunctions, especially such as proximal tubular acidosis and proteinuria.     

Nitric oxide and superoxide impair human placental amino acid uptake and increase Na+ permeability: implications for fetal growth

Based on evidence that thiol and tyrosine reagents inhibit some amino acid transporters, we tested the hypothesis that NO- and O2- -derived free radicals would impair nutrient uptake by the human placenta. Syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments were exposed to the drug SIN-1 in the presence or absence of superoxide dismutase (SOD) and hemoglobin (Hb). The uptake of [3H]arginine, [3H]taurine, and [3H]leucine; [14C]MeAIB; and 22Na was studied in MVM, whereas the uptake of [3H]taurine was examined in villous fragments. Nitrotyrosine formation was assessed by Western blotting and quantified by ELISA. In MVM, SIN-1 caused an inhibition of [3H]arginine, [3H]taurine, and [14C]MeAIB uptake but had no significant effect on equilibrium [3H]leucine uptake. These effects were prevented by SOD or Hb, implying that both NO and O2- radicals were essential. In contrast, 22Na+ uptake was significantly increased, and this effect was prevented by SOD. In villous fragments, SIN-1 impaired Na+-dependent [3H]taurine uptake, with no effect on Na+-independent uptake. Increased nitrotyrosine formation was observed in MVM after SIN-1 treatment. Endogenous NO- and O2- -derived free radicals may alter human placental nutrient transfer in vivo, with implications for fetal growth.     

Effect of dietary polyamines and alpha-difluoromethylornithine on regulation of intestinal Na+/H+ exchanger

Polyamines are compounds required for initiation of rapid cellular growth and differentiation in many cell types. Ornithine decarboxylase is the rate limiting enzyme in polyamine synthesis. Fasting and refeeding regulates the activity of ornithine decarboxylase and polyamine content in the intestinal tract. We tested the hypothesis that polyamines regulate cell growth via the Na+/H+ exchanger which is believed to be intimately involved in cell growth. Ileal Na+/H+ activity was therefore examined in control, fasted, refed fasted, and in rats given the specific inhibitor of ornithine decarboxylase alpha-difluoromethylornithine. A well-validated ileal brush border membrane vesicles for the study of Na+/H+ exchange activity was utilized. Fasting markedly decreased while refeeding stimulated Na+/H+ exchange activity at all times studied (P less than 0.05-0.001). Maximal uptake of Na+ at 5 min was 3.12 +/- 0.05, 2.5 +/- 0.05 and 2.22 +/- 0.05 nmol/mg protein in refed, control and fasted rats respectively. Kinetics of amiloride sensitive Na+/H+ exchanger showed a Vmax of 17.1 +/- 3.5, 8.0 +/- 0.64 and 4.7 +/- 1.1 nmol/mg protein per 5 s in refed fasted, control and fasted rats respectively Km values were not significantly different between the groups studied. 2% alpha-difluoromethylornithine given in the drinking water abolished the stimulation in Na+/H+ exchange activity in refed fasted rats. These results suggest a close relationship between polyamines and Na+/H+ activity in the intestinal mucosa of rats.     

Na+/H+ antiporters

Na+/H+ antiports or exchange reactions have been found widely, if not ubiquitously, in prokaryotic and eukaryotic membranes. In any given experimental system, the multiplicity of ion conductance pathways and the absence of specific inhibitors complicate efforts to establish that the antiport observed actually results from the activity of a specific secondary porter which catalyzes coupled exchanged of the two ions. Nevertheless, a large body of evidence suggests that at least some prokaryotes possess a delta psi-dependent, mutable Na+/H+ antiporter which catalyzes Na+ extrusion in exchange for H+; in other bacterial species, the antiporter my function electroneutrally, at least at some external pH values. The bacterial Na+/H+ antiporter constitutes a critical limb of Na+ circulation, functioning to maintain a delta mu Na+ for use by Na+-coupled bioenergetic processes. The prokaryotic antiporter is also involved in pH homeostasis in the alkaline pH range. Studies of mutant strains that are deficient in Na+/H+ antiporter activity also indicate the existence of a relationship, e.g., a common subunit or regulatory factor, between the Na+/H+ antiporter and Na+/solute symporters in several bacterial species. In eukaryotes, an electroneutral, amiloride-sensitive Na+/H+ antiport has been found in a wide variety of cell and tissue types. Generally, the normal direction of the antiport appears to be that of Na+ uptake and H+ extrusion. The activity is thus implicated as part of a complex system for Na+ circulation, e.g., in transepithelial transport, and might have some role in acidification in the renal proximal tubule. In many experimental systems, the Na+/H+ antiport appears to influence intracellular pH. In addition to a role in general pH homeostasis, such Na+-dependent changes in intracellular pH could be part of the early events in a variety of differentiating and proliferative systems. Reconstitution and structural studies, as well as detailed analysis of gene loci and products which affect the antiport activity, are in their very early stages. These studies will be important in further clarification of the precise structural nature and role(s) of the Na+/H+ antiporters. In neither prokaryotes nor eukaryotes systems is there yet incontrovertible evidence that a specific protein carrier, that catalyzes Na+/H+ antiport, is actually responsible for any of the multitude of effects attributed to such antiporters. The Na+-H+ exchange might turn out to be side reactions of other porters or the additive effects of several conductance pathways; or, as appears most likely in at least some bacteria and in renal tissue, the antiporter may be a discrete, complex carr