Der Einfluß von Natrium- und Kaliumionen auf die Phosphataufnahme bei Ankistrodesmus braunii

Summary The uptake of phosphate as influenced by sodium and potassium ions was investigated in the light and in the dark. It was found to be a function of the external phosphate concentration. At a low concentration (up to 10^–5 mol/l) in the presence of Na⁺ phosphate is quickly absorbed and hence phosphate is the limiting factor for further labelling. In the presence of K⁺ phosphate uptake is constant over a long period.The enhancement of phosphate uptake by Na⁺ is also found when the external concentration of P is raised up to 10^–4 mol/l. Then the gross uptake proceeds over six hours, with the greatest Na⁺-dependent increase occurring in the label of the TCA-insoluble phosphate fraction (P_u).The phosphate uptake is strongly dependent on the pH of the reaction mixture. In the presence of Na⁺ it is highest between pH 5.6 and 7. As the uptake in the presence of K⁺ parallels the dissociation curve of the dihydrogen form H₂PO ₄ ^– , the Na⁺-enhancement is optimal in the alkaline pH range (pH 8).On the basis of a comparison between the pH-dependence of phosphate uptake and the dependence of the uptake on the external phosphate concentration analysed by a method of enzyme kinetics, it is suggested that Ankistrodesmus metabolically transports H₂PO ₄ ^– but not HPO ₄ ⁼ . Moreover, it is concluded from the absence of light stimulation and the weak inhibition of the uptake by DCMU or CCCP in the presence of K⁺ that at low P-concentrations the diffusion is limiting the uptake. Only at higher concentrations is an active phosphate uptake measured.Furthermore it is concluded that the observed Na⁺-stimulation of the ³²P-labelling of the TCA-soluble and insoluble compounds inside the cell is indirect and depends only on the action of Na⁺ and K⁺ ions at the first transport site in the plasmalemma.     

Efficiency,Na+/K+ selectivity and temperature dependence of ion transport through lipid membranes by (221)C10-Cryptand,an ionizable mobile carrier

Summary The kinetics of Na⁺ and K⁺ transport across the membrane of large unilamellar vesicles (LUV) were determined at two pH's when transport was induced by (221)C₁₀-cryptand (diaza-1,10-decyl-5-pentaoxa-4,7,13,16,21-bicyclo [8.8.5.] tricosane) at various temperatures, and by nonactin at 25°C and (222)C₁₀-cryptand at 20 and 25°C. The rate of Na⁺ and K⁺ transport by (221)C₁₀ saturated with the cation and carrier concentrations. Transport was noncooperative and exhibited selectivity for Na⁺ with respect to K⁺. The apparent affinity of (221)C₁₀ for Na⁺ was higher and less pH-dependent than that for K⁺, and seven times higher than that of (222)C₁₀ for K⁺ ions (20.5vs. 1.7 kcal·mole^–). The efficiency of (221)C₁₀ transport of Na⁺ was pH-and carrier concentration-dependent, and was similar to that of nonactin; its activation energy was similar to that for (222)C₁₀ transport of K⁺ (35.5 and 29.7 kcal · mole^–1, respectively). The reaction orders in cationn(S) and in carrierm(M), respectively, increased and decreased as the temperature rose, and were both independent of carrier or cation concentrations; in most cases they varied slightly with the pH.n(S) varied with the cation at pH 8.7 and with the carrier for Na⁺ transport only, whilem(M) always depended on the type of cation and carrier. Results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes.     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

pH Modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistiguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pH_int/pH_ext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2m). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pH_ext 5.8 (K _m =1.33vs. 1.70 m;V _max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect thatK _m, for carrier-mediated F transport. These data are consistent with similarK _i's for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H⁺ K _m (or OH^– IC₅₀) corresponding to pH 4.89. External pH affects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺) rather than competitive binding that is mutually exclusive.     

Stimulation by HCO 3 − of Na+ transport in rabbit gallbladder

Summary Bicarbonate presence in the bathing media doubles Na⁺ and fluid transepithelial transport and in parallel significantly increases Na⁺ and Cl^– intracellular concentrations and contents, decreases K⁺ cell concentration without changing its amount, and causes a large cell swelling. Na⁺ and Cl^– lumen-to-cell influxes are significantly enhanced, Na⁺ more so than Cl^–. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO ₃ ^– -ATPase in the brush border. The stimulation goes together with a large increase in a Na⁺-dependent H⁺ secretion into the lumen. All of these data suggests that HCO ₃ ^– both activates Na⁺–Cl^– cotransport and H⁺–Na⁺ countertransport at the luminal barrier.Thiocyanate inhibits Na⁺ and fluid transepithelial transport without affecting H⁺ secretion and HCO ₃ ^– -dependent Na⁺ influx. It reduces Na⁺ and Cl^– concentrations and contents, increases the same parameters for K⁺, causes a cell shrinking, and abolishes the lumen-to-cell Cl^– influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na⁺-dependent and HCO ₃ ^– -activated. Thus, SCN^– is likely to compete for the Cl^– site on the cotransport carrier and to be slowly transferred by the cotransport system itself.     

pH modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an outwardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistinguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pH_int/pH_ext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2 M). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at low pH (monovalent F^– predominates) and at least 2 H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pH_ext5.8 (K _m =1.33vs. 1.70 M;V _max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect theK _m for carrier-mediated F transport. These data are consistent with similarK _i ^' s for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H⁺ K _m (or OH^– lC₅₀) corresponding to pH 4.89. External pH effects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺),rather than competitive binding that is mutually exclusive.     

Functional roles of Na+ and H+ in SO 4 2− transport by rabbit ileal brush border membrane vesicles

Summary Sulphate uptake by rabbit ileal brush border membrane vesicles was stimulated by a transmembrane sodium gradient ([Na⁺] _o >[Na⁺] _i ), but not by a similar potassium gradient.³⁵SO ₄ ^2– influx (J _oi ^SO4 ) from outside (o) to inside (i) these vesicles was a hyperbolic function of [SO ₄ ^2– ] _o and the affinity constant for anion transport was strongly influenced by [Na⁺] _o (100mm Na⁺,K _t ^SO4 =0.52mm SO ₄ ^2– ; 10mm Na⁺,K _t ^SO4 =4.32mm SO ₄ ^2– ).J _t ^SO4 was a sigmoidal function of [Na⁺] _o at pH 7.4 for both low (0.2m) and high (4.0mm) [SO ₄ ^2– ] _o . The Na⁺-dependency ofJ _t ^SO4 was examined at pH 6.0, 7.4, and 8.0 (same pH inside and outside). At pH 6.0 and 7.4 sigmoidal Na⁺-dependentJ _t ^SO4 exhibited nonlinear Eadie-Hofstee plots indicative of a transport mechanism capable of binding a variable number of sodium ions over the [Na⁺] _o range used. Hill plots of anion transport under these conditions displayed slopes near unity at low [Na⁺] _o and slopes approximating 2.0 at higher cation concentrations. At pH 8.0, Na⁺-dependentJ _t ^SO4 was hyperbolic and showed linear Eadie-Hofstee and Hill plots, the latter with a single slope near 1.0. When a H⁺ gradient was imposed across the vesicle wall (pH _i =8.0, pH _o =6.0), Na⁺-dependentJ _t ^SO4 was hyperbolic and significantly increased at each [Na⁺] _o over values observed using bilateral pH 8.0. In contrast, a H⁺ gradient oriented in the opposite direction (pH _i =6.0, pH _o =8.0) led to Na⁺-dependentJ _t ^SO4 that was sigmoidal and significantly lower at each [Na⁺] _o than values found using bilateral pH 6.0. Electrogenicity ofJ _t ^SO4 at pH 8.0 for both high and low [Na⁺] _o was demonstrated by using a valinomycin-induced transmembrane electrical potential difference. At pH 6.0, electrogenicJ _t ^SO4 occurred only at low [Na⁺] _o (5mm); anion transfer was electroneutral at 50mm Na⁺. A model is proposed for proton regulation of sodium sulphate cotransport where flux stoichiometry is controlled by [H⁺] _i and sodium binding affinity is modified by [H⁺] _o . Preliminary experiments with rabbit proximal tubular brush border membrane vesicles disclosed similarJ _t ^SO4 kinetic properties and a common transport mechanism may occur in both tissues.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Summary Rabbit erythrocytes are well known for possessing highly active Na⁺/Na⁺ and Na⁺/H⁺ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na⁺ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na⁺/Na⁺ exchange activity, determined as the Na _o ⁺ -dependent²²Na efflux or Na _i ⁺ -induced²²Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H _i ⁺ -induced Na⁺ entry sensitive to amiloride (Na⁺/H⁺ exchange activity). Similarly, EIPA, a strong inhibitor of the Na⁺/H⁺ exchanger, did not inhibit Na⁺/Na^– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na⁺/Na⁺ exchanger could be involved in Na⁺/H⁺ countertransport was suggested by studies in which the net Na⁺ transport sensitive to NEM was determined. As expected, net Na⁺ transport through this transport system was zero at different [Na⁺] _i /[Na⁺] _o ratios when intracellular pH was 7.2. However, at pH _i =6.1, net Na⁺ influx occurred when [Na⁺] _i was lower than 39mm. Valinomycin, which at low [K⁺] _o was lower than 39mm. Valinomycin, which at low [K⁺] _o clamps the membrane potential close to the K⁺ equilibrium potential, did not affect the net NEM-sensitive Na⁺ entry but markedly stimulated, the EIPA-and NEM-resistant Na⁺ uptake. This suggest that the net Na⁺ entry through the NEM-sensitive pathway at low pH _i , is mediated by an electroneutral process possibly involving Na⁺/H⁺ exchange. In contrast, the EIPA-sensitive Na⁺/H⁺ exchanger is not involved in Na⁺/Na⁺ countertransport, because Na⁺ transport through this mechanism is not affected by an increase in cell Na^– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na⁺/Na⁺ and Na⁺/H⁺ exchangers, are mediated by distinct transport proteins.     

Characterization of MgATP-Driven H+ uptake in to a microsomal vesicle franction from rat pancreatic acinar cells

Summary In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H⁺ transport was evaluated by measuring H⁺-dependent accumulation of acridine orange (AO). Active H⁺ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (K _m for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H⁺ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H⁺ accumulatioin was abolished by protonophores. such as nigericin (10^–6 mol/liter) or CCCP (10^–5 mol/liter), and by inhibitors of nonmitochondria H⁺ ATPase, such as NEM or NBD-Cl, at a concentration of 10^–5 mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H⁺ pumps, such as DCCD (10^–5 mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H⁺ transport by about 90%. Vanadate (2×10^–3 mol/liter). a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H⁺ transport. The stilbene derivative DIDS (10^–4 mol/liter), which inhibits anion transport systems, abolished H⁺ transport completely. MgATP-dependent H⁺ transport was found to be anion dependernt in the sequence Cl^–>Br^–>gluconate^–; in the presence of SO ₄ ^–2 . CH₃COO^– or No ₃ ^– , no H⁺ transport was observed. MgATP-dependent H⁺ accumulation was also cation dependent in the sequence K⁺>Li⁺>Na⁺=choline⁺, As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl^– and a K⁺ conductance, as well as a small H⁺ conductance. were found in the microsomal membranes. When membranes containing the H⁺ pump wer further purified by Percoll gradient centrifugatin (ninefold enrichment comparad to homogenate), no correlation with markers for endoplasmic reticulum., mitochondria, plasma membranes, zymogen graules or Golgi membranes was found.The present data indicate that the H⁺ pump located in microsomes from rat exocrine pancreas is a vacuolar-or V-type H⁺ ATPase and has most similarities to that described in endoplasmic reticulum. Golgi apparatus or endosomes.     

Transport of alkali cations through thin lipid membranes by (222)C10-Cryptand,an ionizable mobile carrier

Summary The kinetics of K⁺ and Na⁺ transport across the membrane of large unilamellar vesicles (L.U.V.) were compared at two pH's, with two carriers: (222)C ₁₀-cryptand (diaza-1, 10-decyl-5-hexaoxa-4,7,13,16,21,24-bicyclo[8.8.8.]hexacosane) and valinomcyin, i.e. an ionizable macrobicyclic amino polyether and a neutral macrocyclic antibiotic. The rate of cation transport by (222)C₁₀ saturated as cation and carrier concentrations rose. The apparent affinity of (222)C₁₀ for K⁺ was higher and less pH dependent than that for Na⁺ but resembled the affinity of valinomycin for K⁺. The efficiency of (222)C₁₀ transport of K⁺ decreased as the pH fell and the carrier concentration rose, and was about ten times lower than that of valinomycin. Noncompetitive K⁺/Na⁺ transport selectivity of (222)C₁₀ decreased as pH, and cation and carrier concentrations rose, and was lower than that of valinomycin. Transport of alkali cations by (222)C₁₀ and valinomycin was noncooperative. Reaction orders in cationn(S) and carrierm(M) varied with the type of cation and carrier and were almost independent of pH;n(S) andm(M) were not respectively dependent on carrier or cation concentrations. The apparent estimated constants for cation translocation by (222)C₁₀ were higher in the presence of Na⁺ than of K⁺ due to higher carrier saturation by K⁺, and decreased as pH and carrier concentration increased. Equilibrium potential was independent of the nature of carrier and transported cation. Results are discussed in terms of the structural, physicochemical and electrical characteristics of carriers and complexes.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Transport and utilization of methionine sulfoxide in the rabbit

Methionine sulfoxide is transported into purified intestinal and renal brush border membrane vesicles from rabbit by an Na⁺-dependent mechanism and is accumulated inside the vesicles against the concentration gradient. Both in intestine and kidney, the rate of transport is enhanced with increasing concentrations of Na⁺ in the external medium. Increasing the Na⁺ gradient reduces the apparent K_t for methionine sulfoxide without causing any change in V_max. With an outward K⁺ gradient (vesicle > medium), valinomycin stimulates the Na⁺-gradient-dependent transport of methionine sulfoxide in the kidney, showing the electrogenicity of the transport process. A number of amino acids inhibit methionine sulfoxide transport in both the intestine and kidney. An enzymatic activity capable of reducing methionine sulfoxide to methionine is present in the intestinal mucosa, renal cortex and liver. The activity is highest in renal cortex and lowest in intestine. The methionine sulfoxide-reducing activity is stimulated by NADH, NADPH, glutathione and dithiothreitol and the potency of the stimulation is in the order: dithiothreitol > NADPH > glutathione > NADH.     

Relations between intracellular ion activities and extracellular osmolarity inNecturus gallbladder epithelium

Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K⁺ and Cl^– in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K⁺, Cl^– and some unidentified anions. Surprisingly, the apparent K⁺ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K⁺ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na⁺, K⁺ and Cl^– were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na⁺ and Cl^– were equal. The data were consistent with Na⁺ and Cl^– ions initially remaining inside the cell and a cell membraneL _p of 10^–3 cm sec^–1 osm^–1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K⁺ was only 0.1–0.2 times the change observed in Na⁺ and Cl^– activities, and suggests that K⁺ ions leave the cell during the osmotically induced H₂O efflux and enter with an induced H₂O influx. The coupling is between 98 and 102 mmoles liter^–1. Various explanations for the anomalous behavior of intracellular K⁺ ions are considered. A discussion of the apparent coupling between K⁺ and H₂O, observed in nonsteady states, and its effects on the distribution of K⁺ and H₂O across the cell membrane in the steady states, is presented.     

HCO 3 − -coupled Na+ influx is a major determinant of Na+ turnover and Na+/K+ pump activity in rat hepatocytes

Summary Recent studies in hepatocytes indicate that Na⁺-coupled HCO ₃ ^– transport contributes importantly, to regulation of intracellular pH and membrane HCO ₃ ^– transport. However, the direction of net coupled Na⁺ and HCO ₃ ^– movement and the effect of HCO ₃ ^– on Na⁺ turnover and Na⁺/K⁺ pump activity are not known. In these studies, the effect of HCO ₃ ^– on Na⁺ influx and turnover were measured in primary rat hepatocyte cultures with²²Na⁺, and [Na⁺] _i was measured in single hepatocytes using the Na⁺-sensitive fluorochrome SBFI. Na⁺/K⁺ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na⁺-dependent or ouabain-suppressible⁸⁶Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K⁺ on membrane potential difference (PD) and [Na⁺] _i . In hepatocyte monolayers, HCO ₃ ^– increased²²Na⁺ entry and turnover rates by 50–65%, without measurably altering²²Na⁺ pool size or cell volume, and HCO ₃ ^– also increased Na⁺/K⁺ pump activity by 70%. In single cells, exposure to HCO ₃ ^– produced an abrupt and sustained rise in [Na⁺] _i , from 8 to 12mm. Na⁺/K⁺ pump activity assessed in single cells by PD excursions during transient K⁺ removal increased 2.5-fold in the presence of HCO ₃ ^– , and the rise in [Na⁺] _i produced by inhibition of the Na⁺/K⁺ pump was similarly increased 2.5-fold in the presence of HCO ₃ ^– . In intact perfused rat liver, HCO ₃ ^– increased both Na⁺/K⁺ pump activity and O₂ consumption. These findings indicate that, in hepatocytes, net coupled Na⁺ and HCO ₃ ^– movement is inward and represents a major determinant of Na⁺ influx and Na⁺/K⁺ pump activity. About half of hepatic Na⁺/K⁺ pump activity appears dedicated to recycling Na⁺ entering in conjunction with HCO ₃ ^– to maintain [Na⁺] _i within the physiologic range.     

Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

Summary The substrate stoichiometry of the intestinal Na⁺/phosphate cotransporter was examined using two measures of Na⁺-dependent phosphate uptake: initial rates of uptake with [³²P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC₃(5). Isotopic phosphate measures electrogenic and electroneutral Na⁺-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na⁺/phosphate cotransport was found to have similar Na⁺ activations (apparentK _0.5's of 28 and 25mm), apparentK _m 's for phosphate (100 and 410 m), andK _0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na⁺-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na⁺ with apparentK _0.5 for phosphate of 200 m; similar to the apparentK _m for phosphate uptake, andK _0.5 for phosphate protection against FITC-PG inhibition of Na⁺-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na⁺/phosphate cotransporter is electrogenic at pH 7.4, that H₂PO ₄ ^– is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na⁺/phosphate cotransporter.     

Potassium-proton symport inNeurospora: kinetic control by pH and membrane potential

Summary Active transport of potassium in K⁺-starvedNeurospora was previously shown to resemble closely potassium uptake in yeast,Chlorella, and higher plants, for which K⁺ pumps or K⁺/H⁺-ATPases had been proposed. ForNeurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H⁺ to 1 K⁺ taken inward so that K⁺, but not H⁺, moves against its electrochemical gradient (Rodriguez-Navarro et al.,J. Gen. Physiol. 87:649–674).In the present experiments, the current-voltage (I–V) characteristic of K⁺–H⁺ cotransport in spherical cells ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 M K⁺ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 S/cm²) for voltages of –300 to –100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–V curve of the K⁺-H⁺ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of theI–V difference method under reversing conditions.In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora also promptly inhibit K⁺ transport and K⁺-H⁺ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K⁺-H⁺ currents. Thus, the inhibition of K⁺ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action on the cotransport system itself.Detailed study of the effects of [K⁺]_o and pH_o on theI–V curve for K⁺-H⁺ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K⁺, butincreases affinity for protons (K _m range: for [K⁺]_o, 15–45 M; for [H⁺]_o, 10–35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K⁺ first and H⁺ last in the forward direction of transport, or (ii) a negatively charged carrier (–2) binds H⁺ first and K⁺ last.     

Effects of pH,potential, chloride and furosemide on passive Na+ and K+ effluxes from human red blood cells

Summary Ouabain-resistant effluxes from pretreated cells containing K⁺/Na⁺=1.5 into K⁺ and Na⁺ free media were measured.Furosemide-sensitive cation effluxes from cells with nearly normal membrane potential and pH were lower in NO ₃ ^– media than in Cl^– media; they were reduced when pH was lowered in Cl^– media. When the membrane potential was positive inside furosemide increased the effluxes of Na⁺ and K⁺ (7 experiments). With inside-positive membrane potential thefurosemideinsensitive effluxes were markedly increased, they decreased with decreasing pH at constant internal Cl^– and also when internal Cl^– was reduced at constant pH. The correlation between cation flux and the membrane potential was different for cells with high or low internal chloride concentrations. The data with chloride47mm showed a better fit with the single-barrier model than with the infinite number-of-barriers model. With low chloride no significant correlation between flux and membrane potential was found. The data are not compatible with pure independent diffusion of Na⁺ and K⁺ in the presence of ouabain and furosemide.     

Reconstitution of cAMP-Dependent protein kinase regulated renal Na+−H+ exchanger

Summary Studies were performed to determine if the Na⁺–H⁺ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited²²Na⁺ uptake in the absence of a pH gradient (pH_in 6.0. pH_out 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity.These studies indicate that Na⁺–H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na⁺–H⁺ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.