Dependence Of Na Research Articles

Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective.

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1.

The emergence of a basolateral 1-deoxymannojirimycin-sensitive mannose carrier is a function of intestinal epithelial cell differentiation. Evidence for a new inhibitory effect of 1-deoxymannojirimycin on facilitative mannose transport.

We have previously reported that 1-deoxymannojirimycin (dMM), a specific alpha-mannosidase I inhibitor interfered with the uptake of D-[2-3H]mannose in differentiated HT-29 cells (a cell line derived from a human colon adenocarcinoma) (Ogier-Denis, E., Trugnan, G., Sapin, C., Aubery, M., and Codogno, P. (1990) J. Biol. Chem. 265, 5366-5369). In the present work, we have used another cell line derived from a human colon adenocarcinoma, Caco-2 cells, which has the capacity to grow and to differentiate on porous filters. We have determined that mannose could enter the cells by two distinct transporters. One sensitive to dMM, present at the basolateral membrane of differentiated Caco-2 cells, and one insensitive to the drug localized at the brush border membrane of these cells. The basolateral mannose uptake is mediated by a Na(+)-independent transporter whereas the apical entry of mannose is under the dependence of Na+. We have focused our studies on the basolateral dMM-sensitive mannose carrier. Kinetic studies indicated that this facilitative mannose transporter has a Km and a Vmax of 55 +/- 8 microM and 0.144 +/- 0.005 mumol/mg of protein/min, respectively. This basolateral transporter is clearly distinct from facilitative glucose transporters. Moreover, this dMM-sensitive mannose transport accurately follows the differentiation process of intestinal epithelial cells as well in vitro as shown using Caco-2 cells as in vivo when experiments were done on crypt cells and villus cells isolated from rat jejunum.

Ion transport across leech integument. I. Electrogenic Na+ transport and current fluctuation analysis of the apical Na+ channel.

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5 +/- 0.5 k omega.cm-2) with an initial short-circuit current of 29.0 +/- 2.9 microA.cm-2. Removal of Na+ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50 mumol.l-1) added to the basolateral solution, depressed the short-circuit current completely. The Na+ current saturated at a concentration of 90 mmol Na+.l-1 in the apical solution (KM = 11.2 +/- 1.8 mmol.l-1). Amiloride (100 mumol.l-1) on the apical side inhibited ca. 40% of the Na+ current and indicated the presence of Na+ channels. The dependence of Na+ current on the amiloride concentration followed Michaelis-Menten kinetics (Ki = 2.9 +/- 0.4 mumol.l-1). The amiloride analogue benzamil had a higher affinity to the Na+ channel (Ki = 0.7 +/- 0.2 mumol.l-1). Thus, Na+ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na+.l-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8 +/- 2.3% of total short-circuit current. Higher Na+ concentrations lead to a decrease in amiloride-blockade short-circuit current. Stimulation of the tissue with cyclic adenosine monophosphate (100 mumol.l-1) and isobutylmethylxanthine (1 mmol.l-1) nearly doubled short-circuit current and increased amiloride-sensitive Na+ currents by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

The Na(+)-dependence of Na(+)-activated K(+)-channels (IK(Na)) in guinea pig ventricular myocytes, is different in excised inside/out patches and cell attached patches.

Single Na(+)-activated K(+)-channels were recorded from intact guinea-pig ventricular myocytes perfused with Ca-free, Mg-free Tyrode, to increase [Na+]i. The activity of these channels increases with the time of perfusion with this solution and decreases when the bathing sodium is reduced. The activity of the Na(+)-activated K(+)-channel is greater in the cell attached patches than from excised cell free patches, at the same [Na+]. It is suggested that this K+ channel is significantly active in intact cells within the physiological range of [Na]i.

Monovalent cation activated ouabain-insensitive ATPase in the gills of freshwater mussel Anodonta cygnea

Abstract 1. 1. The ouabain-insensitive Na + -ATPase of gill microsomes of the freshwater mussel Anodonta cygnea is activated similarly also by K + , Li + , Tl + , Rb + , Cs + and NH 4 + . 2. 2. Na + -ATPase also hydrolyses UTP and ADP, but with lower activity. 3. 3. The Na + -ATPase activity is maximal at pH 6.5. 4. 4. Na + -ATPase activity is inhibited by furosemide, but by ethacrynic acid and vanadate only at high concentrations. 5. 5. The temperature dependence of Na + -ATPase activity of Anodonta gill microsomes is linear between 5 and 50°C without the discontinuities usually observed in the function of this enzyme from other sources. 6. 6. Cd 2+ , but not Ca 2+ , may substitute Mg 2+ as the necessary cofactor. 7. 7. No significant ouabain-sensitive (Na + + K + )-ATPase activity has been detected in Anodonta gill microsomes, but this and the relative unspecificity of Na + -ATPase do not at present allow for evolutionary conclusions.

Characterization of Na(+)-H+ exchange in segments of rat mesenteric artery.

To develop a technique for measuring Na(+)-H+ exchange activity and intracellular pH (pH(i)) "on line" in resistance vessels, we utilized strips of rat mesenteric arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Strips were held at a fixed length within a 3-ml cuvette, and fluorescence emission was monitored at 530 nm. The spectrofluorimeter was monitored in the ratio mode, and the excitation wavelength was alternated between 440 and 505 nm. Tissues were maintained by perfusing with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid containing buffers. The introduction of ammonium chloride produced a rapid alkalinization. Washout of ammonium caused rapid acidification. Restoration of pH(i) was Na+ dependent and inhibited by dimethyl amiloride (concentration that produces half-maximal inhibition, K0.5 = 30 microM), features characteristic of Na(+)-H+ exchange. Further studies assessed the transport rate of the exchanger, which averaged 0.19 +/- 0.02 pH U/min (means +/- SE, n = 8). An estimate of the dependence of Na(+)-H+ exchange on external Na+ gave an apparent Michaelis constant for external Na+ of 10 mM and an apparent maximal velocity of 0.1 mM H+/s. Intracellular H+ was found to have a cooperative effect (Hill coefficient = 4) on Na(+)-H+ exchange.

Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that the apparent affinity of the Na(+)-K+ pump towards K+ in cardiac Purkinje cells depends on both the membrane potential and the extracellular Na+ concentration. 11. The region of negative slope of the Ip-V curve observed in cells which were superfused with media containing low concentrations of K+ or its congeners strongly suggests the existence of at least two voltage-sensitive steps in the cardiac Na(+)-K+ pump cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiotensin II-induced activation of Na(+)-H+ exchange in adrenal glomerulosa cells is mediated by protein kinase C.

Modulation of Na(+)-H+ exchange (Na/H EXCH) by hormones and growth factors may be important in cell growth. We have previously shown that Na/H EXCH in adrenal glomerulosa cells is activated by angiotensin II (ANG II), a potent stimulator of aldosterone secretion. In the present paper, we have investigated the role of protein kinase C (PKC) in the activation of Na/H EXCH by ANG II. To accomplish this, we monitored cytosolic pH (pHi) in cells loaded with 2,7-biscarboxyethyl-5 (6)-carboxyflourescein and measured initial rates off 22Na uptake into glomerulosa cells in the presence or absence of dimethylamiloride. Both phorbol 12-myristate 13-acetate (PMA) and ANG II increased the activity of Na/H EXCH similarly when studied under basal conditions (pH 7.1). This was accompanied by alkalinization of pH and an increase in dimethylamiloride-sensitive Na+ influx. Study of kinetics of the antiporter activation showed that both ANG II and PMA led to an increase in the maximal rate (Vmax) and a decrease in the Michaelis-Menten constant (Km) for external Na+. The pHi dependence of Na+ influx was half-minimal (pK) at pHi 7.09 and remained unchanged in the presence of ANG II (pK 7.03) and PMA (pK 7.14). Depleting the cells of PKC by exposing them to PMA (1 microM) for 3 h caused a marked reduction in control and ANG II-stimulated Na+ influx and control pK (7.09 to 6.85, P less than 0.05). However, with PKC depletion, the kinetics of Na/H EXCH (Vmax, Km for external Na+, and pK) were unaffected by ANG II. Thus, Na/H EXCH in adrenal glomerulosa cells functions under basal conditions, and its relatively alkaline pK (7.09) is dependent upon PKC activity. In addition, the ANG II-induced activation of Na/H EXCH is modulated by PKC. These effects suggest an important role for PKC in pHi regulation of adrenal glomerulosa cells, particularly during ANG II stimulation of aldosterone secretion.

Kinetic properties and Na+ dependence of rheogenic Na(+)‐HCO3‐ co‐transport in frog retinal pigment epithelium.

1. Na(+)-HCO3- co-transport across the retinal membrane of the frog retinal pigment epithelium was studied by means of double-barrelled pH-selective microelectrodes. Transient changes in the intracellular pH were monitored in response to abrupt changes in the Na+ concentration on the retinal side of the epithelium. 2. The experiments were performed as follows. The Na(+)-HCO3- co-transport was inhibited by perfusing the retinal side of the epithelium with a Na(+)-free solution. The co-transport was then stimulated by changing the perfusate from the Na(+)-free solution to a solution which contained from 5 to 110 mM-Na+. The resulting inward Na(+)-HCO3- co-transport produced an intracellular alkalinization, the initial rate of which was used to calculate the initial rate of Na(+)-HCO3- co-transport, JHCO3-. 3. The Na+ dependence of the Na(+)-HCO3- co-transport was studied at two different values of extracellular pH (7.40 and 7.10), at constant extracellular HCO3- concentration (27.5 mM) and at two different extracellular HCO3- concentrations (27.5 mM and 55 mM) at constant extracellular pH (7.40). In these experiments, the calculated values of JHCO3- followed single Michaelis-Menten kinetics with respect to the extracellular Na+ concentration. 4. The data are consistent with a model in which the co-transporter has a single binding site for the Na+ ion with an apparent affinity constant (apparent Km) of 37 mM. The apparent affinity constant for Na+ was independent of the extracellular concentration of CO3(2-) in the range of 16-65 microM, and of the extracellular HCO3- concentration in the range 27.5-55 mM. 5. The NaCO3- ion-pair hypothesis, in which sodium binds to the co-transporter and is translocated across the cell membrane as the NaCO3- ion pair, was analysed. For stoichiometries 1:2 and 1:3 of the Na(+)-HCO3- co-transport, the NaCO3- ion-pair hypothesis was found incompatible with the data. 6. The intracellular buffer capacity as measured by the CO2 method was 15 mM.

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.

Essential activation of Na(+)-H+ exchange by [H+]i in HL-60 cells

The intracellular pH (pHi) dependence of the rate of Na(+)-H+ exchange was determined in undifferentiated promyelocytic HL-60 cells by measuring alkalinization rates using the fluorescent pHi indicator 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). BCECF was calibrated in the pH range from 5 to 7 using the nigericin technique of Thomas and co-workers (J. A. Thomas, R. N. Buchsbaum, A. Zimniak, and E. Racker. Biochemistry 18: 2210-2218, 1979). Exchange rate increases as pHi is lowered below pH 7.00. At low pH (pH below 6.3), the dependence of Na(+)-H+ exchange rate on intracellular proton activity is well fitted by the Michaelis-Menten equation with a maximum exchange velocity of 33.7 +/- 2.4 mmol H(+).1 cell water-1.min-1 and a half-saturation constant of 1.35 +/- 0.28 microM (corresponding to a minus log of the Michaelis constant of 5.89). However, a Hill plot reveals that the Hill coefficient changes gradually from one to two when pH is changed from 5 to 7, ruling out Michaelian kinetics. The dependence of exchange flux on internal protons is well fit in the full pH range from 5 to 7 by a simple kinetic model (essential activation) with modifier and transport sites for internal proton binding. At low pH, failure to correct BCECF measurement of pHi for contribution to fluorescence signal from extracellular dye and for quenching of intracellular BCECF leads to an artifactual increase in the measured Hill coefficient. These two findings (increase in Hill coefficient as pHi is increased and artifactual increase in Hill coefficient because of methodological reasons) provide a good explanation for the wide range of Hill coefficients reported in the literature.

Sulfate inhibits [14C]phosphonoformic acid binding to renal brush-border membranes

To examine the specificity of the phosphonoformic acid (PFA) interaction with the Na(+)-dependent phosphate transporter of mouse renal brush-border membrane vesicles, we compared the effects of anions on Na(+)-dependent [14C]PFA binding and Na(+)-dependent phosphate transport. Inhibition of PFA binding was achieved by PFA (% control = 0 +/- 1), sulfate (15 +/- 2), arsenate (35 +/- 1), phosphate (59 +/- 2), and nitrate (68 +/- 4), whereas inhibition of phosphate transport was only apparent with phosphate (0 +/- 1), PFA (22 +/- 4), and arsenate (37 +/- 5). Succinate and gluconate had no effect on either Na(+)-dependent process. Under conditions where Na(+)-dependent PFA binding was maximally inhibited by phosphate (% control = 65 +/- 4), further inhibition could be achieved by sulfate (26 +/- 5%). Na(+)-dependent PFA binding was competitively inhibited by phosphate (apparent Ki = 8.9 +/- 1.2 mM) and noncompetitively inhibited by sulfate (apparent Ki = 2.6 +/- 0.5 mM). We found that PFA inhibited Na(+)-dependent sulfate transport (50% inhibition at 9 mM PFA) as well as Na(+)-dependent phosphate transport (50% inhibition at 0.5 mM PFA). We also examined the pH dependence of Na(+)-dependent PFA binding and Na(+)-dependent phosphate and sulfate transport. PFA binding was optimal at pH = 7.4, whereas phosphate transport increased with increasing pH, and sulfate transport increased with decreasing pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Apical membrane Na+/H+ exchange in Necturus gallbladder epithelium. Its dependence on extracellular and intracellular pH and on external Na+ concentration.

Intracellular microelectrode techniques and extracellular pH measurements were used to study the dependence of apical Na+/H+ exchange on mucosal and intracellular pH and on mucosal solution Na+ concentration ([Na+]o). When mucosal solution pH (pHo) was decreased in gallbladders bathed in Na(+)-containing solutions, aNai fell. The effect of pHo is consistent with titration of a single site with an apparent pK of 6.29. In Na(+)-depleted tissues, increasing [Na+]o from 0 to values ranging from 2.5 to 110 mM increased aNai; the relationship was well described by Michaelis-Menten kinetics. The apparent Km was 15 mM at pHo 7.5 and increased to 134 mM at pHo 6.5, without change in Vmax. In Na(+)-depleted gallbladders, elevating [Na+]o from 0 to 25 mM increased aNai and pHi and caused acidification of a poorly buffered mucosal solution upon stopping the superfusion; lowering pHo inhibited both apical Na+ entry and mucosal solution acidification. Both effects can be ascribed to titration of a single site; the apparent pK's were 7.2 and 7.4, respectively. Diethylpyrocarbonate (DEPC), a histidine-specific reagent, reduced mucosal acidification by 58 +/- 4 or 39 +/- 6% when exposure to the drug was at pHo 7.5 or 6.5, respectively. Amiloride (1 mM) did not protect against the DEPC inhibition, but reduced both apical Na+ entry and mucosal acidification by 63 +/- 5 and 65 +/- 9%, respectively. In the Na(+)-depleted tissues mean pHi was 6.7. Cells were alkalinized by exposure to mucosal solutions containing high concentrations of nicotine or methylamine. Estimates of apical Na+ entry at varying pHi, upon increasing [Na+]o from 0 to 25 mM, indicate that Na+/H+ exchange is active at pHi 7.4. Intracellular H+ stimulated apical Na+ entry by titration of more than one site (apparent pK 7.1, Hill coefficient 1.7). The results suggest that external Na+ and H+ interact with one site of the Na+/H+ exchanger and that cytoplasmic H+ acts on at least two sites. The external titratable group seems to be an imidazolium, which is apparently different from the amiloride-binding site. The dependence of Na+ entry on pHi supports the notion that the Na+/H+ exchanger is operational under normal transport conditions.

Ca2+ dependency of Na+ transport by rabbit renal brush border membrane

Intracellular Ca2+ has been suggested to play an important role in the regulation of epithelial Na+ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca2+-free medium enhanced Na+ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na+ entry across the apical membrane by intracellular Ca2+. In the present study, we have examined the effect of Ca2+-free preincubation on apical membrane Na+ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1 mM Ca2+ medium exhibited higher Na+ uptake than those derived from proximal tubules incubated in Ca2+-free EGTA medium. Such effect of Ca2+ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation. Ba2+ (1 mM). Ca2+ in the preincubation medium did not affect Na+-dependent BBM glucose uptake, and its effect on BBM Na+ uptake was pH gradient dependent and amiloride (10(-3) M) sensitive, suggesting the involvement of Na+/H+ antiport system. Addition of verapamil (10(-4) M) to 1 mM Ca2+ preincubation medium abolished while ionomycin (10(-6) M) potentiated the effect of Ca2+ in the preincubation medium is likely to be mediated by Ca2+-dependent cellular pathways and not due to a direct effect of extracellular Ca2+ on BBM.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

We have investigated the kinetic properties of the human red blood cell Na+/H+ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H+ (Hi) and external Na+ (Nao) in red blood cells of normal subjects. Red blood cells with different cell Na+ (Nai) and pH (pHi) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na+ influx were measured by varying pHi (from 5.7 to 7.4), external pH (pHo), Nai and Nao and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na+ influx (Nai less than 2.0 mmol/liter cell, Nao = 150 mM) increased sigmoidally (Hill coefficient 2.5) when pHi fell below 7.0 and the external pHo was 8.0, but increased linearly at pHo 6.0. The net Na+ influx driven by an outward H+ gradient was estimated from the difference of Na+ influx at the two pHo levels (pHo 8 and pHo 6). The H+-driven Na+ influx reached saturation between pHi 5.9 and 6.1. The Vmax had a wide interindividual variation (6 to 63 mmol/liter cell.hr, 31.0 +/- 3, mean +/- SEM, n = 20). The Km for Hi to activate H+-driven Na+ influx was 347 +/- 30 nM (n = 7). Amiloride (1 mM) or DMA (20 microM) partially (59 +/- 10%) inhibited red cell Na+/H+ exchange. The stoichiometric ratio between H+-driven Na+ influx and Na+-driven H+ efflux was 1:1. The dependence of Na+ influx from Nao was studied at pHi 6.0, and Nai lower than 2 mmol/liter cell at pHo 6.0 and 8.0. The mean Km for Nao of the H+-gradient-driven Na+ influx was 55 +/- 7 mM. An increase in Nai from 2 to 20 mmol/liter cell did not change significantly H+-driven net Na+ influx as estimated from the difference between unidirectional 22Na influx and efflux. Na+/Na+ exchange was negligible in acid-loaded, DIDS-treated cells. Na+ and H+ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na+ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na+/H+ exchanger. It is concluded that human red cell Na+/H+ exchange performs 1:1 exchange of external Na+ for internal protons, which is partially amiloride sensitive.(ABSTRACT TRUNCATED AT 400 WORDS)

Dependence of Na+/H+ antiport activation in cultured rat aortic smooth muscle on calmodulin, calcium, and ATP. Evidence for the involvement of calmodulin-dependent kinases.

The role of Ca2+/calmodulin-dependent processes in the activation of the Na+/H+ antiport of primary cultures of rat aortic smooth muscle was studied using 22Na+ uptake and measurement of intracellular pH (pHi) with the fluorescent pH dye 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein. Antiport activation following exposure to serum and by the induction of an intracellular acidosis could be markedly attenuated by calmodulin antagonists. Ionomycin also transiently elevated pHi and 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ influx, effects consistent with activation of the antiport; these effects were abolished in cells exposed to calmodulin antagonists or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. Activation of the antiport following intracellular acidosis was markedly affected by cellular ATP depletion. A comparison of the abilities of control and 2-deoxy-D-glucose-treated cells to increase 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ influx in response to graded acidifications indicated that attenuation of Na+/H+ antiport activity was due to both a shift of its pHi dependence and to a reduction in maximal activity. The results suggest that the Na+/H+ antiport of rat aortic smooth muscle is dependent on Ca2+/calmodulin-dependent processes, presumably phosphorylation, which influences its activity by modulating (i) an intracellular proton dependent regulatory mechanism (allosteric site) and (ii) the maximum activity of the antiport.

LLC-PK1 mutant with increased Na+-H+ exchange and decreased sensitivity to amiloride.

LLC-PK1 cells contain a well-characterized Na+-H+ antiporter that is sensitive to ethylisopropylamiloride (EIPA) in the submicromolar range. Using a modification of the method of Franchi et al. (J. Biol. Chem. 261: 14614-14620, 1986), we have selected mutants that can recover from an acid load in the presence of 100 microM EIPA. One such mutant, designated PKE20, has been studied in detail. The maximal velocity (Vmax) for the Na+-H+ antiporter, assayed as EIPA-sensitive 22Na+ uptake, has increased from 44 nmol.min-1.10(6) cells-1 in the parent cells to 106 nmol.min-1.10(6) cells-1 in PKE20. No detectable change has occurred in the Km for Na+ (118 mM in the parent, 111 mM in the mutant) or in the dependence of Na+ uptake on intracellular pH. However, the PKE20 antiporter exhibits a greatly decreased sensitivity to amiloride and its derivatives, with drops in inhibitory potency ranging from 25-fold (amiloride) to 100-fold (EIPA). The mutation is specific for the antiporter; measurements of Na+-K+ pump and Na+-dependent amino acid uptake show only small changes, which appear to result from minor antiporter-induced alterations in internal Na+ concentration. PKE20 cells should prove useful in experiments to identify and isolate the antiporter protein.

Magnesium ion activity in the mammalian endolymph measured with ion-selective microelectrodes.

The free Mg++ concentration in endolymph was measured with Mg++-selective microelectrodes based on the neutral ligand ETH 1117. The property of Mg++ microelectrodes was obtained from calibration solutions, containing various Mg++ concentrations with the background electrolytes resembling endolymph. The range between 10 and 0.1 mM Mg++ concentrations changed the potentials of Mg++ microelectrodes by 14.4 +/- 3.0 mV. The endocochlear potential and the Mg++ concentration in the endolymph were 82.0 +/- 5.0 mV and 0.77 +/- 0.29 mM in the guinea pig, and 84.4 +/- 4.9 mV and 1.12 +/- 0.24 mM in the chinchilla, respectively. These results are discussed in the light of the dependence of Na+, K+-ATPase and its interaction with Ca++.

Physiological Correlates of Interspecific Variation in Acid Tolerance in Fish

ABSTRACT This study investigated ion regulation in relation to water pH in three species of fish of differing tolerance to low pH (common shiners, Notropis cornutus, most sensitive; rainbow trout, Salmo gairdneri, intermediate; yellow perch, Perea flavescens, least sensitive). Increasing sensitivity to exposure to low pH was characterized by shorter survival times, greater losses of whole-body ions, more complete inhibition of Na+ uptake, and greater stimulation of Na+ efflux, the latter being the most important factor in determining survival. Interspecific variations in acid tolerance were also correlated with Na+ transport characteristics at circumneutral pH; Km was directly correlated Vmax and inversely correlated with acid tolerance. In addition, there were large qualitative differences among the species in the Ca2+-dependence of Na+ efflux. Sodium efflux induced by low pH was markedly Ca2+-dependent in both trout and shiners in a manner consistent with a simple competition between Ca2+ and H+ for gill binding sites. The increased sensitivity of shiners relative to trout was related to lowered Cam binding activity. In contrast, Na+ efflux in perch was virtually unaffected by water [Ca2+], Similarly, La3+ (a Ca2+ antagonist) stimulated higher Na+ losses from shiners than from trout, but had little effect upon perch. Ionic losses produced by saturating La3+ concentrations were generally lower than those produced by H+, suggesting that Ca2+ displacement is not the only mechanism for increased gill permeability at low pH. Nonetheless, the results obtained are consistent with the notion that acid tolerance may be related to Ca2+-binding activity in some species (e.g. trout and shiners) although not in others (e.g. perch).

The secretory mechanism in adrenal chromaffin cells by nitrophenol compounds: possible involvement of the change in the surface potentials.

The rabbit adrenal gland was perfused with a modified Locke's medium and the mechanism of adrenaline secretion induced by nitrophenol compound was investigated. The secretory response to 2,4-dinitrophenol (DNP) or trinitrophenol (TNP) showed the following peculiar features. (1) Prolonged exposure to DNP or TNP caused an immediate, and long-lasting increase of secretion only in the presence of Ca. (2) The response to DNP depended on the concentration of Ca, but that to TNP was largest in the presence of 0.5 mM Ca. (3) Re-addition of 2 mM Ca during prolonged exposure to DNP or TNP produced a larger response than did the simultaneous addition of Ca and either DNP or TNP. (4) The response to DNP in the presence of 2 mM Ca was markedly reduced by removal of external Na, but the Na dependency became less marked in the presence of 0.1 mM Ca. The response to TNP in the presence of 0.5 mM Ca was slightly reduced in the absence of Na. (5) DNP or TNP caused a much larger response during depolarization with a high K, Rb, or Cs medium than that with a 5 mM KCl medium. (6) The response to DNP was largely potentiated in the absence of most ions in the presence of only 0.1 mM Ca and that to TNP was potentiated in the presence of only 0.5 mM Ca under the same condition. (7) Re-addition of Ca 5 min after the removal DNP or TNP still caused substantial secretion. These results suggest that nitrophenol compounds stimulate secretion by two independent mechanisms: one is related to its effect on the surface potentials of the plasma membrane and the other unknown except for the possible dependence of Na and Ca.