4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 5-dimethylamiloride

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. We reported that pHi in primary cultured vascular smooth muscle cells from canine femoral artery is 7.26, a value maintained via HCO3- influx by the Na(+)-dependent C1(-)-HCO3-exchanger but not via H+ efflux by the Na(+)-H+ exchanger [A. M. Kahn, E. J. Cragoe, Jr., J. C. Allen, R. D. Halligan, and H. Shelat. Am. J. Physiol 259 (Cell Physiol. 28): C134-C143, 1990]. To explain these findings, in the present study, we determined the pHi activity profile of these two transport systems. Although both were active at acidic pHi, Na(+)-H+ exchange activity was very low at and above pHi 7.0, while Na(+)-dependent C1(-)-HCO3-exchange activity maintained near-maximal activity up to pHi 7.26 but fell to undetectable levels by pHi 7.4. A Na(+)-independent C1(-)-HCO3-exchanger was present, which mediated HCO3-efflux after an acute alkaline load. The activity of this system was negligible at pHi 7.2 and was stimulated at alkaline pHi. In conclusion, the pHi of these vascular smooth muscle cells at rest is maintained via HCO3-influx by the Na(+)-dependent C1(-)-HCO3-exchanger. After acute acidic or alkaline loads, correction of pHi is mediated by activation of the normally quiescent Na(+)-H+ and Na(+)-independent C1(-)-HCO3-exchangers, respectively. All three acid-base transport systems have pHi set points that protect the cell from overcorrecting pHi after a disturbance in either direction.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Carrier Proteins; Cells, Cultured; Chloride-Bicarbonate Antiporters; Dogs; Female; Fluoresceins; Hydrogen-Ion Concentration; Male; Muscle, Smooth, Vascular; Sodium; Sodium-Hydrogen Exchangers

In blood vessels in the systemic circulation, the plasmalemmal Na+/H+ exchanger has been implicated in a variety of cellular functions, including the regulation of intracellular pH (pHi) and cell volume, and the response to smooth muscle mitogens. The role of this transport system in pulmonary vascular smooth muscle has not been explored. The present study examined the characteristics of Na+/H+ exchange in cultured guinea pig pulmonary artery smooth muscle cells. These cells were subjected to an acid load, and the recovery from acid loading was monitored using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In the absence of HCO3-, pHi recovery from acid loading was dependent on external Na+ and was inhibited by the Na+/H+ exchange inhibitor dimethylamiloride (DMA) (recovery rate was reduced from 54.4 +/- 5.5 to 12.8 +/- 2.0 mmol H+/liter.min). This exchanger was also active in the presence of HCO3-; DMA reduced resting pHi and slowed the rate of recovery from acid loading in HCO3- buffers. However, in the presence of HCO3-, other transport systems, presumably HCO3-/Cl- exchange, also contribute to the regulation of pHi. In HCO3- buffers, the rate of recovery from acid load averaged 40.8 +/- 1.8 mmol H+/liter.min. Addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of HCO3-/Cl- exchange, slowed this recovery to 25.5 +/- 1.6 mmol H+/liter.min. A combination of DIDS and DMA further slowed the recovery to 19.7 +/- 1.5 mmol H+/liter.min. These findings indicate that the Na+/H+ exchanger plays a significant role in the regulation of pHi in pulmonary artery smooth muscle cells, even in HCO(3-)-containing buffers.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Bicarbonates; Carrier Proteins; Cells, Cultured; Fluoresceins; Guinea Pigs; Hydrogen; Hydrogen-Ion Concentration; Kinetics; Male; Muscle, Smooth, Vascular; Pulmonary Artery; Sodium; Sodium-Hydrogen Exchangers

We microperfused the loop of Henle (LOH) to assess its contribution to urine acidification in vivo. Under control conditions (Na HCO3- = 13 mM, perfusion rate approximately 17 nl/min-1) net bicarbonate transport (JHCO3-) was unsaturated, flow- and concentration-dependent, and increased linearly until a bicarbonate load of 1,400 pmol.min-1 was reached. Methazolamide (2 x 10(-4) M) reduced JHCO3 by 70%; the amiloride analogue ethylisopropylamiloride (EIPA) (2 x 10(-4) M) reduced JHCO3 by 40%; neither methazolamide nor EIPA affected net water flux (Jv). The H(+)-ATPase inhibitor bafilomycin A1 (10(-5) M) reduced JHCO3 by 20%; the Cl- channel inhibitor 5-nitro-2'-(3-phenylpropylamino)-benzoate (2 x 10(-4) M) and the Cl(-)-base exchange inhibitor diisothiocyanato-2,2'-stilbenedisulfonate (5 x 10(-5) M), had no effect on fractional bicarbonate reabsorption. Bumetanide (10(-6) M) stimulated bicarbonate transport (net and fractional JHCO3-) by 20%, whereas furosemide (10(-4) M) had no effect on bicarbonate reabsorption; both diuretics reduced Jv. In summary: (a) the LOH contributes significantly to urine acidification. It normally reabsorbs an amount equivalent to 15% of filtered bicarbonate; (b) bicarbonate reabsorption is not saturated; (c) Na(+)-H+ exchange and an ATP-dependent proton pump are largely responsible for the bulk of LOH bicarbonate transport.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Absorption; Acid-Base Equilibrium; Amiloride; Animals; Anti-Bacterial Agents; Bicarbonates; Biological Transport; Bumetanide; Furosemide; Loop of Henle; Macrolides; Male; Methazolamide; Nitrobenzoates; Perfusion; Permeability; Rats; Rats, Inbred Strains

We have studied the kinetic properties of rabbit red cell (RRBC) Na+/Na+ and Na+/H+ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na+ and H+ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Nai and Hi were prepared by nystatin and DIDS treatment of acid-loaded cells. Na+/Na+ EXC was measured as Nao-stimulated Na+ efflux and Na+/H+ EXC as Nao-stimulated H+ efflux and delta pHo-stimulated Na+ influx into acid-loaded cells. The activation of Na+/Na+ EXC by Nao at pHi 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (Km 2.2 mM) and low affinity (Km 108 mM) sites for a single- or two-carrier system. The activation of Na+/H+ EXC by Nao (pHi 6.6, Nai less than 1 mM) also showed high (Km 11 mM) and low (Km 248 mM) affinity sites. External H+ competitively inhibited Na+/Na+ EXC at the low affinity Nao site (KH 52 nM) while internally H+ were competitive inhibitors (pK 6.7) at low Nai and allosteric activators (pK 7.0) at high Nai. Na+/H+ EXC was also inhibited by acid pHo and allosterically activated by Hi (pK 6.4). We also established the presence of a Nai regulatory site which activates Na+/H+ and Na+/Na+ EXC modifying the affinity for Nao of both pathways. At low Nai, Na+/Na+ EXC was inhibited by acid pHi and Na+/H+ stimulated but at high Nai, Na+/Na+ EXC was stimulated and Na+/H+ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Nao sites, cis-inhibited by external Ho, allosterically modified by the binding of H+ to a Hi regulatory site and regulated by Nai. These findings are consistent with Na+/Na+ EXC being a mode of operation of the Na+/H+ exchanger. Na+/H+ EXC was partially inhibited (80-100%) by dimethyl-amiloride (DMA) but basal or pHi-stimulated Na+/Na+ EXC (pHi 6.5, Nai 80 mM) was completely insensitive indicating that Na+/Na+ EXC is an amiloride-insensitive component of Na+/H+ EXC. However, Na+ and H+ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA; this also indicates that the Na+/H+ EXC might operate in reverse or uncoupled modes in the absence of Na+/Na+ EXC.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Biological Transport; Carrier Proteins; Erythrocytes; Hydrogen-Ion Concentration; Kinetics; Male; Protons; Rabbits; Sodium; Sodium-Hydrogen Exchangers

We have estimated the changes in cytosolic pH (pHi) that occur when human platelets are stimulated by thrombin. Changes in pHi were estimated (i) from the H+ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H+ efflux that averaged 7.7 +/- 1.6 mumol/10(11) platelets (means +/- SD) leading to an increase in pHi, from 7.05 +/- 0.04 to 7.45 +/- 0.05. Both H+ efflux and pHi changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4'-acetamido 4'-isothiostilbene-2,2'-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO3-/Cl- exchange. Removal of HCO3- or Cl- from the suspending buffer had no effect on the extent of the rise in pHi. After blockade of Na+/H+ exchange by 100 microM ethylisopropylamiloride (EIPA), thrombin induced a decrease in pHi the rate of which averaged 0.39 unit/min in HCO3(-)-containing medium, and 0.57 unit/min in HCO3(-)-free medium. The cytosolic buffer capacity for H+ was determined by the nigericin/NH4Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO3-, but only 17.2 mmol/(1xpH) in HCO3(-)-free buffer. The total amount of H+ transferred by Na+/H+ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO3- and to 14 mmol/l platelet cytosol in the presence of HCO3-, and is in good agreement with the estimated amount of Na+ uptake by ADP-stimulated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Bicarbonates; Blood Platelets; Bumetanide; Cytosol; Humans; Hydrogen-Ion Concentration; Kinetics; Thrombin

During the early period after poliovirus infection of HeLa cells, cellular Na+/K+ ATPase activity is transiently activated. We investigated the possibility that Na+/K+ ATPase activation is a consequence of Na+/H+ antiporter activation. Increased uptake of the weak organic acid 5,5-dimethyloxazolidine-2,4-dione by infected cells around 2 h after infection suggested cytoplasmic alkalinization equivalent to pH 7.7 during the biosynthetic phase of viral replication. Consistent with the involvement of Na+/H+ antiporter activation in this phenomenon, it was found to be [Na+]-dependent and inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). However, the pH increase was not associated with an increase in amiloride-sensitive Na+ uptake by infected cells predicted by this mechanism. By contrast, the alkalinization could be abolished with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), implicating an anion-exchange mechanism, such as Cl-/HCO3- exchange, in this process. In addition to abolishing virus-induced intracellular alkalinization, both EIPA and DIDS moderately inhibited viral replication. Manipulation of intracellular pH with nigericin in the incubation medium revealed that maximum viral replication required a pH of about 7.7 and that replication was significantly inhibited even at pH 7.3. Thus, the pH increase in infected cells appeared to be physiologically relevant. These findings represent the first demonstration of a biologically meaningful pH increase in cells infected with a lytic virus.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Biological Transport; Carrier Proteins; Cytoplasm; Dimethadione; HeLa Cells; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Nigericin; Poliovirus; Sodium-Hydrogen Exchangers; Virus Replication

Growth factors raise intracellular pH (pHi) by stimulating Na+/H+ exchange in the absence of HCO3-. In mutant cells that lack the Na+/H+ exchange activity, this alkalinization does not occur, and the cells do not proliferate without artificial elevation of pHi. It has therefore been widely suggested that an early pHi increase is a necessary signal for mitogenesis. In the presence of HCO3- however, growth factors fail to raise pHi in A431 cells, renal mesangial cells and 3T3 fibroblasts. In mesangial cells, arginine vasopressin (AVP) raises pHi in the absence of HCO3-, but lowers it when HCO3- is present; growth is stimulated under both conditions. We report here that, in the presence of HCO3-, AVP stimulates two potent HCO3- transporters, as well as the Na+/H+ exchanger. These are the Na+-dependent and Na+-independent Cl-/HCO3- exchangers. Our results indicate that AVP causes acidification in the presence of HCO3- because, at the resting pHi, it stimulates Na+-independent Cl-/HCO3- exchange (which lowers pHi) more than it stimulates the sum of Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange (both of which raise pHi). The stimulation of three acid-base transporters by the growth factor AVP greatly enhances the ability of the cell to regulate pHi.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonia; Animals; Arginine Vasopressin; Bicarbonates; Biological Transport; Cells, Cultured; Glomerular Mesangium; Hydrogen-Ion Concentration; Kinetics; Male; Rats

The biochemical and Ca2+ transport pathways involved in generating the hormone-evoked Ca2+ signal are reported to be influenced by pH. The present study was designed to determine the effect of extracellular pH (pHo) and intracellular pH (pHi) on hormone-stimulated Ca2+ transport. We used rat pancreatic acini and measured free cytosolic Ca2+ concentration ([Ca2+]i) with fura-2, pHi with 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and Ca2+ fluxes with 45Ca2+. In the presence of external Ca2+, increasing pHo increased steady-state [Ca2+]i during sustained agonist stimulation; in the absence of external Ca2+, this increase in [Ca2+]i did not occur. The addition of an antagonist or blocking plasma membrane Ca2+ influx with La3+ in stimulated cells suspended at pHo 8.2 resulted in a reduction in [Ca2+]i. Increasing pHo increased the rate and extent of 45Ca2+ uptake into stimulated cells and the rate and extent of Ca2+ reloading of intracellular stores. The increased Ca2+ content of the intracellular stores with increased pHo indicated that at physiological pHo and pHi the agonist-mobilizable internal stores are not saturated with Ca2+. Changes in pHo affected pHi. However, changes in pHi at constant pHo had no effect on hormone-evoked [Ca2+]i increase, reduction in [Ca2+]i after hormone stimulation, or reloading of intracellular stores. We conclude that the hormone-activated plasma membrane Ca2+ entry pathway responsible for Ca2+ reloading is directly modulated by external H+.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetates; Acetic Acid; Amiloride; Ammonium Chloride; Animals; Atropine; Calcium; Calcium Radioisotopes; Carbachol; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Lanthanum; Pancreas; Rats; Rats, Inbred Strains; Sincalide

The mechanism of chloride uptake at the basal membrane (stromal side) of rabbit corneal epithelium was examined by observing the effects of ion transport inhibitors and ion concentrations on the stimulated epithelial short-circuit current (Isc). Loop diuretics inhibited the theophylline-stimulated peak and sustained Isc. Treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.2 mM) and/or 5-(N,N-dimethyl)amiloride (0.1 mM) as well as the potent anion exchange inhibitor, 5c(+)[(2,3,9,9a-tetrahydro-1H-fluoren-7-yl)oxy]acetic acid (0.01 mM), had no significant effect on Isc. These results are consistent with Cl- uptake by a Na+-Cl- or Na+-K+-2Cl- cotransport mechanism rather than Cl(-)-HCO3(-)-OH- exchange coupled to Na+-H+ exchange. Incubation in low [Na+] or [Cl-] before stimulation with forskolin (0.1 mM) reduced both peak and sustained Isc, and saturation kinetics were exhibited. Hill coefficients for [Na+] and [Cl-] were 0.99 and 1.04, respectively, for peak Isc and 0.66 and 1.18, respectively, for sustained Isc. Apparent ion affinities for Na+ and Cl- were 13.5 and 18 mM, respectively, for peak Isc and 15 and 22 mM, respectively, for sustained Isc. These results favor Cl- uptake by a 1 Na+:1 Cl- cotransport mechanism for the rabbit corneal epithelium, but involvement of K+ in this process has not been eliminated.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Barium; Barium Compounds; Bicarbonates; Biological Transport, Active; Cell Membrane; Chlorides; Colforsin; Cornea; Epithelium; Furosemide; In Vitro Techniques; Kinetics; Potassium; Rabbits; Sodium; Theophylline

1. The pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF) was used to measure intracellular pH (pHi) in segments of rat resistance vessels (internal diameter about 200 microns) with the vessels mounted in a myograph for simultaneous measurements of isometric contraction. 2. BCECF loaded slowly into the vessels over 1 h and did not affect the maximal contractility of the vessels. There was a loss of dye with time which, however, was very slow when the segments were only excited for 2 s/min, suggesting that the loss was mainly due to dye bleaching with only a very slow leak. 3. The ratio of the emissions (at 540 nm) with excitation at 495 and 450 nm was calibrated in terms of pH using the K+-H+ ionophore nigericin. This calibration gave a pHi value of 7.15 +/- 0.02 (n = 20), suggesting that hydrogen ions are not in electrochemical equilibrium in these vascular smooth muscles which have a membrane potential of about -60 mV. 4. Addition of 10 mM-NH4Cl caused a transient alkalinization and wash-out of 10 mM-NH4Cl a transient acidification. Increasing CO2 with maintained bicarbonate caused a rapid acidification followed by an incomplete recovery. Removal of CO2 and bicarbonate (HEPES-buffered solution) with constant extracellular pH caused a transient alkalinization but steady-state pHi was not significantly altered. 5. In bicarbonate-free buffer the Na+-H+ exchange blocker 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and sodium-free conditions caused a slow acidification. In bicarbonate buffer (PSS) EIPA had no detectable effect after 10 min but the anion exchange blocker diisothio-cyanatostilbenedisulphonic acid (DIDS) caused a small acidification over that time course. 6. The rate of recovery after an acid load was about 50% lower in HEPES buffer compared to PSS and it was inhibited by EIPA. In PSS amiloride and EIPA each had a small inhibitory effect on the pH recovery after an acid load. DIDS also inhibited the recovery from an acid load in PSS and this effect was additive to that of EIPA. DIDS and EIPA also had additive inhibitory effects on the 22Na+ influx stimulated by the acid loading, while in HEPES buffer DIDS had no effect on either pH recovery or 22Na+ influx. These results suggest that a Na+-H+ exchange and an influx of bicarbonate coupled to sodium influx are of importance for pHi control in these vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonium Chloride; Animals; Fluoresceins; Hydrogen-Ion Concentration; In Vitro Techniques; Isometric Contraction; Mesenteric Arteries; Muscle, Smooth, Vascular; Nigericin; Norepinephrine; Rats; Vascular Resistance; Vasoconstriction

Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in the cytosol. The ability of this cell type to regulate cytosolic pH (pHi) was examined as a function of stimulation of acid secretion by histamine or forskolin. The pHi was estimated from the change of fluorescence of the trapped dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-bis-carboxyethylcarbo xy fluorescein in a purified cell suspension of rabbit parietal cells. Stimulation of the cell suspension raised pHi by an average of 0.13 +/- 0.038 pH units. The H+,K+-ATPase inhibitor, SCH28080 (2-methyl-8-[phenyl-methoxy]-imidazo-(1,2)-pyridine-3-acetonitrile) had only a small effect on the increase of pHi, therefore, was largely independent of H+,K+-ATPase activity. In Na+-free medium, where Na+/H+ exchange would be absent, the rise of pHi was only 0.03 pH units. This increase was blocked by SCH28080, showing that this small increment was the result of acid secretion. In Na+-containing medium, 90% of the increase was inhibited by an inhibitor of Na+/H+ exchange, dimethyl amiloride (DMA). This compound also blocked changes in pHi due to changes in extracellular Na+. Accordingly, most of the change in pHi upon stimulation of acid secretion by histamine and forskolin is due to activation of Na+/H+ exchange in the parietal cell basal-lateral membrane. The addition of DMA to stimulated, but not resting cells, gave a rapid acidification that was blocked by inhibition of anion exchange by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), showing that anion exchange was also activated by stimulation. In single cell recording, canalicular and cytosolic pH were monitored simultaneously using 9-amino acridine and dimethyl carboxyfluorescein, respectively. Cytosolic alkalinization correlated with acid accumulation in the secretory canaliculus until a set point was reached. Thereafter, acidification continued without further change in pHi. To determine the role of Na+/H+ and Cl-/HCO3- exchange in acid secretion, Cl(-)-depleted cells were suspended in medium containing 40 mM Cl-. DMA and DIDS each blocked acid secretion by about 40%, but in combination, acid secretion was blocked by more than 90%. Thus, basal-lateral Na+/H+ and Cl-/HCO3- exchange activities are necessary for acid secretion across the apical membrane of the parietal cell.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Aminacrine; Animals; Anti-Ulcer Agents; Bicarbonates; Cell Membrane; Chlorides; Cimetidine; Cytosol; Gastric Acid; Histamine; Hydrogen-Ion Concentration; Imidazoles; In Vitro Techniques; Kinetics; Parietal Cells, Gastric; Rabbits; Sodium

The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 microM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pH0 = 8.0, in the presence and absence of 60 microM 5,N,N-dimethyl-amiloride (DMA). The H+ efflux rate in a K+-containing medium was 116.38 +/- 4.5 mmol/l cell X hr. Substitution of Nao+ for Ko+ strongly stimulated H+ efflux to 177.89 +/- 7.9 mmol/l cell X hr. The transtimulation of H+ efflux by Nao+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 X 10(-7) M. The sequence of substrate selectivities for the external transport site were Na greater than greater than greater than Li greater than choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3-. The DMA sensitive H+ efflux was a saturable function of [Na+]o, with an apparent Km and Vmax of about 14.75 +/- 1.99 mM and 85.37 +/- 7.68 mmol/l cell X hr, respectively. However, the Nao+-dependent and DMA-sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 +/- 3.0 mmol/l cell X hr) which was abolished when pHo was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetazolamide; Amiloride; Animals; Bumetanide; Carrier Proteins; Cesium; Choline; Drug Resistance; Erythrocytes; Hydrogen-Ion Concentration; Lithium; Male; Ouabain; Phloretin; Potassium; Rabbits; Sodium; Sodium-Hydrogen Exchangers