pH sensitivity of the Na+:HCO3- cotransporter in basolateral membrane vesicles isolated from rabbit kidney cortex.

HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. We have studied the effect of pH on the activity of this cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. At constant internal pH 6.0, increasing the external pH and [HCO3-] increased the rate of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 22Na+ influx into the vesicles. To determine the role of internal pH on the activity of the Na+:HCO3- cotransport system, the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange was measured in the absence of an initial pH and [HCO3-] gradient (pH(i) = pH(o), 5% CO2). Increasing the pH from 6.8 to 7.2 increased whereas, increasing the pH from 7.4 to 8.0 decreased the rate of 22Na+ influx via this exchange. Increasing pH at constant [HCO3-] (pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 10% CO2) reduced the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange. Increasing pH at constant [CO3(2-)](pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 60% CO2) was associated with reduced 22Na+ uptake. Decreasing the pH (pH(i) = pH(o) = 6.3, 60% CO2 versus pH(i) = pH(o) = 7.2, 5% CO2) was associated with a reduced rate of HCO3(-)-dependent Na(+)-Na+ exchange. We conclude that the Na+:HCO3- cotransporter displays a significant pH sensitivity profile with the cotransporter being more functional at pH 7.0-7.4 and less active at more acid or alkaline pH. In addition, the results suggest that the pH sensitivity arises at the inner surface of the basolateral membrane.     

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium

BACKGROUND: The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. METHOD/PRINCIPAL FINDINGS: Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. CONCLUSIONS: The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.     

Electrogenic sodium-dependent bicarbonate secretion by glial cells of the leech central nervous system

The ability to move acid/base equivalents across the membrane of identified glial cells was investigated in isolated segmental ganglia of the leech Hirudo medicinalis. The intracellular pH (pHi) of the glial cells was measured with double-barreled, neutral-ligand, ion-sensitive microelectrodes during step changes of the external pH (pHo 7.4-7.0). The rate of intracellular acidification after the decrease in extracellular pH (pHo) was taken as a measure of the rate of acid/base transport across the glial membrane. Taking into account the total intracellular buffering power, the maximum rate of acid/base flux was 0.4 mM/min in CO2/HCO3-free saline, and 3.92 mM/min in the presence of 5% CO2/10 mM HCO-3, suggesting that the acid/base flux was dependent upon HCO3-. The rate of acid influx/base efflux increased both with the external HCO3- concentration and with increasing pHi (and hence HCO3-i). This suggested that the decrease in pHi was due to HCO3- efflux. The rapid decrease of pHi was accompanied by a HCO3--dependent depolarization of the glial membrane from -74 +/- 5 mV (n = 20) to -54 +/- 7 mV (n = 13). Both this depolarization and the rate of intracellular acidification were greatly reduced by the anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.3-0.5 mM), but were not affected by the removal of external Cl-. Reduction of the external Na+ concentration to one-tenth normal affected the rate of intracellular acidification only in the presence of CO2/HCO3-: the rate increased within the first 3-5 min after lowering external Na+; after longer exposures in low external Na+ the rate decreased, presumably due to depletion of intracellular Na+. Amiloride (1 mM), which inhibits the Na+-H+ exchange in these cells, had no effect on the rate of intracellular acidification. The intracellular Na activity (aNai) of the glial cells was measured to be 5.2 +/- 1.0 mM (n = 8) in CO2/HCO3-free saline; aNai increased to 7.3 +/- 2.2 mM (n = 8) after the addition of 5% CO2/24 mM HCO3-. Upon a change in pHo to 7.0 in the presence of CO2/HCO3-, aNai decreased by an average of 2 +/- 1.1 mM (n = 5); in CO2/HCO3--free saline external acidification produced a transient increase in aNai. It is concluded that, in the presence of CO2/HCO3-, the rate of intracellular acidification in glial cells is dominated by an outwardly directed, electrogenic Na+-HCO3-cotransport. Neurons, which do not possess this cotransporter, acidify at much lower rates under similar conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder

The hypothesis of Cl-/HCO3- exchange across the apical membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pHo), intracellular pH (pHi), and Cl- activity (alpha Cli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na+/H+ exchange inhibitor amiloride (10(-3) M) to the superfusate, alkalinization was observed. During prolonged (10 min) exposure to amiloride, no significant pHo change occurred. Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na+ in the superfusate (TMA+ substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of Cl- (cyclamate or sulfate substitution) caused a short-lived increase in the rate of luminal acidification, followed by a return to control values (10-30 min). Upon re-exposure to Cl-, there was a transient reduction of luminal acidification. The initial increase in acidification produced by Cl- removal was partially inhibited by SITS (0.5 mM). The pHi increased rapidly and reversibly when the Cl- concentration of the mucosal bathing solution was reduced to nominally 0 mM. The pHi changes were larger in 10 mM HCO3-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO3- is transported in exchange for Cl-. In both HEPES- and HCO3-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of alpha Cli. These results support the hypothesis of apical membrane Cl-/HCO3- exchange, which can be dissociated from Na+/H+ exchange and operates under control conditions. The coexistence at the apical membrane of Na+/H+ and Cl-/HCO3- antiports suggests that NaCl entry can occur through these transporters.     

Voltage and cosubstrate dependence of the Na-HCO3 cotransporter kinetics in renal proximal tubule cells.

The voltage dependence of the kinetics of the sodium bicarbonate cotransporter was studied in proximal tubule cells. This electrogenic cotransporter transports one Na+, three HCO3-, and two negative charges. Cells were grown to confluence on a permeable support, mounted on a Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, di-nitro-stilbene-di-sulfonate-sensitive current was shown to be sodium and bicarbonate dependent and therefore was taken as flux through the cotransporter. Voltage-current relations were measured as a function of Na+ and HCO3- concentrations between -160 and +160 mV under zero-trans and symmetrical conditions. The kinetics could be described by a Michaelis-Menten behavior with a Hill coefficient of 3 for HCO3- and 1 for Na+. The data were fitted to six-state ordered binding models without restrictions with respect to the rate-limiting step. All ordered models could quantitatively account for the observed current-voltage relationships and the transinhibition by high bicarbonate concentration. The models indicate that 1) the unloaded transporter carries a positive charge; 2) the binding of cytosolic bicarbonate to the transporter "senses" 12% of the electric field in the membrane, whereas its translocation across the membrane "senses" 88% of the field; 3) the binding of Na+ to the cotransporter is voltage independent.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K+, Na+ and ATP on the gastric (H+ + K+)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min-1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min-1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min-1. Additions of 5 mM K+ or Na+, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K+, but not on Na+. Alkaline pH increased the rate of dephosphorylation. K+ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K+ was inhibitory. Below pH 7.0 Na+ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na+ inhibited both activities. The effect of extravesicular pH on transport of H+ was investigated. At pH 6.5 the apparent Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km for ATP. Extravesicular K+ and Na+ inhibited the transport of H+. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H+-producing reactions as possible candidates as physiological regulators of (H+ + K+)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO2 and the subsequent formation of H+ and HCO3-. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H+/ATP ratio was about 1 at pH 8.0. When CO2 was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO2. The results indicate a possible co-operation in the production of acid between the H+ + K+-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Ambroxol-induced modification of ion transport in human airway Calu-3 epithelia

Ambroxol is often used as a mucolytic agent in various lung diseases. However, it is unclear how ambroxol acts on bronchial epithelial cells. To clarify the action of ambroxol, we studied the effects of ambroxol on the ion transport in human Calu-3 cells, a human submucosal serous cell line, measuring the transepithelial short-circuit current and conductance across monolayers of Calu-3 cells. Ambroxol of 100 microM diminished the terbutaline (a beta2-adrenergic agonist)-stimulated Cl-/HCO3(-)-dependent secretion without any decreases in the conductance of cystic fibrosis transmembrane conductance regulator (CFTR) channel locating on the apical membrane. On the other hand, under the basal (unstimulated) condition ambroxol increased the Cl(-)-dependent secretion with no significant change in the apical CFTR channel conductance and decreased the HCO3- secretion associated with a decrease in the apical CFTR channel conductance. Ambroxol had no major action on the epithelial Na+ channel (ENaC) or the ENaC-mediated Na+ absorption. These results indicate that in Calu-3 cells: (1) under the basal (unstimulated) condition ambroxol increases Cl- secretion by stimulating the entry step of Cl- and decreases HCO3- secretion by diminishing the activity of the CFTR channel and/or the Na+/HCO3(-)-dependent cotransporter, (2) under the adrenergic agonist-stimulated condition, ambroxol decreases Cl- secretion by acting on the Cl-/HCO3- exchanger, and (3) ambroxol has a more powerful action than the adrenergic agonist on the Cl-/HCO3- exchanger, leading fluid secretion to a moderately stimulated level from a hyper-stimulated level.     

Intracellular pH-regulating mechanism of the squid axon. Relation between the external Na+ and HCO-3 dependences

The intracellular pH-regulating mechanism of the squid axon was examined for its dependence on the concentrations of external Na+ and HCO3-, always at an external pH (pHo) of 8.0. Axons having an initial intracellular pH (pHi) of approximately 7.4 were internally dialyzed with a solution of pH 6.5 that contained 400 mM Cl- and no Na+. After pHi had fallen to approximately 6.6, dialysis was halted, thereby returning control of pHi to the axon. With external Na+ and HCO-3 present, intracellular pH (pHi) increased because of the activity of the pHi-regulating system. The acid extrusion rate (i.e., equivalent efflux of H+, JH) is the product of the pHi recovery rate, intracellular buffering power, and the volume-to-surface ratio. The [HCO3-]o dependence of JH was examined at three fixed levels of [Na+]o: 425, 212, and 106 mM. In all three cases, the apparent Jmax was approximately 19 pmol X cm-2 X s-1. However, the apparent Km (HCO3-) was approximately inversely proportional to [Na+]o, rising from 2.6 to 5.4 to 9.7 mM as [Na+]o was lowered from 425 to 212 to 106 mM, respectively. The [Na+]o dependence of JH was similarly examined at three fixed levels of [HCO3-]o: 12, 6, and 3 mM. The Jmax values did not vary significantly from those in the first series of experiments. The apparent Km (Na+), however, was approximately inversely related to [HCO3-]o, rising from 71 to 174 to 261 mM as [HCO3-]o was lowered from 12 to 6 to 3 mM, respectively. These results agree with the predictions of the ion-pair model of acid extrusion, which has external Na+ and CO3= combining to form the ion pair NaCO3-, which then exchanges for internal Cl-. When the JH data are replotted as a function of [NaCO3-]o, data from all six groups of experiments fall along the same Michaelis-Menten curve, with an apparent Km (NaCO3-) of 80 microM. The ordered and random binding of Na+ and CO3= cannot be ruled out as possible models, but are restricted in allowable combinations of rate constants.     

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter.     

Cation specificity and modes of the Na+:CO3(2-):HCO3- cotransporter in renal basolateral membrane vesicles

The cation specificity and possible exchange modes of the Na+:CO3(2-):HCO3- cotransporter were evaluated by use of basolateral membrane vesicles isolated from rabbit renal cortex. External Li+ inhibited HCO3- gradient-stimulated 22Na uptake, indicating that Li+ interacts with the Na+:CO3(2-):HCO3- cotransporter. No interaction with K+, choline, Rb+, Cs+, or NH4+ could be similarly detected. Imposing an outward Li+ gradient caused quenching of acridine orange fluorescence in the presence but not in the absence of HCO3-, suggesting that Li+:base cotransport takes place via the Na+:CO3(2-):HCO3- cotransporter. Imposing an outward gradient of unlabeled Na+ stimulated the initial rate of 22Na uptake and induced its transient uphill accumulation, indicating Na(+)-Na+ exchange. Na(+)-Na+ exchange was observed in the presence but not in the absence of HCO3- and was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), suggesting that it occurs via the Na+:CO3(2-):HCO3- cotransporter. Similarly, an outward Li+ gradient stimulated uphill 22Na accumulation, indicating Na(+)-Li+ exchange. Na(+)-Li+ exchange was observed in the presence but not in the absence of HCO3-, and was inhibited by DIDS, suggesting that it also occurs via the Na+:CO3(2-):HCO3- cotransporter. Both Na(+)-Na+ and Li(+)-Na+ exchange modes were sensitive to inhibition by harmaline but not by amiloride. We conclude that Li+ is an alternative substrate for the renal Na+:CO3(2-):HCO3- cotransporter. Transport modes of the system include cation:base cotransport and HCO3-dependent cation-cation exchange.     

Acid pH and weak acids induce Na−Cl contransport in the rabbit urinary bladder

We have described a coupled Na--Cl entry step at the apical membrane of a tight epithelium, the rabbit urinary bladder. Mucosal pH values, more acid than 4.6, stimulate a 20 to 40-fold increase in mucosal-to-serosal Na+ and Cl- flux. The flux increase is almost completely blocked by low concentrations of of bumetanide. The transepithelial movement of Na+ and Cl- is normally electroneutral; however, when weak acids (such as acetate) are present in the mucosal solution, the acid-induced increase in flux is accompanied by a large increase in short-circuit current. Besides blockage by bumetanide, both the increase in flux and short-circuit current are blocked by: (1) Na+-free solutions on the mucosa; (2) Cl--free solutions on the mucosa; (3) phosphodiesterase inhibitors; (4) ouabain in the serosal solution; (5) K+-free solutions on the serosa; and (6) HCO3--free solutions on the serosa. The increase in the fluxes and the short-circuit current is unaffected by: (1) amiloride application in the mucosal solution; (2) mucosally applied stilbene derivatives which block Cl-/HCO3- exchange (SITS); and (3) Cl--free solutions applied to the serosa. We interpret these results to imply a coupled Na--Cl uptake step at the apical membrane which is stimulated by intracellular acetate (or (pH). The uptake step leads to a movement of Na+ and Cl- across the basolateral membrane, which is mediated by the Na+, K+-ATPase and a Na/Cl/HCO3- exchange mechanism. Our results demonstrate that "tight" epithelia may, under appropriate circumstances, demonstrate mechanisms of ion movement which are similar to "leaky" epithelia.     

Cyclic AMP inhibits Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder epithelium

Intracellular microelectrode techniques were employed to study the effect of cyclic AMP on apical membrane Cl-/HCO3- exchange and electrodiffusive HCO3- transport in Necturus gallbladder epithelium. Intracellular cAMP levels were raised by addition of either the phosphodiesterase inhibitor theophylline (3 X 10(-3) M) or the adenylate cyclase activator forskolin (10(-5) M) to the serosal bathing solution. Measurements of pH in a poorly buffered control mucosal solution upon stopping superfusion show acidification, owing to secretion of both H+ and HCO3-. When the same experiment is performed after addition of amiloride or removal of Na+ from the mucosal bathing medium, alkalinization is observed since H+ transport is either inhibited or reversed, whereas HCO3- secretion persists. The changes in pH in both amiloride or Na-free medium were significantly decreased in theophylline-treated tissues. Theophylline had no effect on the initial rates of fall of intracellular Cl- activity (aCli) upon reducing mucosal solution [Cl-] to either 10 or 0 mM, although membrane voltage and resistance measurements were consistent with stimulation of apical membrane electrodiffusive Cl- permeability. Estimates of the conductive flux, obtained by either reducing simultaneously mucosal [Cl-] and [HCO3-] or lowering [Cl-] alone in the presence of a blocker of anion exchange (diphenylamine-2-carboxylate), indicate that elevation of intracellular cAMP inhibited the anion exchanger by approximately 50%. Measurements of net Cl- uptake upon increasing mucosal Cl- from nominally zero to levels ranging from 2.5 to 100 mM suggest that the mechanism of inhibition is a decrease in Vmax. Consistent with these results, the rate of intracellular alkalinization upon reducing external Cl- was also inhibited significantly by theophylline. Reducing mucosal solution [HCO3-] from 10 to 1 mM under control conditions caused intracellular acidification and an increase in aCli. Theophylline inhibited both changes, by 62 and 32%, respectively. These data indicate that elevation of intracellular cAMP inhibits apical membrane anion (Cl-/HCO3-) exchange. Studies of the effects of rapid changes in mucosal [HCO3-] on membrane voltages and the apparent ratio of membrane resistances, both in the presence and in the absence of theophylline, with or without Cl- in the mucosal solution, do not support the hypothesis that cAMP produces a sizable increase in apical membrane electrodiffusive HCO3- permeability.     

Intracellular pH-regulating mechanism of the squid axon. Interaction between DNDS and extracellular Na+ and HCO3-

Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models.     

Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium

Intracellular microelectrode techniques and intracellular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCO3]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (Vap). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCO3]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco2 (and consequently [HCO3]i) by the addition of apical NH4 (20 mM) produced an immediate depolarization of Vap. This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 cotransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCO3]i), indicating that the transporter moves NaHCO3 and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering Vap with transepithelial current and then measuring the DIDS-induced change in Vap. Depolarization of Vap increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond -114 mV caused the DIDS-induced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na).     

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons II. Base influx

We used microelectrodes to determine whether the K/HCO3 cotransporter tentatively identified in the accompanying paper (Hogan, E. M., M. A. Cohen, and W. F. Boron. 1995. Journal of General Physiology. 106:821- 844) can mediate an increase in the intracellular pH (pHi) of squid giant axons. An 80-min period of internal dialysis increased pHi to 7.7, 8.0, or 8.3; the dialysis fluid was free of K+, Na+, and Cl-. Our standard artificial seawater (ASW), which also lacked Na+, K+, and Cl-, had a pH of 8.0. Halting dialysis unmasked a slow pHi decrease. Subsequently introducing an ASW containing 437 mM K+ and 0.5% CO2/12 mM HCO3- had two effects: (a) it caused membrane potential (Vm) to become very positive, and (b) it caused a rapid pHi decrease, because of CO2 influx, followed by a slower plateau-phase pHi increase, presumably because of inward cotransport of K+ and HCO3- ("base influx"). Only extracellular Rb+ substituted for K+ in producing the plateau-phase pHi increase in the presence of CO2/HCO3-. Mean fluxes with Na+, Li+, and Cs+ were not significantly different from zero, even though Vm shifts were comparable for all monovalent cations tested. Thus, unless K+ or Rb+ (but not Na+, Li+, or Cs+) somehow activates a conductive pathway for H+, HCO3-, or both, it is unlikely that passive transport of H+, HCO3-, or both makes the major contribution to the pHi increase in the presence of K+ (or Rb+) and CO2/HCO3-. Because exposing axons to an ASW containing 437 mM K+, but no CO2/HCO3-, produced at most a slow pHi increase, K-H exchange could not make a major contribution to base influx. Introducing an ASW containing CO2/HCO3-, but no K+ also failed to elicit base influx. Because we observed base influx when the ASW and DF were free of Na+ and Cl-, and because the disulfonic stilbene derivatives SITS and DIDS failed to block base influx, Na(+)-dependent Cl-HCO3 exchange also cannot account for the results. Rather, we suggest that the most straightforward explanation for the pHi increase we observed in the simultaneous presence of K+ and CO2/HCO3- is the coupled uptake of K+ and HCO3-.     

NHE3 inhibition activates duodenal bicarbonate secretion in the rat

We examined the effect of inhibition of Na+/H+ exchange (NHE) on duodenal bicarbonate secretion (DBS) in rats to further understand DBS regulation. DBS was measured by using the pH-stat method and by using CO2-sensitive electrodes. 5-(N,N-dimethyl)-amiloride (50 microM; DMA), a concentration that selectively inhibits the NHE isoforms NHE1 and NHE2, but not NHE3, did not affect DBS. Nevertheless, 3 mM DMA, a higher concentration that inhibits NHE1, NHE2, and NHE3, significantly increased DBS. Moreover, S1611 and S3226, both specific inhibitors of NHE3 only, or perfusion with Na+-free solutions, dose dependently increased DBS, as measured by pH-stat and CO2-sensitive electrode, without affecting intracellular pH. Coperfusion with 0.1 microM indomethacin, 0.5 mM DIDS, or 1 mM methazolamide did not affect S3226-induced DBS. Nevertheless, coperfusion with 0.1 and 0.3 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid, which inhibits the cystic fibrosis transmembrane conductor regulator (CFTR), dose dependently inhibited S3226-induced DBS. In conclusion, only specific apical NHE3 inhibition increased DBS, whereas prostaglandin synthesis, Na+-HCO3- cotransporter activation, or intracellular HCO3- formation by carbonic anhydrase was not involved. Because NHE3 inhibition-increased DBS was inhibited by an anion channel inhibitor and because reciprocal CFTR regulation has been previously shown between NHE3 and apical membrane anion transporters, we speculate that NHE3 inhibition increased DBS by altering anion transporter function.     

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pH(i)) and Na(+) activity ([Na(+)](i)) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pH(o)). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pH(o) to 2.0 or 10.3. At constant pH(o), buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO(3)(-)/CO(2) inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO(3)(-)/CO(2) buffer, suggesting a regulatory role for pH(i). In polarized TRCs step changes in apical pH(o) from 10.3 to 2.0 induced a linear decrease in pH(i) that remained within the physiological range (slope = 0.035; r(2) = 0.98). At constant pH(o), perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO(3)(-)/CO(2) decreased resting TRC pH(i), and MK-507 or MK-417 attenuated the decrease in pH(i) in TRCs perfused with HCO(3)(-)/CO(2) buffer. In parallel experiments, TRC [Na(+)](i) decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na(+), and (d) acid loading the cells with NH(4)Cl or sodium acetate at constant pH(o). Diethylpyrocarbonate and Zn(2+), modification reagents for histidine residues in proteins, attenuated the CO(2)-induced inhibition of NaCl CT responses and the pH(i)-induced inhibition of apical Na(+) influx in TRCs. We conclude that TRC pH(i) regulates Na(+)-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.     

Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium

We determined the effects of intracellular respiratory and metabolic acid or alkali loads, at constant or variable external pH, on the apical membrane Na+-specific conductance (ga) and basolateral membrane conductance (gb), principally due to K+, in the short-circuited isolated frog skin epithelium. Conductances were determined from the current-voltage relations of the amiloride-inhibitable cellular current pathway, and intracellular pH (pHi) was measured using double barreled H+-sensitive microelectrodes. The experimental set up permitted simultaneous recording of conductances and pHi from the same epithelial cell. We found that due to the asymmetric permeability properties of apical and basolateral cell membranes to HCO3- and NH+4, the direction of the variations in pHi was dependent on the side of addition of the acid or alkali load. Specifically, changing from control Ringer, gassed in air without HCO3- (pHo = 7.4), to one containing 25 mmol/liter HCO3- that was gassed in 5% CO2 (pHo = 7.4) on the apical side caused a rapid intracellular acidification whereas when this maneuver was performed from the basolateral side of the epithelium a slight intracellular alkalinization was produced. The addition of 15 mmol/liter NH4Cl to control Ringer on the apical side caused an immediate intracellular alkalinization that lasted up to 30 min; subsequent removal of NH4Cl resulted in a reversible fall in pHi, whereas basolateral addition of NH4Cl produced a prolonged intracellular acidosis. Using these maneouvres to change pHi we found that the transepithelial Na+ transport rate (Isc), and ga, and gb were increased by an intracellular alkalinization and decreased by an acid shift in pHi. These variations in Isc, ga, and gb with changing pHi occurred simultaneously, instantaneously, and in parallel even upon small perturbations of pHi (range, 7.1-7.4). Taken together these results indicate that pHi may act as an intrinsic regulator of epithelial ion transport.