Effect of Prediabetes on Membrane Bicarbonate Transporters in Testis and Epididymis

The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. This includes the control of bicarbonate (HCO₃ ^?) concentration, which plays an essential role in the maintenance of extracellular and intracellular pH (pH_i) values. Diabetes mellitus alters pH_i regulation in mammalian cells, mainly by altering the activity of ion transporters, particularly HCO₃ ^?-dependent mechanisms. Yet, little is known about the effects of this pathology and its prodromal stage, prediabetes, on the membrane transport mechanisms of male reproductive tract cells. Herein, we analyzed protein and mRNA levels of the most relevant HCO₃ ^? transporters of the SLC4 family [anion exchanger 2 (AE2), Na⁺-driven Cl^?/HCO₃ ^? exchanger (NDCBE), electrogenic Na⁺/HCO₃ ^? cotransporter 1 (NBCe1), electroneutral Na⁺/HCO₃ ^? cotransporter 1 (NBCn1)] in the testis and epididymis of a prediabetic animal model. Firstly, we identified the HCO₃ ^? transporters of the SLC4 family, in both testicular and epididymal tissue. Secondly, although no alterations were detected in protein expression, mRNA levels of NBCe1, NBCn1 and NDCBE were significantly increased in the testis of prediabetic rats. On the other hand, in the epididymis, prediabetes caused an increase of AE2 and a decrease of NDCBE protein levels. These alterations may be translated into changes of HCO₃ ^? transepithelial epididymal fluxes in vivo, which may represent a threat for sperm survival. Moreover, these results provide evidence of the molecular mechanism that may be responsible for the significant increase in abnormal sperm morphology already reported in prediabetic rats.     

Poliovirus-induced intracellular alkalinization involves a proton ATPase and protein phosphorylation

We reported previously that poliovirus infection induces alkalinization in HeLa cells and that an alkaline intracellular pH (pHi) promoted viral replication. Additional experiments were carried out to understand the underlying mechanism. Virus-infected or control monolayer cultures were incubated with nominally bicarbonate-free Eagle's minimal essential medium (MEM) buffered with N-2-hydroxyethylpiperazine-N-3-ethanesulfonic acid (HEPES), and immediately following preincubations, changes in pHi were monitored via benzoic acid uptake around 2 h postinfection. The absence of pH increase in cells infected with ultraviolet light-inactivated virus (UV-virus) indicated that viral gene expression was required for this effect. On the other hand, lack of effect of 3 mM guanidine, an inhibitor of poliovirus-specific RNA but not protein synthesis, suggested that translation of input viral genome RNA is sufficient for the pH increase. Activation of Na⁺/H⁺ exchange, Cl^?HCO^?₃ exchange, or H⁺-ATPase was considered as possible mechanisms by which alkalinization occurs in virus-infected cells. Na⁺/H⁺ exchange was excluded because the pH effect occurred in a Na⁺/H⁺ exchange deficient HeLa cell mutant. Similarly, Cl^?/HCO^?₃ exchange was excluded because virus-specific alkalinization was evident in the presence of Cl^? or bicarbonate deficient medium and was not associated with an increase in HCO^?₃ uptake or a decrease in Cl^? uptake. Lack of dependence on Na⁺, abrogation by 10 μM 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), and resistance to 1 mM vandate suggested that this effect was due to the activation of a vacuolar-type (V) proton ATPase. Studies using protein kinase inhibitors indicated that activation of the ATPase in virus-infected cells probably involved protein kinase C-mediated phosphorylation. © 1993 Wiley-Liss, Inc.     

Sodium-Dependent Antiporters in Choroid Plexus Epithelial Cultures from Rabbit

Abstract: The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na⁺-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO₃^? plus 95% O₂/5% CO₂. Intracellular pH (pH_i) was determined from the steady-state distribution of [¹⁴C]benzoate. Recovery after acidification with NH₄Cl was rapid (t_1/2= 5 min) and was dependent on external Na⁺ (EC₅₀= 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na⁺/H⁺ antiport, blocked 80% of recovery when [Na⁺] was 5 mM with IC₅₀ values of 100 nM. However, neither drug blocked recovery in normal [Na⁺]. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of Cl^?/HCO₃^? antiports, blocked recovery of pH_i in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na⁺ channel, nor with dimethylbenzamil, an inhibitor of Na⁺/Ca²⁺ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of ²²Na⁺ uptake was very rapid (227 pmol/μg of DNA/min at pH 6.2), was dependent on external [Na⁺] with an EC₅₀ value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of ²²Na⁺ uptake by hexamethyleneamiloride was 60% with an IC₅₀ value of 76 nM. We conclude that both the Na⁺/H⁺ antiport and a DIDS-sensitive bicarbonate-dependent antiport are important mechanisms of regulation of the internal pH of rabbit CPE under acidifying conditions. Further, our data suggest that the rabbit choroid plexus Na⁺/H⁺ exchanger can be classified as amiloride insensitive, suggesting that this antiport may play a greater role in controlling transport mechanisms than does the pH of the CNS.     

Potential difference responses due to K+, Na+ and Cl− changes in Bullfrog antrum with and without HCO3−

The effect of changing [K⁺], [Na⁺] and [Cl^?] in nutrient solution was studied in bullfrog antrum with and without HCO₃^? in nutrient. In 25 mM HCO₃^? (95% O₂/5% CO₂) and in zero HCO₃^? (100% O₂), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K⁺ or from 81 to 8.1 mM Cl^? gave a decrease 10 min later in transmucosal PD (nutrient became more negative) — a normal response. These responses were less in zero than in 25 mM HCO₃^?. A decrease from 102 to 8 mM Na⁺ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO₃^?. In contrast, change from 4 to 40 mM K⁺ gave initial anomalous PD response and change from 102 to 8 mM Na⁺, initial normal PD response with either zero or 25 mM HCO₃^?. Both responses were associated with (Na⁺ + K⁺)-ATPase pump and were greater in zero than in 25 mM HCO₃^?. Initial PD increases in zero HCO₃^? are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO₃^? modifies conductance pathways of nutrient membrane.     

Cytosolic pH regulation in perfused rat liver: role of intracellular bicarbonate production

The contribution of metabolic bicarbonate to cytosolic pH (pH_cyto) regulation was studied on isolated perfused rat liver using phosphorus-31 NMR spectroscopy. Removal of external HCO^?₃ decreased proton efflux from 18.6±5.0 to 1.64±0.29 μmol/min per g liver wet weight (w.w.) and pH_cyto from 7.17±0.06 to 6.87±0.06. In the nominal absence of bicarbonate, inhibition of carbonic anhydrase by acetazolamide induced a further decrease of proton efflux of 0.69±0.26 μmol/min per g liver w.w. reflecting a reduction in metabolic CO₂ hydration, and hence a decrease of H⁺ and HCO^?₃ supplies. Even though 27% of the proton efflux was amiloride-sensitive under bicarbonate-free conditions, amiloride did not change pH_cyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and amiloride since pH_cyto decreased by 0.16±0.05 and proton efflux by 0.60±0.14 μmol/min per g liver w.w. The data suggest that amiloride-sensitive or SITS-sensitive transport activities could achieve, by themselves, pH_cyto regulation. The involvement of two mechanisms, most likely Na⁺/H⁺ antiport and Na⁺:HCO^?₃ symport, was confirmed in the whole organ under intracellular and extracellular acidosis. The evidence of Na-dependent transport of HCO^?₃ in the absence of exogenous bicarbonate implies that the amount of metabolic bicarbonate is sufficient to effectively participate to pH_cyto regulation.     

Cyclic GMP Inhibits a Pharmacologically Distinct Na+/H+ Exchanger Variant in Cultured Rat Astrocytes via an Extracellular Site of Action

Abstract: There is growing evidence that cyclic GMP (cGMP) plays important roles in the brain. In cultured rat astrocytes, we observed that the cGMP-inducing C-type natriuretic peptide (CNP) and cGMP analogues caused a decrease in intracellular pH (pH_i). To examine whether this effect was due to inhibition of an Na⁺/H⁺ exchanger (NHE), we acidified cells by replacing extracellular Na⁺ by choline and examined the kinetics of the pH_i recovery that occurred on reintroduction of Na⁺ in the extracellular medium. Both CNP and amiloride analogues inhibited the Na⁺-dependent pH_i recovery, even in the nominal absence of CO₂/HCO₃^?. This indicated that CNP inhibited the activity of an exchanger that was Na⁺-dependent, HCO₃^?-independent, and sensitive to known inhibitors of NHE. However, comparison of the potencies of four distinct amiloride analogues revealed a pharmacological profile that was different from that of any other NHE characterized to date. cGMP mimicked the effect of CNP on sodium-dependent pH_i recovery, but the native nucleotide was as potent as membrane-permeant analogues. Intracellularly produced cGMP was very rapidly exported out of astrocytes. Probenecid and niflumic acid slowed down the rate of cGMP egression and inhibited the effect of CNP on Na⁺-dependent recovery, but not that of extracellular cGMP. Altogether, our data indicate that cGMP inhibits a novel type of NHE in astrocytes via an extracellular site of action. If these results with primary cultures transfer to brain, this phenomenon may constitute a mechanism by which natriuretic peptides exert some of their actions in the brain, as pH_i transients have been shown to modulate several important astrocytic functions.     

Effect of reduced pH in absence of HCO3− on anomalous and normal potential responses in bullfrog antrum

Effect of changing [K⁺], [Na⁺] and [Cl^?] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO₃^? but with 95% O₂/5% CO₂ in both cases. In both cases, changing from 4 to 40 mM K⁺ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO₃^?. Changing from 102 to 8 mM Na⁺ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO₃^? was insignificant but with 25 mM HCO₃^? the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K⁺ and Na⁺ were largely related to ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ pump. Changes in nutrient Cl^? from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ pump and Na⁺/Cl^? symport. Removal of HCO₃^? and concurrent reduction of pH modify resistance of these pathways.     

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl^? and HCO₃^? across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO₃^? efflux through CFTR often required the intracellular H⁺/HCO₃^? production and/or the Na⁺-dependent basolateral HCO₃^? uptake, the intracellular pH (pH_i) balance might be disturbed, especially as a consequence of increased endogenous H⁺ and HCO₃^? production. However, measurement of pH_i by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO₃^? transport. In addition, although the plasma membrane Na⁺/K⁺-ATPase (NKA) is normally essential for basolateral HCO₃^? uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pH_i or NKA abundance on cell membrane.     

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO3 Cotransport

In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na⁺-driven influx of HCO₃ ⁻ causes the increase in intracellular pH (pH_i) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO₂/17 mM HCO₃ ⁻. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na⁺-driven Cl-HCO₃ exchanger, an electrogenic Na/HCO₃ cotransporter, or an electroneutral Na/HCO₃ cotransporter. To determine if the transporter is a Na⁺-driven Cl-HCO₃ exchanger, we depleted the cells of intracellular Cl⁻ by incubating them in a Cl⁻-free solution for an average of ∼11 min. We verified the depletion with the Cl⁻-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl⁻-depleted cells, the pH_i still increases after one or more exposures to CO₂/HCO₃ ⁻. Furthermore, the pH_i decrease elicited by external Na⁺ removal does not require external Cl⁻. Therefore, the transporter cannot be a Na⁺-driven Cl-HCO₃ exchanger. To determine if the transporter is an electrogenic Na/ HCO₃ cotransporter, we measured pH_i and plasma membrane voltage (V_m) while removing external Na⁺, in the presence/absence of CO₂/HCO₃ ⁻ and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO₂/HCO₃ ⁻ solutions contained 20% CO₂ and 68 mM HCO₃ ⁻, pH 7.3, to maximize the HCO₃ ⁻ flux. In pH_i experiments, removing external Na⁺ in the presence of CO₂/HCO₃ ⁻ elicited an equivalent HCO₃ ⁻ efflux of 281 μM s⁻¹. The HCO₃ ⁻ influx elicited by returning external Na⁺ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V_m change was 3.3 mV. Indeed, we found that removing external Na⁺ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO₂/ HCO₃ ⁻. Thus, the Na/HCO₃ cotransporter is electrogenic. Because a cotransporter with a Na⁺:HCO₃ ⁻ stoichiometry of 1:3 or higher would predict a net HCO₃ ⁻ efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO₃ cotransporter with a stoichiometry of 1:2.     

Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation

Amiloride-sensitive, Na⁺-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na⁺ influx is also observed. This indicates that hypertonic challenge activates a Na⁺/H⁺ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH _i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO⁻ ₃, but not in nominally HCO⁻ ₃-free medium, Na⁺/H⁺ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na⁺/H⁺ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995     

cAMP activates Cl−/HCO3− exchange for regulation of intracellular pH in renal epithelial cells

The role of cAMP in regulation of intracellular pH in the confluent LLC-PK₁ cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na⁺-independent Cl^?/HCO₃^? exchange, and by removal of extracellular Cl^?. In addition, Bt₂ cAMP causes Cl^? entry into LLC-PK₁ cells. These results suggest that cAMP activates Cl^? transport, namely Na⁺-independent Cl^?/HCO₃^? exchange, which participates in pH_i regulation.     

Potassium-Induced Stimulation of Glutamate Uptake in Mouse Cerebral Astrocytes: The Role of Intracellular pH

Abstract: The Na⁺-glutamate cotransporters are believed to countertransport OH^? and K⁺. Previous evidence that the velocity of glutamate uptake can exceed the acid extrusion capacity of astrocytes raised the question of whether intracellular pH can become rate limiting for glutamate uptake. Cytoplasmic buffering capacity and acid extrusion in astrocytes are partially HCO₃^? dependent. Also, it was reported recently that raising extracellular [K⁺] alkalinizes astrocyte cytoplasm by an HCO₃^?-dependent mechanism. Here, we have compared glutamate uptake in HCO₃^?-buffered and HCO₃^?-depleted solutions at varying [K⁺]. We observed a pronounced stimulation of glutamate uptake by extracellular K⁺ (3–24 mM) that was substantially HCO₃^? dependent and affected preferentially the uptake of high concentrations (>25 µM) of glutamate. Stimulation of uptake by low extracellular [K⁺] (1.5–3 mM) was less dependent on HCO₃^?. Potassium-induced stimulation of uptake was weaker in rat astrocyte cultures than in mouse. The effects of Ba²⁺ and amiloride on glutamate uptake, as well as the HCO₃^?-dependent stimulatory effects of K⁺ and the species difference, all related consistently to effects on intracellular pH. The effects on uptake, however, were much larger than predicted by the associated changes in electrochemical gradient of OH^?. A “bimodal” scheme for glutamate transport can account qualitatively for the observed correlation between intracellular pH and velocity of glutamate uptake.     

Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH_i), high-energy metabolites, and lactate was investigated using ³¹P and ¹H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO₃^?/CO₂ in the presence of Na⁺ led to a sustained split of the inorganic phosphate (P_i) peak so that the pH_i indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO₃^?. The pH in the compartment with a higher pH_i value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO₃^?-free medium, whereas the pH_i in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl^?, removal of HCO₃^? caused alkalinization without split of the P_i peak. Both treatments reduced the rate of pH_i normalization following alkalinization. Simultaneous omission of external HCO₃^? and Na⁺ did not inhibit alkalinization of the pH_i following CO₂ exit. All these data show that the acid loading mechanism at neutral pH_i is mediated by an Na⁺-independent anion transport. During severe hypoxia, pH_i dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO₃^?, respectively, in Na⁺-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO₃^?-free medium supplemented with 1 mM amiloride, the pH_i fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO₃^? and Cl^?, as the pH_i was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO₃^? but <47% in those preparations superfused without HCO₃^? and Cl^?. This indicates that either neutral pH_i or absence of Cl^? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl^?/HCO₃^? exchange mechanisms have distinct roles in cerebral H⁺ homeostasis depending on the level of pH_i and energy state.     

The ascidian sperm reaction: evidence for Cl- and HCO3- involvement in acid release

Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na⁺ concentration of the medium. The optimal [Na⁺] for acid release is 20 mM with excellent correlation between acid release and initiation of morphological changes. Increasing the [K⁺] to around 20 mM inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K⁺ inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl^?-free (NO^?₃ or glutamate substituted) seawater (SW). Cl^? efflux accompanies H⁺ efflux with twice as many Cl^? being released as H⁺. Both H⁺ and Cl^? release in Cl^?-free SW are dependent upon CO₂ being present in HCO^?₃-free medium, suggesting that H⁺ efflux is in part Cl^? and HCO^?₃-mediated. However, the chloride channel blocking agent SITS has no effect on H⁺ release and augments Cl^? release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na⁺-dependent acidification system of unreacted sperm and the Cl^?- and HCO^?₃-mediated H⁺ release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process.     

Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line che)

The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K⁺] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na⁺] was slightly lower than the extracellular [Na⁺] and was maintained at this level through a vanadate-sensitive transport system. Na⁺ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na⁺ uptake when compared with furosemide-sensitive K⁺ uptake indicated that these cations are not cotransported. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) inhibited Na⁺, K⁺, and Cl^? uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na⁺, K⁺, Cl^?, H⁺ and HCO₃ ^? fluxes are regulated in these cells.     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

Renal cortical basolateral Na+/HCO 3 − cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na⁺/ HCO ₃ ^? cotransporter. We investigated whether the activity of Na⁺/HCO ₃ ^? cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased ²²Na uptake in the presence of HCO₃ but not in the presence of gluconate, indicating that trypsin does not alter diffusive ²²Na uptake but directly stimulates the Na⁺/HCO ₃ ^? cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na⁺/HCO ₃ ^? cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na⁺/HCO ₃ ^? cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na⁺/HCO ₃ ^? cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na⁺/HCO ₃ ^? cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S?). With the addition of fraction S?, the CSU of cAMP-PKA failed to inhibit the Na⁺/HCO ₃ ^? cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na⁺/HCO ₃ ^? cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na⁺/HCO ₃ ^? cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.     

Chloride-bicarbonate exchange in the urinary bladder of the turtle. Independence from sodium ion

The rates of Cl^? absorption and HCO^?₃ secretion were not different in turtle urinary bladders bathed in Na⁺-containing and solutions.These results in turtle bladder are inconsistent with Na⁺-anion cotransport but can be accounted for by a Cl^?/HCO^?₃ exchange system.     

The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30)

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na⁺ and HCO₃^? into the cytoplasm upon intracellular acidosis, restoring the acidic pH_i to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na⁺:1HCO₃^?) and the electrogenic NBCe1 (1Na⁺:2HCO₃^?). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.