Governing effect of regulatory proteins for Cl−/HCO3− exchanger 2 activity

Anion exchanger 2 (AE2) has a critical role in epithelial cells and is involved in the ionic homeostasis such as Cl^? uptake and HCO₃^? secretion. However, little is known about the regulatory mechanism of AE2. The main goal of the present study was to investigate potential regulators, such as spinophilin (SPL), inositol-1,4,5-trisphosphate [IP₃] receptors binding protein released with IP₃ (IRBIT), STE20/SPS1-related proline/alanine-rich kinase (SPAK) kinase, and carbonic anhydrase XII (CA XII). We found that SPL binds to AE2 and markedly increased the Cl^?/HCO₃^? exchange activity of AE2. Especially SPL 1–480 domain is required for enhancing AE2 activity. For other regulatory components that affect the fidelity of fluid and HCO₃^? secretion, IRBIT and SPAK had no effect on the activity of AE2 and no protein-protein interaction with AE2. It has been proposed that CA activity is closely associated with AE activity. In this study, we provide evidence that the basolateral membrane-associated CA isoform CA XII significantly increased the activity of AE2 and co-localized with AE2 to the plasma membrane. Collectively, SPL and CA XII enhanced the Cl^?/HCO₃^? exchange activity of AE2. The modulating action of these regulatory proteins could serve as potential therapeutic targets for secretory diseases mediated by AE2.     

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.     

Evidence for HCO3− conductance pathways in nutrient membrane of bullfrog antrum

The effect of changing the nutrient-side HCO₃⁻ concentration on potential difference (PD) and resistance in bullfrog antrum bathing in Cl⁻ media was determined. Changes in HCO₃⁻ concentration were from 25 mM to several lower concentrations and back to 25mM. A plot of 6ΔPD6 versus log [HCO₃⁻] gave a linear relation for changes of HCO₃ concentration from 25 down to 3.1 mM and back to 25 mM but deviated to some extent for changes to 1.6 mM. In these experiments, changes from higher to lower HCO₃⁻ concentrations gave a less rapid initial PD response than those in the reverse direction. This result eliminated H⁺ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in nutrient solution from 5% CO₂ and 25 mM HCO₃⁻ to 0.6% CO₂ and 3 mM HCO₃⁻ and in the second part the same changes with a simultaneous change of secretory solution from 5% to 10% CO₂. The magnitude of PD decrease was greater by 4.5 mV in the second part. This result indicated that HCO₃⁻ conductance pathways rather than OH⁻ conductance pathways predominated. There was no evidence of HCO₃⁻, OH⁻ and H⁺ conductance pathways in secretory membrane.     

Evidence for HCO3− conductance pathways in nutrient membrane of resting frog fundus

The effect on potential difference (PD) and resistance in Cl⁻ media bathing the resting fundus of Rana pipiens was determined for nutrient HCO₃⁻ changes from 25 mM to several lower concentrations and back to 25 mM. The graph of |vbΔPD|vb versus log[HCO₃⁻] was linear for changes from 25 down to 3.1 mM and also back to 25 mM, but deviated considerably for changes to 1.6 mM. The fact that changes from higher to lower HCO₃⁻ gave a less rapid initial PD response than the reverse direction eliminated H⁺ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in the nutrient solution from 5% CO₂ and 25 mM HCO₃⁻ to 0.6% CO₂ and 3 mM HCO₃⁻ and in the second part, the same changes with the simultaneous change of secretory solution from 5% to 10% CO₂. The magnitude of the PD decrease was greater by 4.0 mV in the second part. This result indicated that HCO₃⁻ rather than OH⁻ conductance pathways predominated. On the secretory side, the change from 25 to 3.1 mM HCO₃⁻ gave a small but significant change in PD. The latter effect was too small to determine whether HCO₃⁻ pathways existed in the secretory membrane.     

Evidence for the phosphorylation of serine259 of histone deacetylase 5 by protein kinase Cδ

Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser₂₅₉ and Ser₄₉₈), resulting in nuclear export of HDAC5 and de-repression of downstream target genes. In the previous paper we reported the important role of PKD isozymes in the regulation of HDAC5 by phosphorylating Ser₄₉₈ of HDAC5 [Q.K. Huynh, T.A. Mckinsey, Arch. Biochem. Biophys. 450 (2006) 141–148]. In the present paper, we provide evidence that PKCδ can directly phosphorylate Ser₂₅₉ of HDAC5. The evidence is based on the following facts (a) isolated kinase fraction from human failing heart tissues contained PKCδ that phosphorylated HDAC5 Ser₂₅₉ peptide and no significant activity was found for the unbound fraction after they were immunoprecipitated with PKCδ specific antibody; (b) specific inhibitors for PKCδ inhibited kinase activity from isolated fraction and recombinant human PKCδ with similar IC₅₀ values; (c) recombinant human PKCδ can directly phosphorylate full length Ser₂₅₉ HDAC5 protein and HDAC5 Ser₂₅₉ peptide. The results suggest that in addition to activation of protein kinase D isozymes by phosphorylating Ser₇₄₄ and Ser₇₄₈ at their activation sites, PKCδ may also play a role in the regulation of HDAC5 by phosphorylation of Ser₂₅₉.     

Negative feedback regulation of Gq signaling by protein kinase C is disrupted by diacylglycerol kinase ζ in COS-7 cells

Cellular response to G(q)-linked agonists is shaped by regulatory inputs which determine signal strength and duration. Stimulation of phospholipase C-β (PLC-β) lipase activity results in an increase in the levels of diacylglycerol (DAG) and activation of protein kinase C (PKC) activity. PKC has been implicated in the feedback regulation of G(q) signaling through actions on PLC-β and phospholipase D (PLD) lipase activity. As PKC activity is modulated by multiple layers of regulation, the physiological impact of PKC on G(q) signaling is unclear. PKC signaling can be terminated by diacylglycerol kinases (DGKs) which are regulated in a cell-specific manner. The present studies investigated the contribution of the ubiquitously expressed DGKζ isoform in the regulation of PKC signaling and G(q) response in transfected COS-7 cells. Genetic depletion of DGKζ protein with antisense oligonucleotides dramatically reduced DAG metabolism. The sustained increase in PKC signaling was associated with a pronounced inhibition of carbachol-stimulated lipase activity in cells co-transfected with m1 muscarinic receptor, Gα(q) and either with or without PLC-β(1). The data also reveal that sustained activation of PKC alone does not increase cellular PLD1 activity. Therefore, G(12)-activated RhoA is physiologically important for adequate stimulation of PLD1 activity. These data show that the impact of PKC on G(q) signal transduction is determined by the background of cell-specific processes.     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

Evidence against H+−HCO 3 − symport as the mechanism for HCO 3 − transport in the cyanobacteriumAnabaena variabilis

Summary The rate of inorganic carbon uptake and its steadystate accumulation ratio (intracellular/extracellular concentration) was determined in the cyanobacteriumAnabaena variabilis as a function of extracellular pH. The free energy of protons ( ) across the plasmalemma was calculated from determinations of membrane potential, and intracellular pH, as a function of the extracellular pH. While inward proton motive force decreased with increasing extracellular pH from 6.5 to 9.5, rate of HCO ₃ ^– influx and its accumulation ration increased. The latter is several times larger than would be expected should HCO ₃ ^– influx be driven by . It is concluded that HCO ₃ ^– transport in cyanobacteria is not driven by the proton motive force.     

Evidence for Cl− exchangers in perfused Carcinus gills

• 1.1. The effects of inhibitors and ion substitution on TBP (transbranchial potentials) and unidirectional ²²Na and ³⁶Cl fluxes from the bathing medium across the apical and basolateral sides into the perfusion solution J_a→b of isolated Carcinus gill epithelia were studied. Identical, diluted sea-water (DSW) and artificial solutions were used in the bathing solution and perfusate (202 ± 5 mM Na).
• 2.2. Externally applied bumetanide (10⁻³ M) did not affect ³⁶Cl and ²²Na fluxes. In addition, ³⁶Cl and ²²Na unidirectional J_a→b fluxes obtained by isosmotic substitution of co-ions did not provide evidence for the presence of a Na-K-2Cl co-transporter on the apical membrane.
• 3.3. 10⁻³ M 4-acetamido-4 isothiocyanostilbene 2,2 disulphonic acid (SITS) in the bathing solution and on both sides (apical and basolateral surfaces) effected a rapid, reversible and statistically significant inhibition of ³⁶Cl J_a→b, fluxes of 27.6 and 39.4%, respectively. In addition, 10 mM acetazolamide reduced Cl⁻ (external and both sides) and Na⁺ J_a→b, fluxes. Furthermore, Cl⁻ influxes were stimulated by addition of NaHCO₃ (15.5mM) on both epithelial sides by 64%.
• 4.4. On the basis of the experimental evidence described, it is suggested that there is a Cl⁻/HCO₃ (or OH⁻) antiporter located on the apical side of the gill surface.

     

Differential regulation of Na+/H+ exchange and H+ -ATPase by pH and HCO 3 − in kidney proximal tubules

This study examines the effects of acute in vitro acid-base disorders on Na⁺/H⁺ and H⁺-ATPase transporters in rabbit kidney proximal tubules (PT). PT suspensions were incubated in solutions with varying acid base conditions for 45 min and utilized for brush border membrane (BBM) vesicles preparation. BBM vesicles were studied for Na⁺/H⁺ exchange activity (assayed by ²²Na⁺ influx) or abundance (using NHE-3 specific antibody) and H⁺-ATPase transporter abundance (using antibody against the 31 kDa subunit). The Na⁺/ H⁺ exchanger activity increased by 55% in metabolic acidosis (pH 6.5, HCO ₃ ^– 3 mm) and decreased by 41% in metabolic alkalosis (pH 8.0, HCO ₃ ^– 90 mm). The abundance of NHE-3 remained constant in acidic, control, and alkalotic groups. H⁺-ATPase abundance, however, decreased in metabolic acidosis and increased in metabolic alkalosis by 57% and 42%, respectively. In PT suspensions incubated in isohydric conditions (pH 7.4), Na⁺/H⁺ exchanger activity increased by 29% in high HCO ₃ ^– group (HCO ₃ ^– 96 mm) and decreased by 16% in the low HCO ₃ ^– groups (HCO ₃ ^– 7mm. The NHE-3 abundance remained constant in high, normal, and low [HCO ₃ ^– ] tubules. The abundance of H⁺-ATPase, however, increased by 82% in high [HCO ₃ ^– ] and decreased by 77% in the low [HCO ₃ ^– ] tubules. In PT suspensions incubated in varying pCO₂ and constant [HCO ₃ ^– ], Na⁺/H⁺ exchanger activity increased by 35% in high pCO₂ (20% pCO₂, respiratory acidosis) and decreased by 32% in low pCO₂ (1.5% pCO₂, respiratory alkalosis) tubules. The NHE-3 abundance remained unchanged in high, normal, and low pCO₂ tubules. However, the H⁺-ATPase abundance increased by 74% in high pCO₂ and decreased by 69% in low pCO₂ tubules.The results of these studies suggest that the luminal Na⁺/H⁺ exchanger is predominantly regulated by pH whereas H⁺-ATPase is mainly regulated by [HCO ₃ ^– ] and/ or pCO₂. They further suggest that the adaptive changes in H⁺-ATPase transporter are likely mediated via endocytic/exocytic pathway whereas the adaptive changes in Na⁺/H⁺ exchanger are via the nonendocytic/exocytic pathway.The excellent technical assistance of Yollanda J. Hattabaugh, Gwen L. Bizal, and L. Yang is greatly appreciated. Portions of these studies were presented at the annual meeting of the American Society of Nephrology, Boston, MA, November 1993, and published in abstract form (J.Am.Soc.Neph. 4:840A, 1993)These studies were supported by a Merit Review Grant from the Department of Veterans Affairs and a grant-in-aid from the American Heart Association (to M.S.), a Baxter Health Care Grant (to B.B.), and the National Institute of Health Grants DK 38510 (to E.B.C. and M.C.R.) and DK 42086 (to E.B.C.).     

Control of Cl− influx inChara by internal pH

Summary The possible regulation of Cl^– influx inChara by the cytoplasmic Cl^– concentration and cytoplasmic pH was investigated using both intact and intracellularly perfused cells. In perfused cells Cl^– influx was sensitive to changes in the internal Cl^– concentration but only when the concentration was less than 1mm.In intact cells the metabolic inhibitors, CCCP, DCMU, and oligomycin which inhibit Cl^– influx also reduced the cytoplasmic pH. A correlation between ATP concentration and cytoplasmic pH was shown to apply when the ATP concentration was lowered using these inhibitors. The possible relationships between ATP status, cytoplasmic pH, and Cl^– influx are discussed.     

Activation of Cl−/OH− exchange by parachloromercuribenzoic acid in rabbit renal brush-border membranes

Summary The effect of the sulfhydryl reagent parachloromercuribenzoic acid (PCMB) on chloride transport was examined in rabbit renal brush-border membrane vesicles (BBMV). PCMB had no effect on the chloride conductive pathway. In the presence of an inside-alkaline pH gradient and a K⁺/valinomycin voltage clamp, the addition of PCMB stimulated³⁶Cl uptake and induced a threefold overshoot above the equilibrium value, indicating Cl/OH exchange. The effect of PCMB was reversed by dithiothreitol. Cl/OH exchange was not observed in the absence of PCMB. PCMB-activated Cl/OH exchange persisted even when the membrane potential was made inside-negative relative to the controls, thus, demonstrating that PCMB's effect on³⁶Cl uptake under pH-gradient conditions is not mediated by parallel Cl^– and H⁺ conductive pathways. PCMB-activated Cl/OH exchange was inhibited by 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) with IC₅₀ values of 290 and 80 m, respectively. These results demonstrate that modification of sulfhydryl groups by PCMB activates Cl/OH exchange in BBMV.     

HCO 3 − /Cl− exchange across the human erythrocyte membrane: Effects of pH and temperature

Summary Changes in extracellular pH (pH_o) in red cell suspensions were monitored in a stopped-flow rapid reaction apparatus under conditions wheredpH_o/dt was determined by the rate of HCO ₃ ^– /Cl^– exchange across the membrane. Experiments were performed at 5°C<T<40°C using either untreated cells or cells exposed to 0.11mm SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid). Although SITS exposure reduced the rate of exchange by 90%, both untreated and SITS-treated cells are similarly affected by changes in pH₀ and temperature. The rate of HCO ₃ ^– /Cl^– exchange exhibits a minimum at about pH_o 5 and a maximum at about pH₀ 7.4 at all temperatures. A transition temperature of 17°C was observed in the Arrhenius relationship for all pH₀. The activation energies (E _a) in kcal/mol are 19.6 below and 11.7 above 17°C for 50<8. These findings, similar to those reported for Cl^– self-exchange, suggest that: (i) a change in the rate-limiting step for HCO ₃ ^– /Cl^– exchange occurs at 17°C, possibly due to an altered interaction between the transport pathway and membrane lipids; (ii) the carrier system can be titrated by either H⁺ or SITS from the outside of the membrane, but the untitrated sites continue to transport normally; (iii) the pH₀ dependence of the rate of exchange is consistent with the titratable carrier having its most alkaline pK in the range expected for amino groups; and (iv) below pH₀ 5, the nature of the exchange is markedly altered.     

Cl− channels in basolateral renal medullary membrane vesicles: IV. Analogous channel activation by Cl− or cAMP-dependent protein kinase

Summary We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl^– concentrations in modulating the activity of Cl^– channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, thecis andtrans solutions face the extracellular and intracellular aspects of these Cl^– channels, respectively. Raising thetrans Cl^– concentration from 2 to 50mm increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990.J. Membrane Biol. 118:269–278). With 2mm trans KCl,trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage-independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C-PKA plus ATP) addition tocis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50mm)trans Cl^– concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl^– channel activity by phosphorylation at or near the Cl^–-sensitive site on the intracellular face of these channels.     

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl−/HCO3− exchanger,AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO₂ to HCO₃^?+H⁺. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl^?/HCO₃^? exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl^?/NO₃^? exchange assays, which were independent of CA activity, and in Cl^?/HCO₃^? exchange assays. Transport was measured by following changes of intracellular [Cl^?] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl^?/NO₃^? exchange activity with EC₅₀ values in the range 0.22–2.8 μM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 μM of each compound was only 22–53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl^?/HCO₃^? exchange assays, which depend on functional CA to produce transport substrate, 40 μM celecoxib inhibited AE1 by 62±4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.     

12-O-tetradecanoylphorbol-1, 3-acetate induces the negative regulation of protein kinase B by protein kinase Cα during gastric cancer cell apoptosis

The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCα, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCα into the nucleus, where PKCα exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCα in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.     

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

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

Regulation of intracellular pH by electrogenic Na+/HCO3– co-transporters in embryonic neural stem cell-derived radial glia-like cells

A stroke causes a hypoxic brain microenvironment that alters neural cell metabolism resulting in cell membrane hyperpolarization and intracellular acidosis. We studied how intracellular pH (pH_i) is regulated in differentiated mouse neural progenitor cells during hyperpolarizing conditions, induced by prompt reduction of the extracellular K⁺ concentration. We found that the radial glia-like population in differentiating embryonic neural progenitor cells, but not neuronal cells, was rapidly acidified under these conditions. However, when extracellular calcium was removed, an instant depolarization and recovery of the pH_i, back to normal levels, took place. The rapid recovery phase seen in the absence of calcium, was dependent on extracellular bicarbonate and could be inhibited by S0859, a potent Na/HCO3 cotransporter inhibitor. Immunostaining and PCR data, showed that NBCe1 (SLC4A4) and NBCn1 (SLC4A7) were expressed in the cell population and that the pH_i recovery in the radial glial-like cells after calcium removal was mediated mainly by the electrogenic sodium bicarbonate transporter NBCe1 (SLC4A4). Our results indicate that extracellular calcium might hamper pH_i regulation and Na/HCO3 cotransporter activity in a brain injury microenvironment. Our findings show that the NBC-type transporters are the main pH_i regulating systems prevailing in glia-like progenitor cells and that these calcium sensitive transporters are important for neuronal progenitor cell proliferation, survival and neural stem cell differentiation.     

Intestinal HCO 3 − secretion inAmphiuma: Stimulation by mucosal Cl− and serosal Na+

Summary The requirement for Na⁺ and Cl^– in the bathing media to obtain a maximal HCO ₃ ^– secretory flux ( ) across isolated short-circuitedAmphiuma duodenum was investigated using titration techniques and ion substitution. Upon substitution of media Na⁺ with choline, HCO ₃ ^– secretion was markedly reduced. Replacement of media Cl^– produced a smaller reduction of . The presence of Cl^– enhanced HCO ₃ ^– secretion only if Na⁺ was also in the media. Elevation of media Na⁺ or Cl^– in the presence of the other ion produced a saturable increase of . In the presence of Na⁺, Cl^– stimulated when added to the mucosal but not the serosal medium. In the presence of Cl^–, Na⁺ elevated when added to the serosal but not the mucosal medium. The ability of mucosal Cl^– to stimulate was not apparently dependent on mucosal Na⁺. Simultaneous addition of 10mm Cl^– to the Na⁺-free mucosal medium and 10mm Na⁺ to the Cl^–-free serosal medium stimulated above levels produced by serosal Na⁺ alone. In conclusion, intestinal HCO ₃ ^– secretion required mucosal Cl^– and serosal Na⁺ and did not involve mucosal NaCl cotransport. The results are consistent with a mucosal Cl^– absorptive mechanism in series with parallel basolateral Na⁺–H⁺ and Cl^––HCO ₃ ^– exchange mechanisms.