Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996     

Renal cortical basolateral Na+/HCO 3 − cotransporter: I. Partial purification and reconstitution

The renal basolateral Na⁺/HCO ₃ ^– cotransporter is the main system responsible for HCO ₃ ^– transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na⁺/HCO ₃ ^– cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l--phosphatidylcholine (proteoliposomes). Na⁺/HCO ₃ ^– cotransporter activity was assessed as the difference in ²²Na uptake in the presence of HCO ₃ ^– and gluconate. The activity of the Na⁺/HCO ₃ ^– cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na⁺/HCO ₃ ^– cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na⁺/HCO ₃ ^– cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na⁺/HCO ₃ ^– cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na⁺/HCO ₃ ^– cotransporter and suggest that the 56 kD doublet protein may represent the Na⁺/HCO ₃ ^– cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

Renal cortical basolateral Na+/HCO 3 − cotransporter: II. Detection of conformational changes with fluorescein isothiocyanate labeling

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups and has been useful in detecting conformational changes in transport proteins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral membrane proteins, enriched in Na⁺/HCO ₃ ^– cotransporter activity, were reconstituted into liposomes and treated with FITC or its nonfluorescent analogue PITC (phenyl isothiocyanate). In the absence of Na⁺ and HCO ₃ ^– , incubation of proteoliposomes with PITC or FITC significantly inhibited cotransporter activity. However, in the presence of Na⁺ and HCO ₃ ^– during labeling both agents failed to inhibit cotransporter activity, indicating that these probes interact specifically with the cotransporter. In the presence of the substrates Na⁺ and HCO ₃ ^– , PITC binds covalently to amino groups unprotected by substrates leaving the Na⁺/HCO ₃ ^– cotransporter available for specific labeling with FITC. Addition of NaHCO₃ to FITC-labeled proteoliposomes resulted in a concentration-dependent enhancement of the fluorescence signal which was inhibited by pretreatment with 4,4-diisothiocyanostilbene 2,2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteoliposomes showed the presence of two distinct fluorescent bands (approximate MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurement of gel slice fluorescence. Thus, FITC detects conformational changes of the Na⁺/HCO ₃ ^– cotransporter and labels proteins which may represent the cotransporter or components of this cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

Effect of 3-isobutyl-1-methylxanthine on HCO3− transport in turtle bladder: Evidence for electrogenic HCO3− secretion

Ouabain-treated turtle bladders bathed on both surfaces by identical HCO₃^?/CO₂-containing, Cl^?-free Na⁺ media exhibit a short-circuit current (I_sc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the I_sc and p.d.. The IBMX-induced reversal of I_sc and p.d. is (1) dependent on the presence of HCO₃^? (and CO₂) in the serosal bathing fluid, (2) independent of Na⁺ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed I_sc elicited by IBMX represents electrogenic secretion of HCO₃^?.     

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.     

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.     

HCO 3 − -coupled Na+ influx is a major determinant of Na+ turnover and Na+/K+ pump activity in rat hepatocytes

Summary Recent studies in hepatocytes indicate that Na⁺-coupled HCO ₃ ^– transport contributes importantly, to regulation of intracellular pH and membrane HCO ₃ ^– transport. However, the direction of net coupled Na⁺ and HCO ₃ ^– movement and the effect of HCO ₃ ^– on Na⁺ turnover and Na⁺/K⁺ pump activity are not known. In these studies, the effect of HCO ₃ ^– on Na⁺ influx and turnover were measured in primary rat hepatocyte cultures with²²Na⁺, and [Na⁺] _i was measured in single hepatocytes using the Na⁺-sensitive fluorochrome SBFI. Na⁺/K⁺ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na⁺-dependent or ouabain-suppressible⁸⁶Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K⁺ on membrane potential difference (PD) and [Na⁺] _i . In hepatocyte monolayers, HCO ₃ ^– increased²²Na⁺ entry and turnover rates by 50–65%, without measurably altering²²Na⁺ pool size or cell volume, and HCO ₃ ^– also increased Na⁺/K⁺ pump activity by 70%. In single cells, exposure to HCO ₃ ^– produced an abrupt and sustained rise in [Na⁺] _i , from 8 to 12mm. Na⁺/K⁺ pump activity assessed in single cells by PD excursions during transient K⁺ removal increased 2.5-fold in the presence of HCO ₃ ^– , and the rise in [Na⁺] _i produced by inhibition of the Na⁺/K⁺ pump was similarly increased 2.5-fold in the presence of HCO ₃ ^– . In intact perfused rat liver, HCO ₃ ^– increased both Na⁺/K⁺ pump activity and O₂ consumption. These findings indicate that, in hepatocytes, net coupled Na⁺ and HCO ₃ ^– movement is inward and represents a major determinant of Na⁺ influx and Na⁺/K⁺ pump activity. About half of hepatic Na⁺/K⁺ pump activity appears dedicated to recycling Na⁺ entering in conjunction with HCO ₃ ^– to maintain [Na⁺] _i within the physiologic range.     

Na+-dependent HCO 3 − transport and Na+/H+ exchange regulate pH i in human ciliary muscle cells

Summary We investigated intracellular pH (pH _i ) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4,5-dimethylfluorescein (CDMF). The steady-state pH _i was 7.09±0.04 (n = 12) in CO₂/ HCO ₃ ^– -buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO₂/ HCO ₃ ^– and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH _i recovery, which was almost completely amiloride-sensitive in the absence of CO₂/ HCO ₃ ^– but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH _i recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH _i recovery was almost completely mediated by Na⁺/H⁺ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH _i recovery was observed in CO₂/HCO ₃ ^– -buffered solution which was mediated by chloride-independent and chloride-dependent Na⁺ HCO ₃ ^– cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH _i recovery after NH₄Cl prepulse revealed V _max = 0.57 pH units/min, K _m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V _max = 0.19 pH units/min, K _m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na⁺/H⁺ exchange and chloride-independent and chloride-dependent Na⁺HCO ₃ ^– cotransport are involved in the pH _i regulation of cultured human ciliary muscle cells.The expert technical assistance of Astrid Krolik is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft grant DFG Wi 328/11.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

Properties of soluble ATPase of gastric mucosa II Effect of HCO3−

An ATPase has been solubilized from dog gastric mucosa which was localized to the smooth vesicular fraction of the tissue homogenate. With solubilization, there was a marked increase in the HCO₃⁻ activation of the enzyme, and several other oxyanions were found to stimulate ATPase activity, such as borate, selenite, arsenite, arsenate and sulfite. The stimulation was a function of the concentration and pK of the base. Additionally, methane sulfonyl chloride inhibited the enzyme, particularly the base activated component. Based on these observations a tentative model for the HCO₃⁻ activation mechanism is suggested.     

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.).     

Amiloride-induced stimulation of HCO3− reabsorption in turtle bladder

Amiloride in the mucosal fiuid (at concentrations of 5 · 10^?6 M to 10^?4 M) reversibly stimulates the HCO₃^?-dependent moiety of the short-circuiting current (I_sc) in ouabain-treated turtle bladders bathed by Na-free Ringer solutions with or without Cl_?.This effect is uniquely different from the known inhibitory effect of this agent on Na⁺ transport. Thus, any comprehensive hypothesis on the action of amiloride over a wide dosage-response fange should take into account its effect on HCO₃^? transport.     

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.     

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.     

Secretion of HCO 3 − /OH− in cortical distal tubule of the rat

Secretion of bicarbonate has been described for distal nephron epithelium and attributed to apical Cl^–/HCO ₃ ^– exchange in beta-intercalated cells. We investigated the presence of this mechanism in cortical distal tubules by perfusing these segments with acid (pH 6) 10 mm phosphate Ringer. The kinetics of luminal alkalinization was studied in stationary microperfusion experiments by double-barreled pH (ion-exchange resin)/1 m KCl reference microelectrodes. Luminal alkalinization may be due to influx (into the lumen) of HCO ₃ ^– or OH^–, or efflux of H⁺. The magnitude of the Cl^–/ HCO ₃ ^– exchange component was measured by perfusing the lumen with solutions with or without chloride, which was substituted by gluconate. This component was not different from zero in control and alkalotic (chronic plus acute) Wistar rats. Homozygous Brattleboro rats (BRB), genetically devoid of antidiuretic hormone, were used since this hormone has been shown to stimulate H⁺ secretion, which could mask bicarbonate secretion. In these rats, no evidence for Cl^–/HCO ₃ ^– exchange was found in control BRB and in early distal segments of alkalotic animals, but in late distal tubule a significant component of 0.14±0.033 nmol/cm² · sec was observed, which, however, is small when compared to the reabsorptive flow found in control Wistar rats, of 0.95±0.10 nmol/cm² · sec. In addition, 5×10^–4 m SITS had no effect on distal bicarbonate reabsorption in controls as well as on secretion in alkalotic Wistar and Brattleboro rats, which is compatible with the absence of effect of this drug on the apical Cl^–/HCO ₃ ^– exchange in other tissues. It is concluded that most distal alkalinization is not Cl^– dependent, and that Cl^–/HCO ₃ ^– exchange may be found in cortical distal tubule, but its magnitude is, even in alkalosis, markedly smaller than the reabsorptive flux, which predominates in the rats studied in this paper, keeping luminal pH lower than that of blood.     

The Detailed Localization Pattern of Na+/K+/2Cl? Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na⁺/K⁺/2Cl⁻ cotransporter type 2 (NKCC-2) and Na⁺/K⁺-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na⁺/K⁺-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na⁺/K⁺-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)     

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.