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.     

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.     

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.).     

Role of epithelial HCO3⁻ transport in mucin secretion: lessons from cystic fibrosis

The invitation to present the 2010 Hans Ussing lecture for the Epithelial Transport Group of the American Physiological Society offered me a unique, special, and very surprising opportunity to join in saluting a man whom I met only once, but whose work was the basis, not only for my career, but also for finding the molecular defect in the inherited disease cystic fibrosis (CF). In this context, I will venture to make the tribute with a new explanation of why a mutation in a single gene that codes for an anion channel can cause devastation of multiple epithelial systems with pathogenic mucus. In so doing, I hope to raise awareness of a new role for that peculiar anion around which so much physiology revolves, HCO(3)(-). I begin by introducing CF pathology as I question the name of the disease as well as the prevalent view of the basis of its pathology by considering: 1) mucus, 2) salt, and 3) HCO(3)(-). I then present recent data showing that HCO(3)(-) is required for normal mucus discharge, and I will close with conjecture as to how HCO(3)(-) may support mucus discharge and why the failure to transport this electrolyte is pathogenic in CF.     

Intracellular pH regulation and buffer capacity in CO2/HCO− 3-buffered media in cultured epithelial cells from rainbow trout gills

The influence of a CO₂/HCO⁻ ₃-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH_i) was monitored by continuous spectrofluorometric recording from cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH=7.70 were transferred to normal CO₂/HCO⁻ ₃-buffered medium {P _CO2=3.71 mmHg, [HCO⁻ ₃]= 6.1 mmol l⁻¹, extracellular pH (pH_e)=7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (∼7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5–8.0 slykes) based on intracellular [HCO⁻ ₃], and was unaffected by most drugs and treatments. However, after transfer to high P _CO2=11.15 mmHg, [HCO⁻ ₃]= 18.2 mmol l⁻¹ at the same pH_e=7.70, the final regulated pHi was elevated (∼7.53). The rate of correction of alkalosis caused by washout of this high P _CO2, high-HCO⁻ ₃ medium was unaffected by removal of extracellular Cl⁻. Removal of extracellular Na⁺ lowered resting pH_i and greatly inhibited the rate of pH_i recovery from acidosis. Bafilomycin A₁ (3 μmol l⁻¹) had no effect on these responses. However amiloride (0.2 mmol l⁻¹) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH_i, the latter differing from the response in HEPES where amiloride also lowered resting pH_i. Similarly 4-acetamido-4′- isothiocyanatostilbene-2,2′-disulfonic acid, sodium salt (0.1 mmol l⁻¹) did not affect resting pH_i but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl⁻ also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H⁺ extrusion rate was unaffected. These results are consistent with the presence of Na⁺-(HCO⁻ ₃)_N co-transport and/or Na⁺-dependent HCO⁻ ₃/Cl⁻ exchange, in addition to Na⁺/H⁺ exchange, as mechanisms contributing to “housekeeping” pH_i regulation in gill cells in CO₂/HCO⁻ ₃ media, whereas only Na⁺/H⁺ exchange is seen in HEPES. Both Na⁺-independent Cl⁻/HCO⁻ ₃ exchange and V-type H⁺-ATPase mechanisms appear to be absent from these cells cultured in isotonic media. Accepted: 30 November 1999     

Short-chain fatty acids and CO2 as regulators of Na+ and Cl− absorption in isolated sheep rumen mucosa

Summary Unidirectional ²²Na⁺ and ³⁶Cl^– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO₂/HCO _– ³ -containing and CO₂/HCO _– ³ -free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na⁺ absorption without affecting the Cl^– transport in the same direction. Short-chain fatty acid stimulation of Na⁺ transport was at least partly independent of Cl^– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na⁺ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO₂ in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na⁺ flux. Along with its effect on sodium transport, raising the PCO₂ also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO₂ permeate the cell membrane and produce a raise in intracellular H⁺ concentration. This stimulates an apical Na⁺/H⁺ exchange, leading to increased Na⁺ transport. The stimulatory effect of CO₂ on Cl^– transport is probably mediated by a Cl^–/HCO _– ³ exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid - G _t transepithelial conductance (mS·cm^-2) - HSCFA undissociated short-chain fatty acids - J _ms mucosal-to-serosal flux (Eq · cm^-2 · h^-1) - J _net net flux (Eq · cm^-2 · h^-1) - J _sm serosal-to-mucosal flux (Eq · cm^-2 · h^-1) - PD transepithelial potential difference (mV) - SCFA dissociated short-chain fatty acids - SCFA short-chain fatty acids     

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.     

Role of F-actin in the activation of Na+-K+-Cl− cotransport by forskolin and vasopressin in mouse kidney cultured thick ascending limb cells

The influence of microtubules and F-actin on Na⁺-K⁺-Cl^? cotransport was investigated in cultured cells derived from outer-medullary thick ascending limb tubules microdissected from the mouse kidney. The cultured cells contained Tamm-Horsfall protein, produced cAMP in response to dD-arginine vasopressin (dD-AVP), isoproterenol, prostaglandin E2 and forskolin (FK), and exhibited an ouabain-resistant furosemidesensitive (Or-Fs) component of ⁸⁶Rb⁺ influx mediated by the Na⁺-K⁺-Cl^? cotransporter. Both FK and dD-AVP stimulated the Or-Fs component of Rb⁺ influx. Neither agent altered the tubulin and cytokeratin networks nor the shape of the tight junction using a specific anti-ZO-1 antibody. In contrast, they did induce a marked redistribution of F-actin to the periphery of the cells delineating the tight junctions. Preincubation of the cells with nocodazole, to disrupt microtubules, did not alter the FK-or dD-AVP-elicited Or-Fs Rb⁺ influx. In contrast, phalloidin and NBD-phallicidin, which stabilize F-actin, markedly impaired the stimulation of Na⁺-K⁺-Cl^? cotransport by FK or dD-AVP, without affecting the Na⁺-K⁺ ATPase pumps and the rate constant of ³⁶Cl^? and ⁸⁶Rb⁺ efflux. These results strongly suggested that cAMP-stimulated Na⁺-K⁺-Cl^? cotransport is linked to F-actin in renal TAL cells.     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.     

Bicarbonate transport inChara corallina: evidence for cotransport of HCO 3 − with H+

Summary Experiments were undertaken on the fresh water algaChara corallina to determine the form of inorganic carbon (CO₂ or HCO ₃ ^– ) which enters the cell during photosynthesis at alkaline pH. Recent proposals have centered on the possibility that proton efflux in alkaline solution is able to generate, in the immediate vicinity of the cell, a sufficiently low pH to raise the partial pressure of CO₂, and hence facilitate its passive permeation into the cell. Predictions have been made by modelling this situation (N.A. Walker, F.A. Smith & I.R. Cathers, 1980,J. Membrane Biol. 5751–58, J.M. Ferrier, 1980,Plant Physiol. 661198–1199), and these were tested by placing recessed-tip pH microelectrodes in the unstirred layer surrounding cells in stagnant solution (bulk pH 8.2, buffered only with 1mm HCO ₃ ^– ). Even as close as 2 m from the cell wall, the pH was typically 7.2 to 7.6 in the acid band center — over 1 pH unitgreater than that suggested by the models for CO₂ entry at the necessary rate for C-fixation. Further evidence for the entry of HCO ₃ ^– , rather than CO₂, at high solution pH was obtained from experiments in which the radial pH gradient in the unstirred layer was reduced. Buffer solutions containing 5mm phosphate or 5mm HEPES, raised the pH at the cell surface in the acid regions from around 7.2 to 7.8 or higher. This pH increase (reduction in acid gradient) would have greatly reduced the CO₂ level at the cell surface and should, therefore, have greatly reduced the CO₂-related¹⁴C-influx. However,¹⁴C-fixation was reduced by only 31% (phosphate) or 15% (HEPES), compared with buffer-free controls. Reduction of the unstirred layer thickness by fast solution flow resulted in a stimulation, and not a reduction, of¹⁴C-fixation. The similarity of our radial pH profiles near the wall with that predicted by the model (Walker et al., 1980) assuming H⁺–HCO ₃ ^– cotransport, together with the effects of buffer, and the results of increased solution flow rate, lead to the conclusion that cotransport of HCO ₃ ^– with H⁺ is the likely method of entry of inorganic carbon. Longitudinal pH profiles of theChara cell were obtained at a distance of 25 m from the wall. These revealed much sharper delineation of the acid and alkaline bands than has previously been possible with miniature pH electrodes. Profiles of local electric field, obtained with a vibrating probe, were in excellent agreement with the high resolution pH profiles. This supports the hypothesis that membrane proton transport has a role (direct) in the generation of the extracellular currents.     

Stimulation of duodenal epithelial HCO3− transport in the guinea pig and cat by luminal prostaglandin E2

Segments of guinea pig or cat duodenum distal to the Brunner gland containing area and devoid of bile or pancreatic secretions were cannulated in situ. The unbuffered luminal solution was gassed with 100% O₂ or N₂ and HCO₃⁻ transport titrated at pH 7.40 or 8.00 with solutions containing HCl. Cat duodenum transported HCO₃⁻ at a greater rate (∼17μeq, cm⁻¹, h⁻¹) than did jejunum in the same animals (∼5μeq, cm⁻¹, h⁻¹) and also developed a greater transmucosal electrical potential difference. Luminal application of PGE₂ (1 – 12 μM) in cat duodenum increased HCO₃⁻ transport and the potential difference. HCO₃⁻ transport by guinea pig duodenum (∼27 μeq, cm⁻¹, h⁻¹) was increased by luminal PGE₂ only in animals where transport had been inhibited by pretreatment with aspirin (30 mg/kg intravenously). Exposure of the cat duodenal lumen to HCl (1 – 25 mM, 5 min) stimulated HCO₃⁻ transport and continuous exposure of duodenum in the guinea pig to acid discharged from the stomach may increase endogenous prostaglandin concentrations, resulting in an apparent lack of effect of exogenous prostaglandins. The present results and previous similar findings in amphibians in vitro suggest that surface epithelial transport of HCO₃⁻ protects duodenal mucosa against acid.     

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.     

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     

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

Summary Bidirectional transepithelial K⁺ flux measurements across high-resistance epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K⁺ flux.Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K⁺.K⁺ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain-and furosemide-insensitive component.The action of furosemide upon K⁺ influx is independent of (Na⁺–K⁺)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K⁺, Na⁺ and Cl^– content. Acetate and nitrate are ineffective substitutes for Cl^–, whereas Br^– is partially effective. Partial Cl^– replacement by NO₃ gives a roughly linear increase in the furosemide-sensitive component. Na⁺ replacement by choline abolishes the furosemide-sensitive component, whereas Li⁺ is a partially effective replacement. Partial Na⁺ replacement with choline gives an apparent affinity of 7mm Na, whereas variation of the external K⁺ content gives an affinity of the furosemide-sensitive component of 1.0mm.Furosemide inhibition is of high affinity (K _1/2=3 m). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K⁺ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective.Externally applied furosemide and Cl^– replacement by NO ₃ ^– inhibit K⁺ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of KK exchange.     

Dehydroascorbic acid uptake and intracellular ascorbic acid accumulation in cultured Müller glial cells (TR-MUL)

Vitamin C is mainly transported across the inner blood–retinal barrier (inner BRB) as dehydroascorbic acid (DHA) via a facilitative glucose transporter (GLUT) 1, and accumulates as ascorbic acid (AA) in the retina. Müller cells, huge glial cells, exhibit passive structural and metabolic functions for retinal neurons and the inner BRB. We characterized DHA transport and its corresponding transporter in a rat Müller cell line (TR-MUL5 cells). [¹⁴C]DHA uptake by TR-MUL5 cells took place in a time-dependent and Na⁺-independent manner. [¹⁴C]DHA uptake was inhibited by substrates and inhibitors of GLUTs, suggesting that Müller cells take up DHA via GLUTs. HPLC analysis revealed that most of the DHA taken up by TR-MUL5 cells was converted to AA and accumulated as AA in TR-MUL5 cells. [¹⁴C]DHA uptake by TR-MUL5 cells took place in a concentration-dependent manner with a Michaelis–Menten constant of 198 μM and was inhibited by cytochalasin B in a concentration-dependent manner with a 50% inhibition concentration of 0.283 μM. Although GLUT1, 3, and 4 mRNA are expressed in TR-MUL5 cells, quantitative real-time PCR revealed that GLUT1 mRNA expression was 5.85- and 116-fold greater than that of GLUT3 and 4, respectively. Western blot analysis supports the expression of GLUT1 protein with 45 kDa in TR-MUL5 cells. In conclusion, DHA is taken up by facilitative glucose transporters, most likely GLUT1, and converted to AA in TR-MUL5 cells.     

Nature of the neutral Na+-Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na+/H+, Cl−/HCO 3 − double exchange,hydrochlorothiazide-sensitive Na+-Cl− symport and Na+-K+-2Cl− cotransport are all involved

Transepithelial fluid transport was measured gravimetrically in rabbit gallbladder (and net Na+ transport was calculated from it), at 27 degrees C, in HCO(3-)-free bathing media containing 10(-4) M acetazolamide. Whereas luminal 10(-4) M bumetanide or 10(-4) M 4-acetamido-4'-iso-thiocyanostilbene-2,2'-disulfonate (SITS) did not affect fluid absorption, 25 mM SCN- abolished it; hydrochlorothiazide (HCTZ) in the luminal medium reduced fluid absorption from 28.3 +/- 1.6 (n = 21) to 8.6 +/- 1.6 microliters cm-2 hr-1 (n = 10), i.e., to about 30%. This maximum effect was already obtained at 10(-3) M concentration; the apparent IC50 was about 2 x 10(-4) M. The residual fluid absorption, again insensitive to SITS, was completely inhibited by SCN- or bumetanide. Cl- influx at the luminal border of the epithelium, measured under the same conditions and corrected for the extracellular space and paracellular influx, proved insensitive to 10(-4) M bumetanide, but was slowly inhibited by 10(-3) M HCTZ, with maximum inhibition (about 54%) reached after a 10-min treatment; it subsequently rose again, in spite of the presence of HCTZ. However, if the epithelium, treated with HCTZ, was exposed to 10(-4) M bumetanide during the measuring time (45 sec), inhibition was completed and the subsequent rise of Cl- influx eliminated. Intracellular Cl- accumulation with respect to the predicted activity value at equilibrium decreased significantly upon exposure to 10(-3) M HCTZ, reached a minimum within 15-30 min of treatment, then rose again significantly at 60 min. Simultaneous exposure to HCTZ and bumetanide decreased the accumulation to a significantly larger extent as compared to HCTZ alone, already in 15 min, and impeded the subsequent rise. Intracellular K+ activity rose significantly within 30 min treatment with HCTZ; the increase proved bumetanide dependent. The results obtained show that Na(+)-Cl- symport, previously detected under control conditions, is the HCTZ-sensitive type; its inhibition elicits bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport. Thus, the three forms of neutral Na(+)-Cl(-)-coupled transport so far evidenced in epithelia, Na+/H+, Cl-/HCO3- double exchange (in the presence of exogenous bicarbonate), HCTZ-sensitive Na(+)-Cl- symport and bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport, are all present in the apical membrane of rabbit gallbladder.     

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.     

Functional roles of Na+ and H+ in SO 4 2− transport by rabbit ileal brush border membrane vesicles

Summary Sulphate uptake by rabbit ileal brush border membrane vesicles was stimulated by a transmembrane sodium gradient ([Na⁺] _o >[Na⁺] _i ), but not by a similar potassium gradient.³⁵SO ₄ ^2– influx (J _oi ^SO4 ) from outside (o) to inside (i) these vesicles was a hyperbolic function of [SO ₄ ^2– ] _o and the affinity constant for anion transport was strongly influenced by [Na⁺] _o (100mm Na⁺,K _t ^SO4 =0.52mm SO ₄ ^2– ; 10mm Na⁺,K _t ^SO4 =4.32mm SO ₄ ^2– ).J _t ^SO4 was a sigmoidal function of [Na⁺] _o at pH 7.4 for both low (0.2m) and high (4.0mm) [SO ₄ ^2– ] _o . The Na⁺-dependency ofJ _t ^SO4 was examined at pH 6.0, 7.4, and 8.0 (same pH inside and outside). At pH 6.0 and 7.4 sigmoidal Na⁺-dependentJ _t ^SO4 exhibited nonlinear Eadie-Hofstee plots indicative of a transport mechanism capable of binding a variable number of sodium ions over the [Na⁺] _o range used. Hill plots of anion transport under these conditions displayed slopes near unity at low [Na⁺] _o and slopes approximating 2.0 at higher cation concentrations. At pH 8.0, Na⁺-dependentJ _t ^SO4 was hyperbolic and showed linear Eadie-Hofstee and Hill plots, the latter with a single slope near 1.0. When a H⁺ gradient was imposed across the vesicle wall (pH _i =8.0, pH _o =6.0), Na⁺-dependentJ _t ^SO4 was hyperbolic and significantly increased at each [Na⁺] _o over values observed using bilateral pH 8.0. In contrast, a H⁺ gradient oriented in the opposite direction (pH _i =6.0, pH _o =8.0) led to Na⁺-dependentJ _t ^SO4 that was sigmoidal and significantly lower at each [Na⁺] _o than values found using bilateral pH 6.0. Electrogenicity ofJ _t ^SO4 at pH 8.0 for both high and low [Na⁺] _o was demonstrated by using a valinomycin-induced transmembrane electrical potential difference. At pH 6.0, electrogenicJ _t ^SO4 occurred only at low [Na⁺] _o (5mm); anion transfer was electroneutral at 50mm Na⁺. A model is proposed for proton regulation of sodium sulphate cotransport where flux stoichiometry is controlled by [H⁺] _i and sodium binding affinity is modified by [H⁺] _o . Preliminary experiments with rabbit proximal tubular brush border membrane vesicles disclosed similarJ _t ^SO4 kinetic properties and a common transport mechanism may occur in both tissues.