Anion transport across the erythrocyte membrane, in situ proteolysis of band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate.

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents. Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a cross-linking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport, Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the noninhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control anion transport.     

Mechanism of the increase in cation permeability of human erythrocytes in low-chloride media. Involvement of the anion transport protein capnophorin

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.     

Conformational change of band 3 protein induced by diethyl pyrocarbonate modification in human erythrocyte ghosts

Diethyl pyrocarbonate inhibited the phosphate exchange across the human erythrocyte membrane. The exchange rate was inhibited only when the membranes were modified with the reagent from the cytosolic surface of resealed ghosts. The intracellular modification by diethyl pyrocarbonate inhibited the extracellular binding of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid to band 3 protein. Furthermore, the extracellular 4,4'-dinitrostilbene-2,2'-disulfonic acid protected the membranes from the intracellular modification by diethyl pyrocarbonate. These results suggest that the extracellular binding of 4,4'-dinitrostilbene-2,2'-disulfonic acid to band 3 protein induces the conformational change of the intracellular counterpart of band 3 protein and the diethyl pyrocarbonate susceptible residue(s) is (are) hidden from the cytosolic surface of the cell membrane in connection with the conformational change. Conversely, under the conditions where the diethyl pyrocarbonate modification is confined to the intracellular side of the membrane, the extracellular binding site of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid is hidden from the cell surface.     

The functional role of arginine 901 at the C-terminus of the human anion transporter band 3 protein

To determine which arginine residues are responsible for band 3-mediated anion transport, we analyzed hydroxyphenylglyoxal (HPG)-modified band 3 protein in native erythrocyte membranes. HPG-modification leads to inhibition of the transport of phosphoenolpyruvate, a substrate for band 3-mediated transport. We analyzed the HPG-modified membranes by reverse phase-HPLC, and determined that arginine 901 was modified by HPG. To determine the role of Arg 901 in the conformational change induced by anion exchange, we analyzed HPG-modification of the membranes when 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethypyrocarbonate (DEPC) was present. DNDS and DEPC fix band 3 in the outward and inward conformations, respectively. HPG-modification was unaffected in the presence of DEPC but decreased in the presence of DNDS. In addition to that, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which specifically reacts with the outward conformation of band 3, did not react with HPG-modified membranes. Furthermore, we expressed a band 3 mutant in which Arg 901 was replaced by alanine (R901A) on yeast membranes. The kinetic parameters indicated that the R901A mutation affected the rate of conformational change of the band 3 protein. From these results, we conclude that the most C-terminal arginine, Arg 901, has a functional role in the conformational change that is necessary for anion transport.     

Chinese hamster ovary cell mutants deficient in an anion exchanger functionally similar to the erythroid band 3

Studies in Chinese hamster ovary cells demonstrate the presence of an anion exchanger, which has some of the properties of the band 3 transporter in erythrocytes. 1) Extracellular chloride is a competitive inhibitor of sulfate influx and stimulates sulfate efflux, suggesting that the mechanism of uptake is SO2-(4)/Cl- exchange. 2) The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate uptake in a dose-dependent manner. Half-maximal inhibition is achieved at 0.06 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. 3) Low extracellular pH markedly stimulates sulfate uptake. A 6-fold decrease in the apparent Km is observed at pHout 5.5 as compared to pHout 7.5. However, studies carried out over a broad range of extracellular SO2-(4) concentrations indicate the presence of three components of this transport activity in Chinese hamster ovary cells: two high affinity low capacity systems, one in the range 0.5 microM less than [SO2-(4)]out less than 50 microM and one in the range 50 microM less than [SO2-(4)]out less than 150 microM, and a low affinity, high capacity system (at [SO2-(4)]out greater than 150 microM). These properties have not been previously reported for the erythroid band 3 transporter. The availability of mutants deficient in these activities has enabled us to carry out studies which suggest that the high affinity systems are functionally independent of the low affinity system, but that all systems are dependent on the same anion exchange protein. Studies in a mutant which lacks all components of the transport activity indicates that the anion exchanger may be instrumental in the regulation of the intracellular pH in Chinese hamster ovary cells.     

Inhibition of phosphate transport in human erythrocytes by water-soluble carbodiimides

Phosphate entry into human erythrocytes is irreversibly inhibited by treatment of the cells with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMC) in the absence of added nucleophile. EDC is the more potent inhibitor (40% inhibition, 2 mM EDC, 5 min, 37 degrees C, 50% hematocrit, pH 6.9), while more than 20 mM CMC is required to give the same inhibition under identical conditions. EDC inhibition is temperature-dependent, being complete in 5 min at 37 degrees C, and sensitive to extracellular pH. At pH 6.9 only 50% of transport is rapidly inhibited by EDC, but at alkaline pH over 80% of transport is inhibited. Inhibition is not prevented by modification of membrane sulfhydryl groups but is decreased in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), a reversible competitive inhibitor of anion transport. EDC treatment leads to crosslinking of erythrocyte membrane proteins, but differences between the time course of this action and inhibition of transport indicate that most transport inhibition is not due to crosslinking of membrane proteins.     

Characterization of the chicken erythrocyte anion exchange protein

The avian erythrocyte anion exchange protein (band 3), after labeling with [3H2]4,4'-diisothiocyanodihydrostilbene-2, 2'-disulfonic acid appears as a doublet of polypeptide chains with apparent Mr = 105,000 and 100,000 by sodium dodecyl sulfate gel electrophoresis. The structures of the two species are almost identical as determined by partial proteolysis. The copy number of band 3 molecules per chicken erythrocyte was determined to be 800,000 by quantitating the amount of [3H2]4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid covalently bound to the cell surface. A comparison of human and chicken band 3 has revealed differences in their structure. Chicken band 3 differs from the human polypeptide in isoelectric point and proteolytic patterns. Antisera raised against human and chicken band 3 do not cross-react, implying that the two sera do not recognize any common antigenic determinants. There is a 6.5-fold lower activity per cell in the rate of phosphate exchange in the chicken erythrocyte which can be entirely explained by the 1.5-fold decrease in copy number per cell and the increased size of the chicken erythrocyte. This would suggest that there is no difference in the enzyme turnover number between chicken and human band 3. A major functional difference resulting from the structural differences is the inability to bind glyceraldehyde-3-phosphate dehydrogenase, a function associated with the NH2 terminus of human band 3.     

pH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally HCO3(-)-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10-20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1-2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to HCO3(-)-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in HCO3(-)-buffered media was inhibited by 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid and was slowed after exposure to Cl(-)-free media. Following inhibition of acid extrusion by amiloride, transition to HCO3- media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+-H+ exchange and an independent HCO3(-)-dependent mechanism. Evidence for HCO3(-)-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.     

Haemolysis of various mammalian erythrocytes in sodium chloride, glucose and phosphate-buffer solutions.

Mouse, rat, rabbit, hamster, cow, pig, sheep, guinea-pig, dog and human erythrocytes were studied. A 0.9% or stronger solution of sodium chloride completely prevented haemolysis; sheep and pig erythrocytes appeared the more fragile, while human and dog erythrocytes were not haemolized in concentrations of 0.4% or more. Haemolysis of human, rabbit, cow, hamster, guineapig, pig and sheep erythrocytes was not observed in solutions of 0.4% or more of glucose. Except for sheep, human and dog erythrocytes, haemolysis was depressed in rate but not completely prevented by phosphate-buffer solution of pH 7.0.     

Lysine 539 of human band 3 is not essential for ion transport or inhibition by stilbene disulfonates

The anion transporter from human red blood cells, band 3, has been expressed in Xenopus laevis frog oocytes microinjected with mRNA prepared from the cDNA clone. About 10% of the protein is present at the plasma membrane as determined by immunoprecipitation of covalently bound 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) with anti-DIDS antibody. The expressed band 3 transport chloride at a rate comparable to that in erythrocytes. Transport of chloride is inhibited by stilbene disulfonates, niflumic acid, and dipyridamole at concentrations similar to those that inhibit transport in red blood cells: DIDS and 4,4'-dinitro-2,2'-stilbene disulfonate inhibit chloride uptake with Kiapp of 34 nM and 2.5 microM, respectively. Lysine 539 has been tentatively identified as the site of stilbene disulfonate binding. Site-directed mutagenesis of this lysine to five different amino acids has no effect on transport. Inhibition by stilbene disulfonates or their covalent binding was not affected when Lys-539 was substituted by Gln, Pro, Leu, or His. However, substitution by Ala resulted in weaker inhibition and covalent binding. These results indicate that lysine 539 is not part of the anion transport site and that it is not essential for stilbene disulfonate binding and inhibition.     

Extracellular sodium is required for methacholine-induced secretion of mucus glycoconjugates from canine tracheal explants

Extracellular sodium is known to influence secretion by certain secretory cells, possibly by mobilizing calcium from cellular stores or by altering intracellular pH via regulation of a Na(+)-H+ antiport system. Using canine tracheal explants, we determined whether agents which alter sodium fluxes are capable of modulating basal or cholinergically-induced secretion of mucus glycoconjugates. Methacholine, a cholinergic agonist, increased mucus secretion from explants incubated in the presence or absence of calcium, but had no effect on secretion when incubated in sodium-deficient media, indicating (a) that cholinergically-induced secretion can be mediated by mobilization of cellular calcium and (b) that extracellular sodium was required for this stimulatory effect. Several agents which increase intracellular sodium were tested for their effect on mucus secretion. Ouabain, a sodium pump inhibitor, and veratridine, a sodium channel activator, did not significantly affect control or methacholine-induced secretion; gramicidin, a sodium ionophore, also had no effect on basal release. Tetrodotoxin, a sodium channel inhibitor, was also without effect on basal or methacholine-stimulated mucus release. Agents which alter intracellular pH were also examined for their effects on basal or methacholine-induced glycoconjugate secretion. Amiloride, which decreases intracellular pH by inhibiting Na(+)-H+ exchange, produced a 19 per cent increase in basal secretion (not statistically significant), but had no effect on methacholine-induced secretion. An agent, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which decreases intracellular pH by inhibiting HCO3(-)-Cl- exchange, elicited decreases in both basal and methacholine-induced secretion, but the inhibition did not reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of murine band 3 protein-mediated Cl- transport as measured in mouse red blood cells and in oocytes of Xenopus laevis

Murine band 3 protein was expressed in oocytes of Xenopus laevis after microinjection of the mRNA from the spleens of anemic mice. The 36Cl- efflux from the oocytes was compared with the chloride fluxes measured in murine red cells. In both oocytes and red cells, the band 3-mediated chloride transport showed the following features: the selective inhibitor of band 3-mediated anion transport, 4,4'-dinitrostilbene-2,2'-disulfonate exerts its effects only when applied to the outside and not when applied to the inside of the membrane. The K1/2 for inhibition by external 4,4'-dinitrostilbene-2,2'-disulfonate was of the order of 1.5 to 2.0 mumol/l. Flufenamate and persantine also produce similar inhibitory effects. Decreasing the pH from 7.4 to 6.0 leads to some inhibition. It is concluded that essential features of the mode of action of murine erythroid band 3 protein in the plasma membrane of the oocyte are similar to the mode of action in the bilayer of the red blood cell of the mouse.     

Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.     

Anion transport regulates intracellular pH in renal cortical tissue

The regulation of cell pH by anion transport was examined in suspensions of rabbit renal proximal tubules. Values for cell pH were derived from 14C-labeled 5,5-dimethyloxazolidine-2,4-dione distribution. In buffer with 10 mM/l HCO3-- and gassed with 95% O2/5% CO2, the anion transport inhibitors, 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene and furosemide, raised the cell-to-extracellular pH gradient from 0.23 +/- 0.02 to 0.31 +/- 0.02 and 0.31 +/- 0.03, respectively, but in combination their effects were not additive. Replacement of extracellular Cl-- by NO3-- raised the pH gradient from 0.24 +/- 0.04 to 0.37 +/- 0.05. Neither inhibitor raised the pH gradient in Cl-- -free media. Incubation of suspensions in HCO3-- and CO2-free media raised the pH gradient from 0.18 +/- 0.02 to 0.29 +/- 0.03. Removal of Cl-- in addition to HCO3-- and CO2 raised the pH gradient still further, to 0.36 +/- 0.02. The results demonstrate that two different anion transport inhibitors raise cell pH and the cell-to-extracellular pH gradient in proximal tubules and are consistent with the idea that the mechanism for this effect is inhibition of alkali anion exit from the tubule cell. This process appears to depend on extracellular Cl-- and probably occurs primarily by HCO3-- transport. The results support the concept that alkali anion transport, most probably HCO3-- exit from the peritubular cell border, is an important regulator of cell pH in renal proximal tubule.     

Entrapment of protein protease inhibitors in red blood cells.

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.     

Control of red cell urea and water permeability by sulfhydryl reagents

The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.     

Histidine-834 of human erythrocyte band 3 has an essential role in the conformational changes that occur during the band 3-mediated anion exchange

We have shown that diethyl pyrocarbonate (DEPC) inhibits band 3-mediated anion exchange and that the inhibition occurs only when histidine residue(s) is (are) modified with DEPC from the cytosolic surface of resealed ghosts [Izuhara et al. (1989) Biochemistry 28, 4725-4728]. In the present study, we have identified the DEPC-modified histidine residue as His834 using liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). This mild, rapid, sensitive, and quantitative method was successfully applied to analysis of the unstable DEPC-histidine adduct. The DEPC modification of His834 was pH dependent and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) sensitive as previously shown. After DEPC modification, band 3-mediated anion exchange is inhibited. Consistent with previous results, we confirmed that His834 was located on the cytosolic side of the membrane and the DEPC modification of His834 had allosteric effects on the extracellular DNDS-binding site of band 3. Therefore, we conclude that His834 is located at the cytosolic surface of band 3 and is an essential residue for band 3-mediated anion exchange. We will discuss important roles of the region from TM12 to TM14 in the conformational changes that occur during the band 3-mediated anion exchange.     

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.     

Effects of 4-acetamido-4'-isothiocyano-2,2-disulfonic stilbene on ion transport in turtle bladders.

The disulfonic stilbene (4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO-3,Cl-) moiety of the short-circuiting current is eliminated by 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene, but only after its addition to the serosal bathing fluid. Whereas 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene has no effect on Na+ transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na+ + K+)-ATPase of both turtle bladder and eel electric organ.     

The role of HCO3- and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation

The intracellular pH (pHi) of a rat parotid acinar preparation was monitored using the pH-sensitive fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Under resting (unstimulated) conditions both Na+/H+ exchange and CO2/HCO3- buffering contribute to the regulation of pHi. Muscarinic stimulation (carbachol) of the acini produced a gradual rise in pHi (approximately 0.1 unit by 10 min) possibly due to activation of the Na+/H+ exchanger. When the exchanger was blocked by amiloride or sodium removal, carbachol induced a dramatic (atropine inhibitable) decrease in pHi (approximately 0.4 pH unit with t1/2 approximately 0.5 min at 1 mM carbachol). The rate of this acidification was reduced by removal of exogenous HCO3- and by the carbonic anhydrase inhibitor methazolamide. Also, acini stimulated with carbachol in Cl- -free solutions showed a more pronounced acidification than in the corresponding Cl- -replete media. Taken together, these data indicate that the carbachol-induced acidification of rat parotid acinar cells unmasked by inhibition of the Na+/H+ exchanger is due to a rapid loss of intracellular HCO3-. Carbachol induced acidification was inhibited by the Cl- channel blocker diphenylamine 2-carboxylate but not by 4-acetomido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Cl-/HCO3- exchange. In addition, this acidification could not be sustained in Ca2+-free media and was totally blocked by chelation of intracellular Ca2+. Interpreted in terms of HCO3- loss, these results closely parallel the pattern of carbachol-induced Cl- release from this same preparation and indicate that HCO3- is secreted in response to muscarinic stimulation via the same or a very similar exit pathway, presumably an apical anion channel. Under normal physiological conditions the intracellular acidification resulting from HCO3- secretion is buffered by the Na+/H+ exchanger.