The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl^–/HCO ₃ ^– exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl^–/HCO ₃ ^– exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl^–/HCO ₃ ^– exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CA carbonic anhydrase - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - E ₀ total concentration of free enzyme - i fractional inhibition of enzyme activity - IU international units - K ₁ inhibition constant - K _M Michaelis constant - NBT nitro blue tetrazolium - NCP nitrocellulose paper - RBC red blood cell - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max maximal velocity of reaction     

Evidence for involvement of protein kinase C in regulation of intracellular pH by Cl−/HCO 3 − antiport

Summary The activity of the main base-extruding mechanism in Vero cells, the Na⁺-independent Cl^–/HCO ₃ ^– antiport, increases 5- to 10-fold when the cytosolic pH (pH _i ) is increased over a narrow range close to neutrality. We have studied the effect on this regulation of stimulation and inhibition of protein kinase C by short-term and long-term treatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). After short-term treatment with TPA to stimulate the kinase, the threshold value for activation of the antiport is shifted to a more acidic pH. After prolonged treatment with TPA to downregulate protein kinase C the sensitivity of the antiport to variation in proton concentration was lowered, possibly by reducing the number of essential protonbinding sites. Concomitantly, the steady state pH _i of the cells was increased. The data indicate that protein kinase C is involved in the regulation of the Na⁺-independent Cl^–/HCO ₃ ^– antiport.     

Characterization of carbonic anhydrase and anion exchange in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish

The purpose of this study was to investigate the characteristics of carbonic anhydrase (CA) and the Cl⁻/HCO₃⁻ exchanger (Band 3; AE1) in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish, in order to further understand the strategies of blood CO₂ transport in lower vertebrates and gain insights into the evolution of the vertebrate erythrocyte proteins, CA and Band 3. A significant amount of CA activity was measured in the erythrocytes of bowfin (70 mmol CO₂ min⁻¹ ml⁻¹), although it appeared to be lower than that in the erythrocytes of teleost fish. The turnover number (K_cat) of bowfin erythrocyte CA was intermediate between that of the slow type I CA isozyme in agnathans and elasmobranchs and the fast type II CA in the erythrocytes of the more recent teleost fishes, but the inhibition properties of bowfin erythrocyte CA were similar to the fast mammalian CA isozyme, CA II. In contrast to previous findings, a plasma CA inhibitor was found to be present in the blood of bowfin. Bowfin erythrocytes were also found to possess a high rate of Cl⁻/HCO₃⁻ exchange (6 nmol HCO₃⁻ s⁻¹ cm⁻²) that was sensitive to DIDS. Visualization of erythrocyte membrane proteins by SDS-PAGE revealed a major band in the 100 kDa range for the trout, which would be consistent with the anion exchanger. In contrast, the closest major band for the membranes of bowfin erythrocytes was around the 140 kDa range. Taken together, these results suggest that the strategy for blood CO₂ transport in bowfin is probably similar to that in most other vertebrates despite several unique characteristics of erythrocyte CA and Band 3 in these primitive fish.     

pHi regulation in Ehrlich mouse ascites tumor cells: Role of sodium-dependent and sodium-independent chloride-bicarbonate exchange

pH _i recovery in acid-loaded Ehrlich ascites tumor cells and pH _i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO ₃ ^– -free media and at 25 mm HCO ₃ ^– , the measured pH _i (7.26 and 7.82, respectively) was significantly more alkaline than the pH _i . value calculated assuming the transmembrane HCO ₃ ^– gradient to be equal to the Cl^– gradient. Thus, pH _i in these cells is not determined by the Cl^– gradient and by Cl^–/HCO ₃ ^– exchange.pH _i recovery following acid loading by propionate exposure, NH ₄ ⁺ withdrawal, or CO₂ exposure is mediated by amiloride-sensitive Na⁺/H⁺ exchange in HCO₃ ^– free media, and in the presence of HCO ₃ ^– (25 mm) by DIDS-sensitive, Na⁺-dependent Cl^–/HCO ₃ ^– exchange. A significant residual pH _i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H⁺ pump in pH _i regulation. pH _i maintenance at steady-state involves both Na⁺/H⁺ exchange and Na⁺-dependent Cl^–/HCO ₃ ^– exchange.Acute removal of external Cl^– induces a DIDS-sensitive, Na⁺-dependent alkalinization, taken to represent HCO ₃ ^– influx in exchange for cellular Cl^–. Measurements of ³⁶Cl^– efflux into Cl^–-free gluconate media with and without Na⁺ and/or HCO ₃ ^– (10 mm) directly demonstrate a DIDS-sensitive, Na⁺ dependent Cl^–/HCO ₃ ^– exchange operating at slightly acidic pH _i (pH_o 6.8), and a DIDS-sensitive, Na⁺-independent Cl^–/HCO ₃ ^– exchange operating at alkaline pH _i (pH _o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).     

The mycotoxin ochratoxin A deranges pH homeostasis in Madin-Darby canine kidney cells

Ochratoxin A (OTA) is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Added to the culture medium, OTA transforms MDCK cells in a manner similar to exposure to alkaline stress. By means of video-imaging and microelectrode techniques, we investigated whether OTA (1 mol/liter) affects intracellular pH (pH.), Cl^– (Cl _i ^– ) or cell volume of MDCK cells acutely exposed to normal (pH_norm=7.4) and alkaline (pH_alk=7.7) conditions. At pH_norm, OTA increased Cl _i ^– by 2.6±0.4 mmol/liter (n=14, P<0.05) but had no effect on pH _i . At pH_alk, application of OTA increased Cl _i ^– by 8.6±2.6 mmol/liter (n=10, P< 0.05) and raised pH _i by 0.11±0.03 (n= 8, P<0.05). The Cl^–HCO ₃ ^– exchange inhibitor DNDS (4,4-dinitro-stilbene-2, 2-disulfonate; 10 mol/liter) eliminated the OTA-induced changes of pH _i and Cl _i ^– . OTA did not affect cell volume under both pH_norm and pH_alk conditions.We conclude that the OTA-induced blockade of plasma membrane anion conductance increases Cl _i ^– without changing cell volume. The driving force of plasma membrane Cl^–/HCO ₃ ^– exchange dissipates, leading to a rise of pH _i when cells are exposed to an acute alkaline load. Thus, OTA interferes with pH _i and Cl _i ^– homeostasis leading to morphological and functional alterations in MDCK cells.The work was supported by the Deutsche Forschungsgemeinschaft (DFG, Si 170/7-1).We thank the Zeiss Company (Oberkochen, Germany) for providing the Attofluor video-imaging system for the intracellular Ca²⁺ measurements.This study was carried out with the technical assistance of Sigrid Mildenberger and Ruth Freudinger.     

Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO ₃ ^– /Cl^– exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO₂ to HCO ₃ ^– . CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.This work was supported in part by a grant-in-aid from the American Heart Association, by the Squibb Institute for Medical Research and by The Council for Tobacco Research.We should like to express our thanks to Dr. I.M. Wiener for kindly supplying us with the impermeable sulfonamide, ZBI, which we used in preliminary experiments and to Dr. T.H. Maren for analysis of a sample of BCA II.     

The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels

Summary— Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO₃^? ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl^?/HCO₃^? antiport or a simple HCO₃^? channel. The stilbene derivatives 4-acetamido-4 isothiocyano-2-2′-disulfonic acid (SITS) and 4,4′ diisothiocyano stilbene-2,2′ disulfonic acid (DIDS) reduced both the number of domes and the Cl^? and HCO₃^? flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membranes using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO₃^? ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO₃^? secretion at the cellular and molecular levels.     

Mechanism of acquisition of exogenous bicarbonate by internodal cells of Chara corallina

Electrophysiological measurements on internodal cells of the alga, Chara corallina Klein ex Willd., em. R.D.W., showed that the potential across the plasmalemma was sensitive to the level of exogenous HCO ₃ ^- . In alkaline solutions (pH 8) the membrane potential depolarized by 50–75 mV when exogenous HCO ₃ ^- was removed from the bathing medium. In the presence of exogenous HCO ₃ ^- , the membrane potential rapidly hyperpolarized when the cell was given a brief dark treatment; in the light the potential was approx.-240 mV; after the cell had been in the dark for 3–6 min the potential was -330 to -350 mV. In the absence of exogenous HCO ₃ ^- the potential only hyperpolarized slowly and to a much smaller extent when cells were placed in the dark. Upon re-illuminating the cell, the potential further hyperpolarized, transiently, and then rapidly depolarized back towards the light-adapted value. (These responses were only obtained when cells were not perturbed by microelectrode insertion into the vacuole.) Analysis of membrane potential and experiments with the extracellular vibrating electrode indicated a high level of correlation between the light- and dark-induced changes in membrane potential and extracellular currents. However, when experiments were conducted in HCO ₃ ^- -free media that contained 1.0 mM phosphate buffer, pH 8, it was found that the dark-induced hyperpolarization of the membrane potential and the light-dependent extracellular currents could be maintained in the absence of exogenous HCO ₃ ^- . These results are interpreted in terms of two basic models by which internodal cells of C. corallina may acquire exogenous HCO ₃ ^- for photosynthesis. They are consistent with HCO ₃ ^- being transported across the plasmalemma via an electrically neutral HCO ₃ ^- –H⁺ cotransport system. The hyperpolarizing response is thought to be the consequence of the operation of an electrogenic H⁺-translocating ATPase that has a transport stoichiometry of 1 H⁺ per ATP hydrolyzed.Abbreviation CPW/B artificial Chara pond water containing exogenous bicarbonate     

pH Regulation in tissue-cultured bovine lens epithelial cells

Summary The intracellular pH (pH _i ) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO ₃ ^- and 5% CO₂, pH 7.25, pH _i was 7.19±0.02 (sem, n = 95). On removing HCO ₃ ^- and CO₂ there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO ₃ ^- and CO₂ led to a rapid and pronounced fall in pH. Exposure to Na⁺-free AAH again led to a marked fall in pH _i , but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl^– in the presence of HCO ₃ ^- led to a rise in pH _i , while substitution in the absence of HCO ₃ ^- led to a fall in pH _i . The above data indicate a significant role for a sodium-dependent Cl^–-HCO ₃ ^- exchange mechanism in the regulation of pH _i . Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO ₃ ^- led to a marked fall in pH _i , indicating that a Na⁺/H⁺ exchange mechanism also has a significant role in the regulation of pH _i . There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH _i . Larger changes in pH _i were observed in control compared to HCO ₃ ^- -free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH _i with either NH ₄ ⁺ or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO ₃ ^- was 5 mm/pH unit (procaine). However, in the presence of HCO ₃ ^- and CO₂ the buffer capacity increases approximately fourfold, indicating that HCO ₃ ^- is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship.     

Rapid increase in pH set-point of the Na+-in-dependent chloride/bicarbonate antiporter in vero cells exposed to heat shock

Internal pH (pH _i ) is in Vero cells regulated mainly by three antiports. Na⁺/H⁺ antiport and Na⁺-dependent Cl⁺/HCO ₃ ⁺ antiport increase pH _i in acidified cells, and Na⁺-independent Cl⁺/HCO ₃ ⁺ antiport lowers pH _i in cells after alkalinization. The activities of the antiporters were altered in cells after exposure to 41–45°C. Under such conditions the Na⁺/H⁺ antiport and the Na⁺-dependent Cl⁺/HCO ₃ ⁺ antiport were both stimulated, whereas the Na⁺-independent Cl⁺/HCO ₃ ⁺ antiport was inhibited in such a way that a higher pH value was required to activate it. This alteration was also induced by some other forms of cellular stress, but did most likely not involve stress proteins as protein synthesis was not required. The possibility of regulation by alteration in protein phosphorylation is discussed.We are grateful to Mrs. Jorunn Jacobsen for her skillful handling of the cell cultures. This work was supported by the Norwegian Research Council for Science and Humanities, the Norwegian Cancer Society and the Lærdal Foundation.     

Evidence for an anion exchanger in the mouse lacrimal gland acinar cell membrane

Summary Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H⁺ (pH_i) and Cl^– (aCl_i) activities with double-barreled ion-selective microelectrodes. In a HCO ₃ ^– -free solution of pH 7.4 (HEPES/Tris buffered), pH_i was 7.25 andaCl_i was 33mm. By an exposure to a HCO ₃ ^– (25mm HCO ₃ ^– /5% CO₂, pH 7.4) solution for 15 min,aCl_i was decreased to 25mm and pH_i was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO ₃ ^– concentration [HCO ₃ ^– ]_i, calculated by the Henderson-Hasselbalch's equation, was 11mm at 1 min after the exposure and then slowly increased to 15mm. Readmission of the HCO ₃ ^– -free solution reversed the changes inaCl_i and pH_i. The intracellular buffering power was about 40mm/pH. An addition of DIDS (0.2mm) significantly inhibited the rates of change inaCl_i, pH_i, and [HCO ₃ ^– ]_i caused by admission/withdrawal of the HCO ₃ ^– , solution and decreased the buffer value. Replacement of all Cl^– with gluconate in the HCO ₃ ^– solution increased pH_i, and readmission of Cl^– decreased pH_i. The rates of these changes in pH_i were reduced by DIDS by 32–45% but not by amiloride (0.3mm). In the HCO ₃ ^– solution, a stimulation of intracellular HCO ₃ ^– production by exposing the tissue to 25mm NH ₄ ⁺ increasedaCl_i significantly. While in the HCO ₃ ^– -free solution or in the HCO ₃ ^– , solution containing DIDS, exposure to NH ₄ ⁺ had little effect onaCl_i. All of these findings were consistent with the presence of a reversible, disulfonic stilbene-sensitive Cl^–/HCO ₃ ^– exchanger in the basolateral membrane of the acinar cells. The possibility of anion antiport different from one-for-one Cl^–/HCO ₃ ^– exchange is discussed.     

Mechanisms of Cl− uptake through brush border membranes isolated from the posterior intestine of the freshwater trout,Oncorhynchus mykiss,R.

Summary This paper presents a study of the mechanisms of Cl^– transport through the brush border membranes of the posterior part of the intestine in the freshwater trout, Oncorhynchus mykiss. The mechanisms for Cl^– transport in the posterior intestine are distinct from those in the middle intestine; an inwardly directed pH gradient stimulates Cl^– uptake by bursh border membrane vesicles, indicating a Cl^–/OH^– exchange. A pH-regulated Cl^– conductance is present, which is not activated at normal intracellular pH. Cl^– uptake is stimulated by an outwardly directed HCO ₃ ^– gradient revealing the presence of a Cl^–/HCO ₃ ^– exchange but, conversely, Cl^– is not exchanged against SO ₄ ^2- . In addition, carbonic anhydrase activities have been detected in both the intracellular and extracellular leaflets of the bursh border membranes which favour the establishment of a bicarbonate gradient. A model of Cl^– transport mechanisms through the brush-border membranes of the posterior intestine of the freshwater trout is proposed.Abbreviations BBM brush border membrane - CA carbonic anhydrase - EGTA ethylene-bis(oxyethylenenitrilo)tetra-acetic acid - FW fresh water - Hepes N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulphonic acid - Mes 2-(N-morpholino)ethane sulphonic acid - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid - TEA triethanolamine - TMA tetramethylammonium - TRIS tris(hydroxymethyl)aminomethane     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Effects of salicylate on HCO 3 − /Cl− exchange across the human erythrocyte membrane

Summary Changes in extracellular pH (pH _o ) in human red cell suspensions were monitored in a stopped-flow rapid reaction apparatus. A 20% suspension of washed human RBC in saline at pH 7 containing NaHCO₃ and extracellular carbonic anhydrase was mixed with an equal volume of buffered saline solution at pH 6.7. Sodium salicylate, when present, was added to both the erythrocyte suspension and the buffer solution. The effects of salicylate in the therapeutic to toxic concentration range on HCO ₃ ^– /Cl^– exchange were studied at 37°C. HCO ₃ ^– /Cl^– exchange flux was estimated using the extracellular buffer capacity and the difference betweendpH _o /dt using a control RBC suspension and that using a suspension of RBC whose anion exchange pathway was markedly inhibited. The results show that salicylate competitively decreases the rate of HCO ₃ ^– /Cl^– exchange, with inhibition increasing as salicylate concentration increases.K _I is 2.4mm. At a salicylate concentration of 10mm, HCO ₃ ^– /Cl^– exchange under the conditions of our experiments was inhibited by more than 70%. These findings are consistent with the possibility that CO₂ transfer in capillary bedsin vivo may be diminished in the presence of salicylate due to slowing of red cell HCO ₃ ^– /Cl^– exchange.     

Effect of anisotonic media on volume,ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture

Summary Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K⁺, Cl^– and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K⁺ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na⁺ and Cl^– increase and followed by a slow recovery of the initial K⁺ and Na⁺ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na⁺ and Cl^– conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K⁺ and Cl^– content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca²⁺ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl^– by nitrate. When cells in a monolayer were incubated in a hypotonic K₂SO₄ medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K⁺ and Na⁺ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca²⁺ concentration.     

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

Mechanisms of Na and Cl absorption across the distal colon epithelium of the pig

Summary Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (I_sc) ranged from-15 to 220 A·cm^-2. Variations in basal I_sc resulted from differences in the amount of amiloride (10M mucosal addition)-sensitive Na⁺ absorption. Ion substitution and transepithelial flux experiments showed that 10 M amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on I_sc was independent of Cl^- and HCO ₃ ^- replacement. When the concentration of mucosal amiloride was increased from 10 to 100 M, little change in I_sc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the I_sc. The decrease in I_sc due to 1 mM amiloride was dependent on both Cl^- and HCO ₃ ^- , and was attributed to reductions in the M-S and net Na⁺ fluxes as well as the M-S unidirectional Cl^- flux. Ion replacement experiments demonstrated that Cl^- substitution reduced the M-S and net Na fluxes, while replacement of HCO ₃ ^- with HEPES abolished net Cl^- absorption by reducing the M-S unidirectional Cl^- flux. From these data it can be concluded that: (1) Na⁺ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl^- absorption is completely HCO ₃ ^- -dependent (presumably mediated by Cl^-/HCO ₃ ^- exchange) and occurs independently of Na⁺ absorption.Abbreviations G_t tissue conductance - HEPES tris (hydroxymethyl) aminomethane - (tris) N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - I_sc short-circuit current - J_r residual flux - M-S mucosal-to-scrosal - S-M serosal-to-mucosal - TTX tetrodotoxin     

Fusion of cultured dog kidney (MDCK) cells: II. Relationship between cell pH and K+ conductance in response to aldosterone

Summary We have chosen the MDCK cell line to investigate aldosterone action on H⁺ transport and its role in regulating cell membrane K⁺ conductance (G _m ^K ). Cells grown in a monolayer respond to aldosterone indicated by the dose-dependent formation of domes and by the alkalinization of the dome fluid. The pH sensitivity of the plasma membrane K⁺ channels was tested in giant cells fused from individual MDCK cells. Cytoplasmic pH (pH _i ) andG _m ^K were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep signoidal relationship between pH _i andG _m ^K (Hill coefficient 4.4±0.4), indicating multiple H⁺ binding sites at a single K⁺ channel. Application of aldosterone increased pH _i within 120 min from 7.22±0.04 to 7.45±0.02 and from 7.15±0.03 to 7.28±0.02 in the absence and presence of the CO₂/HCO ₃ ^– buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO₂/ HCO ₃ ^– is limited by the increased activity of a pH _i -regulating HCO ₃ ^– extrusion system. SinceG _m ^K is stimulated half-maximally at the pH _i of 7.18±0.04, internal H⁺ ions could serve as an effective intracellular signal for the regulation of transepithelial K⁺ flux.     

Pathways for bicarbonate transfer across the serosal membrane of turtle urinary bladder: Studies with a disulfonic stilbene

Summary Bicarbonate is transferred across the serosal (S) membrane of the epithelial cells of the turtle bladder in two directions. Cellular HCO ₃ ^– generated behind the H⁺ pump moves across this membrane into the serosal solution. This efflux of HCO ₃ ^– is inhibited by SITS (4-isothiocyano-4-acetamido-2,2-disulfonic stilbene). When HCO ₃ ^– is added to the serosal solution it is transported across the epithelium in exchange for absorbed Cl^–. This secretory HCO ₃ ^– flow traverses the serosal cell membrane in the opposite direction. In this study the effects of serosal addition of 5×10^–4 m SITS on HCO ₃ ^– secretion and Cl^– absorption were examined. The rate of H⁺ secretion was brought to zero by an opposing pH gradient, and 20mm HCO ₃ ^– was added toS. HCO ₃ ^– secretion, measured by pH stat titration, was equivalent to the increase inMS Cl^– flux after HCO ₃ ^– addition. Neither theSM flux of HCO ₃ ^– nor theMS flux of Cl^– were affected by SITS. In the absence of electrochemical gradients, net Cl^– absorption was observed only in the presence of HCO ₃ ^– in the media; under such conditions, unidirectional and net fluxes of Cl^– were not altered by serosal or mucosal SITS. H⁺ secretion, however, measured simultaneously as the short-circuit current in ouabain-treated bladders decreased markedly after serosal SITS. The inhibition of the efflux of HCO ₃ ^– in series with the H⁺ pump and the failure of SITS to affect HCO ₃ ^– secretion and Cl^– absorption suggest that the epithelium contains at least two types of transport systems for bicarbonate in the serosal membrane.     

Osmolarity-sensitive release of free amino acids from cultured kidney cells (MDCK)

Summary The amino acid pool of MDCK cells was essentially constituted by alanine, glycine, glutamic acid, serine, taurine, lysine, -alanine and glutamine. Upon reductions in osmolarity, free amino acids were rapidly mobilized. In 50% hyposmotic solutions, the intracellular content of free amino acids decreased from 69 to 25mm. Glutamic acid, taurine and -alanine were the most sensitive to hyposmolarity, followed by glycine, alanine and serine, whereas isoleucine, phenylalanine and valine were only weakly reactive. The properties of this osmolarity-sensitive release of amino acids were examined using³H-taurine. Decreasing osmolarity to 85, 75 or 50% increased taurine efflux from 0.6% per min to 1.6, 3.5 and 5.06 per min, respectively. The time course of³H-taurine release closely follows that of the regulatory volume decrease in MDCK cells. Taurine release was unaffected by removal of Na⁺, Cl^– or Ca²⁺, or by treating cells with colchicine or cytochalasin. It was temperature dependent and decreased at low pH. Taurine release was unaffected by bumetanide (an inhibitor of the Na⁺/K⁺/2Cl^– carrier); it was inhibited 16 and 67 by TEA and quinidine (inhibitors of K⁺ conductances), unaffected by gadolinium or diphenylamine-2-carboxylate (inhibitors of Cl^– channels) and inhibited 50% by DIDS. The inhibitory effects of DIDS and quinidine were additive. Quinidine but not DIDS inhibited taurine uptake by MDCK cells.