A pore-forming toxin produced by Aeromonas sobria activates cAMP-dependent Cl- secretory pathways to cause diarrhea

Aeromonas sobria hemolysin (ASH) is one of the major virulence factors produced by A. sobria, a human pathogen that causes diarrhea. We investigated the effects of ASH on Cl(-) transport in human colonic epithelial cells. ASH increased short-circuit currents (Isc) and (125)I efflux from Caco-2 cells, indicating ASH activate Cl(-) secretion. Additions of inhibitors of cyclic AMP dependent Cl(-) channels, glybenclamide and NPPB suppressed the Isc and (125)I efflux increases induced by ASH. And ASH increased the intracellular cyclic AMP concentration. Moreover, ASH stimulated fluid accumulation in the iliac loop test, and glybenclamide and NPPB suppressed this fluid accumulation. Thus, cAMP-dependent Cl(-) secretory pathway could be related with diarrhea induced by A. sobria.     

海洋浮游藻类无机碳利用机理的研究

为了认识海洋浮游藻类在碳充足和碳受限条件下对水体中溶解无机碳(DIC)的利用方式与可能机理,对13种海洋浮游藻类在不同pH和CO2浓度及不同DIC条件下细胞外碳酸酐酶(CA)的活性进行了分析测定.结果显示:13种藻中,只有Amphidinium carterae和Prorocentrum minimum在碳充足条件下具细胞外CA活性.Melosira sp.、Phaeodactylum tricornutum、Skeletonema costatum、Thalassiosira rotula、Emiliania huxleyi和Pleurochrysis carterae则在碳受限条件下才具细胞外CA活性.Chaetoceros compressus、Glenodinium foliaceum、Coccolithus pelagicus、 Gephrocapsa oceanica和Heterosigma akashiwo即使在碳受限条件下也未检测到细胞外CA活性.应用封闭系统中pH漂移技术和阴离子交换抑制剂4′4′-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS)等的研究表明,Coc. pelagicus和G. oceanica可通过阴离子交换机制进行HCO-3的直接利用.H. akashiwo没有潜在的HCO-3直接利用或细胞外CA催化的HCO-3利用.     

The effects of exogenous extracellular carbonic anhydrase on CO2 excretion in rainbow trout (Oncorhynchus mykiss): role of plasma buffering capacity

The buffering capacity (beta) of rainbow trout (Oncorhynchus mykiss) plasma was manipulated prior to intravascular injection of bovine carbonic anhydrase to test the idea that proton (H+) availability limits the catalysed dehydration of HCO3- within the extracellular compartment. An extracorporeal blood shunt was employed to continuously monitor blood gases in vivo in fish exhibiting normal plasma beta (-3.9+/-0.3 mmol 1(-1) pH unit(-1)), and in fish with experimentally (using N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) elevated plasma beta (-12.1+/-1.1 mmol 1(-1) pH unit(-1)). An injection of 5 mg kg(-1) carbonic anhydrase equally reduced (after 90 min) the arterial partial pressure of CO2 in trout with regular (-0.23+/-0.05 Torr) or high (-0.20+/-0.05 Torr) plasma beta; saline injection was without effect. Because ventilation and venous blood gases were unaffected by carbonic anhydrase, the effect of extracellular carbonic anhydrase in lowering arterial partial pressure of CO2 was likely caused solely by a specific enhancement of CO2 excretion owing to acceleration of HCO3- dehydration within the plasma. The lowering of arterial partial pressure of CO2 in trout after injection of exogenous carbonic anhydrase provides the first in vivo evidence that the accessibility of plasma HCO3- to red blood cell carbonic anhydrase constrains CO2 excretion under resting conditions. Because the velocity of red blood cell Cl-/HCO3- exchange governs HCO3- accessibility to red blood cell carbonic anhydrase, the present study also provides evidence that CO2 excretion at rest is limited by the relatively slow rate of Cl-/HCO3- exchange. The effect of carbonic anhydrase in lowering arterial partial pressure of CO2 was unrelated to plasma buffering capacity. While these data could suggest that H+ availability does not limit extracellular HCO3- dehydration in vivo at resting rates of CO2 excretion, it is more likely that the degree to which plasma beta was elevated in the present study was insufficient to drive a substantially increased component of HCO3- dehydration through the plasma.     

Carbonic anhydrase inhibitors: inhibition of the cytosolic human isozyme VII with anions

An inhibition study of the cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozyme VII (hCA VII) with anions has been conducted. Cyanate, cyanide, and hydrogensulfite were weak hCA VII inhibitors (K(I)s in the range of 7.3-15.2 mM). Cl- and HCO3- showed good inhibitory activity against hCA VII (K(I)s of 0.16-1.84 mM), suggesting that this enzyme is not involved in metabolons with anion exchangers or sodium bicarbonate cotransporters. The best inhibitors were sulfamate, sulfamide, phenylboronic, and phenylarsonic acid (K(I)s of 6.8-12.5 microM).     

Control of flatfish sperm motility by CO2 and carbonic anhydrase

Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25-100 mM HCO3-, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3-. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3(-)-arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/HCO3(-)-dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm.     

Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure

We found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3-. This suggests that these carbonic anhydrases are essential because they maintain HCO3- levels at ambient CO2 concentrations.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Enhanced formation of a HCO3- transport metabolon in exocrine cells of Nhe1-/- mice

Cl(-) influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked Cl(-)/HCO(3)(-) (Ae) and Na(+)/H(+) (Nhe) exchange mechanisms. The dependence of this major Cl(-) uptake pathway on Na(+)/H(+) exchanger expression was examined in the parotid acinar cells of Nhe1(-/-) and Nhe2(-/-) mice, both of which exhibited impaired fluid secretion. No change in Cl(-)/HCO(3)(-) exchanger activity was detected in Nhe2-deficient mice. Conversely, Cl(-)/HCO(3)(-) exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in Cl(-)/HCO(3)(-) exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1(-/-) mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced Cl(-)/HCO(3)(-) exchange activity was accompanied by an increased abundance of Car2.Ae2 complexes in the parotid plasma membranes of Nhe1(-/-) mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2.Ae2 HCO(3)(-) transport metabolon in parotid exocrine cell function. Increased abundance of this HCO(3)(-) transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1(-/-) mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.     

Carbonic anhydrase supports electrolyte transport in Drosophila Malpighian tubules. Evidence by X-ray microanalysis of cryosections

Electron probe X-ray microanalytical studies on the role of carbonic anhydrase in electrolyte transport in the cells of Drosophila Malpighian tubules indicate that carbonic anhydrase delivers protons and bicarbonate ions to ion transport systems in the cell membrane. After injection and after feeding acetazolamide or hydrochlorothiazide, known inhibitors of carbonic anhydrase, the contents of potassium, magnesium and chloride in the apical cytoplasm and in the cytoplasm close to the basal plasma membrane decreased. We explain our measurements by the hypothesis of a basal Mg-H-antiport system in parallel with Cl-HCO(3)-antiport, inhibitable by DIDS. Zinc is supposed to enters cells and intracellular Zn storage vacuoles by a negatively charged Zn-anion-complex in exchange for HCO(3)(-) ions. This antiport is inhibitable by SITS. The content of the Zn storage vacuoles is acid, as shown by red fluorescence after incubation of Malpighian tubules with acridine orange. Red fluorescence is absent after preincubation in a medium containing an inhibitor of carbonic anhydrase. Carbonic anhydrase was demonstrated cytochemically in the Golgi-ER complex, Golgi vesicles and intercellular space. We suppose that carbonic anhydrase is synthesized and stored in the Golgi-ER-complex from where it is released into the tubule lumen.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

不同理化因子对雨生红球藻CG-11碳酸酐酶活性的影响

以雨生红球藻CG-11为实验藻株,探讨在不同CO2、HCO3-、Zn2＋浓度以及pH和氮磷比例条件下,藻细胞的碳酸酐酶活性对这些理化因子的响应。结果表明,通入空气实验组的碳酸酐酶活性最高,为（75.20±1.53）U·mg-1（Chla）,通入5%CO2条件下的碳酸酐酶活性为（9.96±1.43）U·mg-1（Chla）;高浓度HCO3-对碳酸酐酶活性亦具有明显抑制作用,培养液中可溶性无机碳的浓度与碳酸酐酶活性呈负相关;在实验设置的pH范围内,pH9.0时碳酸酐酶活性最高,为（62.32±3.25）U·mg-1（Chla）;适当的氮磷比与Zn2＋浓度显著提高了雨生红球藻CG-11的生长速率,碳酸酐酶的活性亦有明显提高。     

Exchange of labeled bicarbonate and carbon dioxide with erythrocytes suspended in an elutriator

An elutriator was used to study exchange of labeled CO2 and bicarbonate with erythrocytes. Rabbit erythrocytes were suspended by centrifugation in a stream of fluid and exposed to transient injections of an extracellular indicator (125I-albumin or 22Na+), a water indicator (3H2O), and H14CO3- and/or 14CO2. Diffusion of indicators into erythrocytes was judged by comparison of initial concentrations of diffusible and extracellular indicators in the elutriator outflow. It was possible to conduct these experiments at normal hematocrits because any carbonic anhydrase released from erythrocytes by hemolysis was washed away in the elutriator flow, and ambient pH, PO2, and PCO2 were kept constant by the inflow of fresh fluid. Equilibration of HCO3- with erythrocytes was complete during the 7- to 10-s transit time through the chamber. After this exchange was irreversibly inhibited by the anion exchange inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), addition of carbonic anhydrase (100 mg/dl) accelerated exchange, but acetazolamide (20 mg/dl) was without effect. These observations were consistent with the absence of carbonic anhydrase on the surface of the erythrocytes.     

The bicarbonate transport metabolon

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane Cl- /HCO3(-) anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 HCO3(-) transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and Na+/HCO3(-) co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) Cl-/HCO3(-) exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAIV, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon."     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.     

Ouabain-sensitive bicarbonate secretion and acid absorption by the marine teleost fish intestine play a role in osmoregulation

The gulf toadfish (Opsanus beta) intestine secretes base mainly in the form of HCO3- via apical anion exchange to serve Cl- and water absorption for osmoregulatory purposes. Luminal HCO3- secretion rates measured by pH-stat techniques in Ussing chambers rely on oxidative energy metabolism and are highly temperature sensitive. At 25 degrees C under in vivo-like conditions, secretion rates averaged 0.45 micromol x cm(-2) x h(-1), of which 0.25 micromol x cm(-2) x h(-1) can be accounted for by hydration of endogenous CO2 partly catalyzed by carbonic anhydrase. Complete polarity of secretion of HCO3- and H+ arising from the CO2 hydration reaction is evident from equal rates of luminal HCO3- secretion via anion exchange and basolateral H+ extrusion. When basolateral H+ extrusion is partly inhibited by reduction of serosal pH, luminal HCO3- secretion is reduced. Basolateral H+ secretion occurs in exchange for Na+ via an ethylisopropylamiloride-insensitive mechanism and is ultimately fueled by the activity of the basolateral Na+-K+-ATPase. Fluid absorption by the toadfish intestine to oppose diffusive water loss to the concentrated marine environment is accompanied by a substantial basolateral H+ extrusion, intimately linking osmoregulation and acid-base balance.     

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.     

Carbonic anhydrases in chick extra-embryonic structures: a role for CA in bicarbonate reabsorption through the chorioallantoic membrane

The villus cavity cells, a specific cell type of the chick chorioallantoic membrane, express both cytosolic carbonic anhydrase in their cytoplasm and HCO3(-)/Cl(-) anion exchangers at their basolateral membranes. By immunohistochemical analysis, we show here that villus cavity cells specifically react with antibodies directed against the membrane-associated form of carbonic anhydrase, CAIV. Staining is restricted to the apical cell membranes, characteristically invaginated toward the shell membrane, as well as to endothelia of blood vessels present in the mesodermal layer. The occurrence of a membrane-associated CA form at the apical pole of villus cavity cells, when definitively confirmed, would be fairly consistent with the role proposed for these cells in bicarbonate reabsorption from the eggshell so to prevent metabolic acidosis in the embryo during development.     

Swelling and ion uptake in cat cerebrocortical slices:

Cat cerebrocortical slices incubating in medium containing normal K+ concentrations were exposed to a number of different transmitters. Norepinephrine, histamine and adenosine or 2-chloroadenosine caused increased swelling of the slices associated with an increased Na+ and Cl- content. These effects were seen only when both Cl- and HCO3- were present in the medium, and were inhibited by a number of anion transport inhibitors. These characteristics were identical to those of the HCO3(-)-dependent component of the swelling induced by high K+ levels in the medium. Other transmitters, namely 5-hydroxytryptamine, dopamine, and gamma-amino butyric acid, were ineffective. The effects of norepinephrine, histamine and 2-chloroadenosine were antagonised by propranolol and phentolamine, chlorpheniramine and diphenhydramine, and theophylline respectively. These antagonists also inhibited HCO3(-)-dependent, K+-stimulated swelling. The transmitters which induced swelling also stimulated the carbonic anhydrase activity of cerebrocortical slices. We conclude from these data that the HCO3(-)-dependent component of K+-stimulated swelling may be due to K+-stimulated release of transmitters. Furthermore, the fact that the transmitters which induce swelling have also been reported to be most effective in increasing cAMP content in both brain slices or cultured astrocytes is consistent with the swelling response being mediated via cAMP-induced changes and being predominantly localized to astrocytes.     

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

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