Role Of Carbonic Anhydrase Research Articles

The roles of carbonic anhydrase in metabolism, cell growth and cancer in animals.

Viewed from the standpoint of chemical reactivity, CO2 would appear to be a more appropriate substrate than bicarbonate for carboxylation reactions, since it would be more susceptible to nucleophilic attack. However, in aqueous medium, the equilibrium between dissolved CO2 and bicarbonate is such that, at physiological pH, bicarbonate is present at some 20-fold higher concentration. Furthermore, bicarbonate probably has greater potential for binding to enzymes since it is a more polar molecule than CO2. It is perhaps not surprising then, that although the product of decarboxylation reactions in catabolic processes is CO2, several carboxylating enzymes have evolved to employ bicarbonate, not CO2, as their substrate. The carboxylating enzymes in animals, which are known to bind bicarbonate as substrate, are the biotin-dependent carboxylases and the carbamoyl phosphate synthetase isozymes. These enzymes bind bicarbonate, but then generally convert it either to CO2 (biotin-dependent carboxylases) or to an activated form of CO2 (carbamoyl phosphate synthetases). Carbonic anhydrase (CA) is perhaps alone among enzymes in being able to bind either of these substrates. (For reviews see Rubio, 1986; Knowles, 1989 and O’Leary, 1992).

Bacterial carbonic anhydrases.

In contrast to animal and plant carbonic anhydrases, relatively little is known about carbonic anhydrases in bacteria. Carbonic anhydrase activity has been well documented in a few bacterial species and its presence has been inferred on the basis of gene sequence homologies in several others, but their functions are generally not known. Two exceptions are the carbonic anhydrase (CA) that is a part of the cyn operon in Escherichia coli and the CA in the cyanobacterium Synechococcus PCC7942, both of which serve known specialized roles, i.e. prevention of CO2 depletion during cyanate degradation and concentration of CO2 for photosynthesis, respectively. The focus of this chapter will be on the properties and function of these two bacterial carbonic anhydrases. Since the function of the carbonic anhydrase in cyanobacteria is presumably closely related to the role of carbonic anhydrase in photosynthesis in higher plants, which is reviewed elsewhere in this book, more emphasis is placed on the carbonic anhydrase in E. coli. Although not eubacterial, a short section is included on the carbonic anhydrase from the Archaebacterium Methanosarcina thermophila. The distribution, properties, and possible functions and wider occurrence of carbonic anhydrases in other bacterial species are reviewed in the last section.

A novel role for carbonic anhydrase: cytoplasmic pH gradient dissipation in mouse small intestinal enterocytes.

1. The spatial and temporal distribution of intracellular H+ ions in response to activation of a proton-coupled dipeptide transporter localized at the apical pole of mouse small intestinal isolated enterocytes was investigated using intracellular carboxy-SNARF-1 fluorescence in combination with whole-cell microspectrofluorimetry or confocal microscopy. 2. In Hepes-buffered Tyrode solution, application of the dipeptide Phe-Ala (10 mM) to a single enterocyte reduced pHi locally in the apical submembranous space. After a short delay (8 s), a fall of pHi occurred more slowly at the basal pole. 3. In the presence of CO2/HCO3--buffered Tyrode solution, the apical and basal rates of acidification were not significantly different and the time delay was reduced to 1 s or less. 4. Following application of the carbonic anhydrase inhibitor acetazolamide (100 microM) in the presence of CO2/HCO3- buffer, addition of Phe-Ala once again produced a localized apical acidification that took 5 s to reach the basal pole. Basal acidification was slower than at the apical pole. 5. We conclude that acid influx due to proton-coupled dipeptide transport can lead to intracellular pH gradients and that intracellular carbonic anhydrase activity, by facilitating cytoplasmic H+ mobility, limits their magnitude and duration.

The Role of Carbonic Anhydrases in Tumors

Ever since Meldrum and Roughton discovered carbonic anhydrase1 (CA; EC 4.2.1.1.), the zinc-containing metalloenzyme that catalyzes the reversible hydration of CO2 (CO2 + H2O ⇆ HCO−3 + H+), the role of the enzyme has been thoroughly investigated. It has become clear that the main functions of the enzyme are to produce HCO−3 for the intermediate metabolism and to maintain pH, water, and ion equilibrium in the body.2 The presence of the enzyme has been proven essential in almost every organ. A series of studies performed over many years described nine isozymes (CA I through IX)3,4 and several CA-related peptides/proteins.

HCO3- absorption in rabbit outer medullary collecting duct: role of luminal carbonic anhydrase.

Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing the dehydration of carbonic acid into CO2 plus water, facilitates H+ secretion in the renal outer medullary collecting duct from the inner stripe (OMCDi). To examine the role of CA IV on H+ secretion, we measured net HCO3- transport in perfused OMCDi segments and examined the effect on transport of two extracellular CA inhibitors, benzolamide and F-3500, aminobenzolamide coupled to a nontoxic polymer, polyoxyethylene bis(acetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg, Chem, 24: 262-272, 1996)]. These agents would inhibit only the luminal CA enzyme. Dose titration curves for net HCO3- flux were performed for each drug. Basal HCO3- absorptive flux was 12 pmol.min-1.mm-1 in control segments and significantly increased to 16 pmol.min-1.mm-1 in segments from 3-day acid-treated animals. The concentrations of benzolamide and F-3500 that inhibited HCO3- absorption by 50% were approximately 0.1 and approximately 5 microM, similar to the Ki for CA IV inhibition by these agents (0.2 and 4.0 microM, respectively; T. Maren, C. W. Conroy, G. C. Wynns, and D. R. Godman. J. Pharmacol. Exp. Ther. 280: 98-105, 1997). Adding exogenous CA to the inhibitor in the perfusate nearly restored basal HCO3- transport, suggesting that cytosolic CA II was not inhibited by these impermeant inhibitors. In OMCDi segments from acidotic rabbits, the concentrations of benzolamide and F-3500 that inhibited HCO3- absorption by 50% were 50 and 500 microM, respectively, > 100 times the Ki for CA IV inhibition and for inhibition of HCO3- transport in control tubules. Thus, in the OMCDi, doses of extracellular CA inhibitors that inhibited approximately 50% of CA IV activity also comparably inhibited HCO3- transport, indicating that H+ secretion depends in part on the availability of luminal CA IV activity. Acidosis substantially decreased the sensitivity of HCO3- transport to CA inhibition.

Physiology and biochemistry of the pseudobranch: an unanswered question?

The structure and function of the pseudobranch has long interested scientists, but its overall role has remained a mystery. Previous studies have attributed respiratory, endocrine, osmoregulatory and sensory roles to the pseudobranch, and the present review concentrates on new findings. Perfusion experiments on the pseudobranch of the rainbow trout (Oncorhynchus mykiss) using both erythrocyte suspensions and Ringer solution have shown that this organ is able to generate values for the respiratory quotient (RQ) greater than 1.0. The release of carbon dioxide into the perfusate was found to be largely independent of flow between perfusion rates of 120-190 microl/min and could be inhibited by acetazolamide (10(-5) M), indicating a role for carbonic anhydrase. Noradrenaline (10(-5) M) had no effect on oxygen consumption or carbon dioxide release of the pseudobranch. The rate of carbon dioxide release was also dependent on the pH of the pre-pseudobranch perfusate, carbon dioxide release being reduced at lower perfusate pH values. Based on the glucose balance of the isolated saline-perfused rainbow trout pseudobranch and on the enzyme profiles for the rainbow trout, cod, swordfish and deep-water grenadier pseudobranch, it is suggested that the pentose phosphate shunt might be a source of carbon dioxide, yielding the high RQ values found for this organ. Most evidence now available indicates that the pseudobranch is integrally linked with the choroid rete and the supply of oxygen to the retina of the fish eye.

Models of CO 2 concentrating mechanisms in microalgae taking into account cell and chloroplast structure

Detailed mathematical models have been developed for the functioning of CO 2 concentration mechanisms in microalgae. The models treat a microalgal cell as several compartments: pyrenoid, chloroplast stroma, cytoplasm and periplasmic space. Cases for both the active bicarbonate transport through the plasmalemma and the passive CO 2 diffusion through it with the subsequent concentrating of CO 2 inside the chloroplast are analyzed. CO 2 evolution from bicarbonate inside the pyrenoid is modeled. The great diffusion resistance for CO 2 flux from the pyrenoid is caused by a starch envelope and the concentric thylakoid membranes surrounding it. The role of carbonic anhydrase in the periplasmic space, cytoplasm and inside the chloroplast is evaluated numerically. The models also offer an explanation for the absence of `short-circuited' inorganic carbon fluxes between the external medium and the cytoplasm under active bicarbonate transport through the plasmalemma and in the presence of carbonic anhydrase in the cytoplasm. If the cytoplasm is driven from the space between a chloroplast envelope and plasmalemma upon the microalgae adaptation to low concentration of the dissolved inorganic carbon, the inorganic carbon leak might be avoided. The models reproduce accurately the majority of known experimental data. The high efficiency of CO 2 concentrating mechanisms in microalgae can be explained by a considerable diffusion resistance for CO 2 flux from the pyrenoid and by the effective scavenging of CO 2 leaking outward from the chloroplast to cytoplasm and from cell to periplasmic space.

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.

Carbonic anhydrase provides bicarbonate for de novo lipogenesis in the locust

The role of carbonic anhydrase (CA) in lipogenesis was investigated in the rat body of the desert locust ( Schistocerca gregaria), an excellent model system for the study of fat metabolism. When reared in appropriate conditions, the locust passes through a period in development when its metabolism is strongly directed towards lipogenesis. This enhanced level of lipogenesis, which occurred in the 4- to 6-day old adult locust, was inhibited by the potent and specific sulphonamide inhibitors of CA, acetazolamide, methazolamide and ethoxzolamide, whilst the low levels of synthesis evident in the insect outside this lipogenic period remained unaffected. The relative effectiveness of the three different sulphonamides in inhibiting lipogenesis correlated well with their effectiveness as inhibitors of carbonic anhydrase activity. Inhibition of lipogenesis by methazolamide was not overcome by the addition of bicarbonate (up of 25 mM) to the incubation medium. Consequently, these data suggest that inhibition is mediated through restriction of bicarbonate provision largely for pyruvate carboxylase in the mitochondrion rather than for acetyl CoA in the cytoplasma. We propose that carbonic anhydrase is necessary for the provision of bicarbonate for elevated levels of lipogenesis.

Do histopathology reports of primary cutaneous melanoma contain enough essential information?

To audit the content of primary cutaneous malignant melanoma histopathology reports with special reference to Breslow thickness and lateral excision margins. The Trent Regional Cancer Registry was asked to provide details of primary cutaneous malignant melanomas for the most recent year available (1990). Histopathology departments were then requested to provide copies of the relevant reports, which were then analysed. In total, 178 reports were obtained from 16 departments. Breslow thickness was present in 87.1% (155/178) and a comment had been made on lateral excision in 85.4% (152/178). A specific clearance measurement was recorded in 5.6% (10/178), and in 9.6% (17/178) tumour was stated to extend to the margin. In 4.5% (8/178) neither thickness nor a comment on excision was recorded. Clinical advice on excision was offered in 12.4% (22/178). A macroscopic description was absent in 6.7% (12/178). Deficiencies were identified in the quality of malignant melanoma histopathology reports in Trent Region. There is no reason to believe that significant improvements have occurred since 1990 or that other regions are performing differently. A national standard for reporting primary cutaneous malignant melanoma is recommended. As a minimum, all reports should include Breslow thickness and a specific measurement of lateral clearance. This will facilitate prognostic evaluation, clinical management and audit. This standard would not exclude the reporting of other information, depending on local policy. As with all standards, continual review must be undertaken and consideration given as to whether other more recent parameters, such as growth phase, also warrant future inclusion.

光合成制御 ４ 光合成器官へのＣＯ２供給 カーボニックアンヒドラーゼの役割

光合成制御 ４ 光合成器官へのＣＯ２供給 カーボニックアンヒドラーゼの役割

Multiple Roles of Carbonic Anhydrase in Cellular Transport and Metabolism

Multiple Roles of Carbonic Anhydrase in Cellular Transport and Metabolism

Regulation of statoconia mineralization in Aplysia californica in vitro.

Statoconia are calcium carbonate inclusions in the lumen of the gravity-sensing organ, the statocyst, of Aplysia californica. The aim of the present study was to examine the role of carbonic anhydrase and urease in statoconia mineralization in vitro. The experiments were performed using a previously described culture system (Pedrozo et al., J. Comp. Physiol. (A) 177:415-425). Inhibition of carbonic anhydrase by acetazolamide decreased statoconia production and volume, while inhibition of urease by acetohydroxamic acid reduced total statoconia number, but had no affect on statoconia volume. Inhibition of carbonic anhydrase initially increased and then decreased the statocyst pH, whereas inhibition of urease decreased statocyst pH at all times examined; simultaneous addition of both inhibitors also decreased pH. These effects were dose and time dependent. The results show that carbonic anhydrase and urease are required for statoconia formation and homeostasis, and for regulation of statocyst pH. This suggests that these two enzymes regulate mineralization at least partially through regulation of statocyst pH.

Role of hepatic carbonic anhydrase in de novo lipogenesis

The role of carbonic anhydrase in de novo lipid synthesis was examined by measuring [1-14C]acetate incorporation into total lipids, fatty acids and non-saponifiable lipids in freshly isolated rat hepatocytes. Two carbonic anhydrase inhibitors, trifluoromethylsulphonamide (TFMS) and ethoxozolamide (ETZ) decreased incorporation of 14C into total lipids. Both fatty acid and non-saponifiable lipid components of the total lipid were inhibited to approximately the same extent by 100 microM TFMS (29 +/- 0.3% and 35 +/- 0.3% of control respectively in replicate studies). However, neither drug significantly affected ATP concentrations or the transport activity of Na+/K(+)-ATPase, two measures of cell viability. To establish the site of this inhibition, water-soluble 14C-labelled metabolites from perchloric acid extracts of the radiolabelled cells were separated by ion-exchange chromatography. TFMS inhibited 14C incorporation into citrate, malate, alpha-oxoglutarate and fumarate, but had no effect on incorporation of 14C into acetoacetate. Since ATP citrate-lyase, the cytosolic enzyme that catalyses the conversion of citrate into acetyl-CoA, catalyses an early rate-limiting step in fatty acid synthesis, levels of cytosolic citrate may be rate controlling for de novo fatty acid and sterol synthesis. Indeed citrate concentrations were significantly reduced to 37 +/- 6% of control in hepatocytes incubated with 100 microM TFMS for 30 min. TFMS also inhibited the incorporation of 14C from [1-14C]pyruvate into malate, citrate and glutamate, but not into lactate. This supports the hypothesis that TFMS inhibits pyruvate carboxylation, i.e. since all of the 14C from [1-14C]pyruvate converted into citric acid cycle intermediates must come via pyruvate carboxylase (i.e. rather than pyruvate dehydrogenase). Our findings indicate a role for carbonic anhydrase in hepatic de novo lipogenesis at the level of pyruvate carboxylation.

Effect of Carbonic Anhydrase Inhibition on Photosynthesis by Leaf Pieces of C3 and C4 Plants

The role of carbonic anhydrase (CA) in the photosynthesis of C3 and C4 plants was investigated by use of the effects of the CA inhibitor ethoxyzolamide on peeled and vacuum infiltrated leaf pieces. Two C4(Zea mays and Amaranthus edulis) and three C3 (Spinacea oleracea, Nicotiana rustica and Hordeum vulgare) species were studied. The results clearly showed that photosynthesis at limiting inorganic carbon was more severely inhibited in the C4 species compared with the C3 species. However, the degree of inhibition in the C4 species was less than might have been expected based on the rate limitation which the conversion of CO2 to HCO3- would impose on the provision of substrate to PEP carboxylase. The reasons for this lower than expected inhibition is discussed in terms of the lack of penetration of ethoxyzolamide, the diffusion of HCO3- through plasmodesmata and the possible existence of a plasma membrane CA activity.

Photobiont-related differences in carbon acquisition among green-algal lichens

The photosynthetic properties of a range of lichens (eight species) containing green algal primary photobionts of either the genus Coccomyxa, Dictyochloropsis or Trebouxia were examined with the aim of obtaining a better understanding for the different CO2 acquisition strategies of lichenized green algae. Fast transients of light/dark-dependent CO2 uptake and release were measured in order to screen for the presence or absence of a photosynthetic CO2-concentrating mechanism (CCM) within the photobiont. It was found that lichens with Trebouxia photobionts (four species) were able to accumulate a small pool of inorganic carbon (DIC; 70–140 nmol per mg chlorophyll (Chl)), in the light, which theoretically may result in, at least, a two to threefold increase in the stromal CO2 concentration, as compared to that in equilibrium with ambient air. The other lichens (four species), which were tripartite associations between a fungus, a cyanobacterium (Nostoc) and a green alga (Coccomyxa or Dictyochloropsis) accumulated a much smaller pool of DIC (10–30 nmol·(mg Chl)−1). This pool is most probably associated with the previously documented CCM of Nostoc, inferred from the finding that free-living cells of Coccomyxa did not show any signs of DIC accumulation. In addition, the kinetics of fast CO2 exchange for free-living Nostoc were similar to those of intact tripartite lichens, especially in their responses to the CCM and the carbonic anhydrase (CA) inhibitor ethoxyzolamide. Trebouxia lichens had a higher photosynthetic capacity at low and limiting external CO2 concentrations, with an initial slope of the CO2-response curve of 2.6–3.9 μmol·(mg Chl)−1·h−1·Pa−1, compared to the tripartite lichens which had an initial slope of 0.5–1.1 μmol-(mg Chl)−1·h−1·-Pa−1, suggesting that the presence of a CCM in the photobiont affects the photosynthetic performance of the whole lichen. Regardless of these indications for the presence or absence of a CCM, ethoxyzolamide inhibited the steady-state rate of photosynthesis at low CO2 in all lichens, indicating a role of CA in the photosynthetic process within all of the photobionts. Measurements of CA activity in photobiont-enriched homogenates of the lichens showed that Coccomyxa had by far the highest activity, while the other photobionts displayed only traces or no activity at all. As the CCM is apparently absent in Coccomyxa, it is speculated that this alga compensates for this absence with high internal CA activity, which may function to reduce the CO2-diffusion resistance through the cell.

Localization of carbonic anhydrase in the salivary glands of the cockroach, Periplaneta americana

Carbonic anhydrase (CA) activity was localized in the salivery glands of the cockroach, Periplaneta americana, by (1) Hansson's histochemical technique, and (2) the use of the fluorescent sulphonamide, 5-dimethyl-amino-naphthalene-1-sulphonamide (DNSA). Both techniques reveal the same distribution pattern of CA in the four morphologically different cell types of the glands: peripheral cells, central cells, inner acinar duct cells, and distal duct cells. Positive reactions with Hansson's cobalt/phosphate technique were found in the apical regions of the peripheral cells and the distal duct cells, and were inhibited by 10(-5) M acetazolamide in control experiments. No staining could be detected in the central cells and the inner acinar duct cells. The fluorescent CA inhibitor DNSA (10(-4)M) specifically stained the peripheral cells and the distal duct cells in methanol-fixed cryostat sections, whereas the central cells and the inner acinar duct cells remained unstained. The role of CA in the peripheral cells is not clear. CA activity in the distal duct cells may provide the protons needed to run the vacuolar-type H(+)-ATPase on the apical infoldings of the cells. This ATPase may be involved in modification of the primary saliva.

Carbonic anhydrases in the human adrenal gland and its disorders: Immunohistochemical and biochemical studies of the enzymes.

To study the possible roles of carbonic anhydrases (CA) in the human adrenal glands, an immunohistochemical study (using polycloncal antibodies against CA I and II), and a biochemical assay of CA activity (based on the change in pH caused by the hydration of CO2) were performed in normal human adrenal tissue and adrenocortical adenomas. All of 10 normal adrenal glands showed positive staining for CA II in the zona glomerulosa, whereas weak CA I-positive staining was detected in only 5 glands. A biochemical study of 3 normal human adrenal specimens also demonstrated CA activity only in the outer adrenal cortex. Of 26 adrenocortical adenoma specimens ( 15 aldosteronomas, 6 nonfunctioning adenomas, and 5 Cushing's adenomas), CA I and II immunoreactivity was observed in only 3 and 5 aldosteronoma samples, respectively. A biochemical analysis of CA in 5 adenoma specimens (3 aldosteronomas, 2 nonfunctioning adenomas) detected enzymatic activity in 2 aldosteronomas. In all 26 specimens of non-neoplastic attached adrenals of adrenocortical adenomas, CA II immunoreactivity was present in the zona glomerulosa cells. These results indicate that CAs are present in the zona glomerulosa cells of the non-neoplastic human adrenals glands (but not in the majority of adrenocortical adenomas) and that CA II may constitute the major form of the enzyme. Exclusive localization of CA II in the zona glomerulosa cells in human adrenal glands may suggest a possible involvement of CAs in aldosterone biosynthesis or secretion through ion transport, or both. Our data also suggest that CA II can be used as an immunochemical marker for the zona glomerulosa cells in human adrenal glands.

The Role of Carbonic Anhydrase in Photosynthesis

INTRODUCTION 369THE REQUIREMENT FOR CARBONIC ANHYDRASES IN DIFFERENTORGANISMS 370CYANOBACTERIA 371Carboxysomal Carbonic Anhydrase 373Is the Ci Pump a Membrane Carbonic Anhydrase? 374MICROALGAE 374Chloroplast Carbonic Anhydrase 375Periplasmie Carbonic Anhydrase 376Cytosolic Carbonic Anhydrase 377Induction of Carbonic Anhydrase at Low-C02 378AQUATIC MACROALGAE AND ANGIOSPERMS 379External Carbonic Anhydrose 379Internal Carbonic Anhydrase 380C4 PLANTS 381CAM PLANTS 382C3 PLANTS 382EVOLUTION OF PHOTOSYNTHETIC CARBONIC ANHYDRASES 385CONCLUSIONS 387

Role of carbonic anhydrase in basal and stimulated bicarbonate secretion by the guinea pig duodenum.

The role of carbonic anhydrase in the process of proximal duodenal mucosal bicarbonate secretion was investigated in the guinea pig. In a series of experiments in vivo, the duodenum was perfused with 24 mmol/liter NaHCO3 solution (+ NaCl for isotonicity) to ensure that active duodenal HCO3- secretion against a concentration gradient was measured. Acetazolamide (80 mg/kg) was infused intravenously to examine the role of carbonic anhydrase on basal and agonist-stimulated HCO3- secretion. Acetazolamide abolished basal HCO3- secretion and significantly decreased HCO3- secretion after stimulation with dibutyryl 5'-cyclic adenosine monophosphate (dBcAMP, 10(-5) mol/kg), dibutyryl 5'-cyclic guanosine monophosphate (dBcGMP, 10(-5) mol/kg), prostaglandin E2 (PGE2, 10(-6) mol/kg), PGF2 alpha (10(-6) mol/kg), tetradecanoyl-phorbol-acetate (TPA, 10(-7) mol/kg), glucagon (10(-7) mol/kg), vasoactive intestinal polypeptide (VIP, 10(-8) mol/kg), and carbachol (10(-8) mol/kg). Utilizing a fluorescence technique, we could detect the enzyme carbonic anhydrase in equal amounts in villous and crypt cells of the proximal duodenal epithelium; no activity was demonstrated in tissues pretreated with acetazolamide. In conclusion, carbonic anhydrase is required for both basal and stimulated duodenal HCO3- secretion.