Pulmonary vasodilation by acetazolamide during hypoxia is unrelated to carbonic anhydrase inhibition

Acute hypoxic pulmonary vasoconstriction can be inhibited by high doses of the carbonic anhydrase inhibitor acetazolamide. This study aimed to determine whether acetazolamide is effective at dosing relevant to human use at high altitude and to investigate whether its efficacy against hypoxic pulmonary vasoconstriction is dependent on carbonic anhydrase inhibition by testing other potent heterocyclic sulfonamide carbonic anhydrase inhibitors. Six conscious dogs were studied in five protocols: 1) controls, 2) low-dose intravenous acetazolamide (2 mg.kg(-1).h(-1)), 3) oral acetazolamide (5 mg/kg), 4) benzolamide, a membrane-impermeant inhibitor, and 5) ethoxzolamide, a membrane-permeant inhibitor. In all protocols, unanesthetized dogs breathed spontaneously during the first hour (normoxia) and then breathed 9-10% O(2) for the next 2 h. Arterial oxygen tension ranged between 35 and 39 mmHg during hypoxia in all protocols. In controls, mean pulmonary artery pressure increased by 8 mmHg and pulmonary vascular resistance by 200 dyn.s.cm(-5) (P <0.05). With intravenous acetazolamide, mean pulmonary artery pressure and pulmonary vascular resistance remained unchanged during hypoxia. With oral acetazolamide, mean pulmonary artery pressure increased by 5 mmHg (P < 0.05), but pulmonary vascular resistance did not change during hypoxia. With benzolamide and ethoxzolamide, mean pulmonary artery pressure increased by 6-7 mmHg and pulmonary vascular resistance by 150-200 dyn.s.cm(-5) during hypoxia (P < 0.05). Low-dose acetazolamide is effective against acute hypoxic pulmonary vasoconstriction in vivo. The lack of effect with two other potent carbonic anhydrase inhibitors suggests that carbonic anhydrase is not involved in the mediation of hypoxic pulmonary vasoconstriction and that acetazolamide acts on a different receptor or channel.     

Bone-targeted carbonic anhydrase inhibitors: effect of a proinhibitor on bone resorption in vitro

Many investigations have indicated a functional role for carbonic anhydrase in the mediation of hormone-stimulated bone resorption. These studies depend heavily on the use of heterocyclic sulfonamide inhibitors of carbonic anhydrase. These drugs have effects on many tissues other than bone, and some of these effects confound the interpretation of studies of the role of carbonic acid in bone metabolism. A novel, "bone-targeted" sulfonamide has been produced to obviate these extraosseous effects. This compound (designated WP-1) is the combination of tetracycline and acetazolamide, such that the acetazolamide is not an active inhibitor. Hydrolysis of WP-1 yields an active carbonic anhydrase inhibitor. WP-1 has a marked affinity for bone mineral, allowing deposition of the drug in bone. At a concentration of 10(-5) M, WP-1 attenuates parathyroid hormone stimulated net release of calcium from neonatal rat calvaria in culture. WP-1 is the first member of a class of drugs which may prove useful as pharmacological probes in the study of bone metabolism.     

Histomorphometric identification of carbonic anhydrase in fetal rat bone embedded in glycolmethacrylate

Carbonic anhydrase was identified in bone-resorbing cells present in sections of fetal rat femur embedded in glycolmethacrylate. Using a slight modification of the Hansson's histochemical method, we demonstrated that most chondroclasts (91.8-95.4%) and osteoclasts (95.1-96.3%) display a positive histochemical reaction for carbonic anhydrase. This staining was consistently inhibited in the presence of very low concentrations (10(-6), 10(-7) M) of the specific inhibitor acetazolamide. The number of chondroclasts reacting for carbonic anhydrase was identical to the number of acid phosphatase-stained chondroclasts determined on adjacent sections. A large majority of osteoclasts (96.3%) stained for carbonic anhydrase and for acid phosphatase (97.2%), with more osteoclasts reacting for the latter enzyme than the former (76.8 +/- 8.5 (SD) vs 85.3 +/- 9.2 cells/mm2 of endosteal bone; p less than 0.01). The observation that acetazolamide at a concentration as low as 10(-7) M inhibited Hansson's reaction, together with our histomorphometric results, validates the use of histochemical staining for carbonic anhydrase to evaluate activity of bone-resorbing cells identified in plastic-embedded fetal bone tissue.     

Affinity chromatography of carbonic anhydrase

An insoluble support for affinity chromatography of carbonic anhydrase has been prepared by coupling Sulfamylon (p-aminomethylbenzene sulfonamide) to Sepharose 4B. Carbonic anhydrase binds to Sulfamylon-Sepharose very strongly and can be eluted under mild conditions by the addition of enzyme inhibitors. The gel was used to purify carbonic anhydrase from human erythrocytes and to separate isozymes B and C. It was also employed to separate native enzyme from modified carbonic anhydrases. The apoenzyme and the carboxymethyl enzyme of human carbonic anhydrase B were both isolated by this method.     

Carbonic anhydrase inhibitors. Inhibition of red blood cell ostrich (Struthio camelus) carbonic anhydrase with a series of aromatic and heterocyclic sulfonamides

The purification of red blood cell carbonic anhydrase (CA, EC 4.2.1.1) from ostrich (scCA) blood is reported, as well as an inhibition study of this enzyme with a series of aromatic and heterocylic sulfonamides. The ostrich enzyme showed a high activity, comparable to that of the human isozyme II, with kcat, of 1.2 x 10(6) s(-1) and kcat/KM of 1.8 x 10(7) M(-1)s(-1), and an inhibition profile quite different from that of the human red blood cell cytosolic isozymes hCA I and II. scCA has generally a lower affinity for sulfonamide inhibitors as compared to hCA I and II. The only sulfonamide which behaved as a very potent inhibitor of this enzyme was ethoxzolamide (KI = 3.9 nM) whereas acetazolamide and sulfanilamide behaved as weaker inhibitors (inhibition constants in the range 303-570 nM). Several other aromatic and heterocyclic sulfonamides, mostly derivatives of sulfanilamide, homosulfanilamide, 4-aminoethylbenzenesulfonamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide, showed good affinities for the ostrich enzyme, with KI values in the range 25-72 nM.     

Invertebrate red blood cell carbonic anhydrase

This is the first report documenting the presence of carbonic anhydrase (CA) for any invertebrate red cells. CA activity was measured in plasma, hemolysates of blood cells, and in hemolymph of selected species of invertebrates. Annelid red blood cells (RBC) and sipunculid pink blood cells both possessed significant levels of CA activity. Molluscan RBC, on the other hand, lacked CA activity. The distribution appears to have fallen along phylogenetic lines, with CA being present only in blood cells of the two more closely related groups. However, the presence of extracellular CA was confirmed in oyster hemolymph. Oyster hemolymph CA showed a similar affinity (Ki) for the sulfonamide inhibitors acetazolamide and ethoxzolamide, as did the vertebrate RBC CA II isozyme, supporting the idea that this isozyme could be the ancestral form of the enzyme.     

The esterase activity of bovine carbonic anhydrase B above pH 9. Reversible and cooalent inhibition by acetozolamide.

The reversible complex between the metalloenzyme bovine carbonic anhydrase B and the sulfonamide inhibitor acetazolamide can be "frozen" irreversibly by the addition of a covalent bond between the methyl group of the inhibitor and the tau-nitrogen of histidine-64. In both cases the inhibited enzyme is inactive as an esterase toward p-nitrophenyl propionate at physiological pH but retains activity controlled by an ionization in the protein exhibiting a pK-a greater than 10. Similarly, both the covalently and reversibly inhibited enzymes in which the catalytically essential Zn(II) ion has been replaced with Co(II) display the same visible absorption spectrum which is invariant over the pH range from 5 to 12. The evidence therefore indicates that the position of the acetazolamide moiety in the active site is independent of both pH and the presence of the covalent bond to histidine-64. Moreover, when reversibly bound, this inhibitor has been shown to replace the water molecule (or hydroxide ion) known to occupy the fourth coordination position of the metal ion and frequently implicated in the catalytic mechanism of carbonic anhydrases. Thus, the activity exhibited by the inhibited enzymes and consequently the second rise observed in the pH rate profile of the native enzyme above pH 0 cannot reflect the ionization of such a water molecule in contrast to what has been postulated previously (Pocker, Y., and Storm, D. R. (1968) Biochemistry 7, 1202-1214). Displacement of the zinc-bound solvent molecule rather than the alkylation of histidine-64 is suggested, however, as the cause of the inactivation of the alkylated enzyme round neutrality. Taken together, the biphasic pH rate profile of native bovine carbonic anhydrase B as well as the activity retained by the alkylated enzyme above pH 9 are best described by a model in which two groups in the enzyme ionize independently, thereby raising the possibility that the high pH activity is controlled by an ionization outside the active site region of the enzyme. Above pH 9.5 the pK; for the reversible interaction between native carbonic anhydrase and acetazolamide falls off linearly with increasing pH. The slope of --1.56 suggests that, among other factors, more than one ionization is responsible for the descending limb of the pH-i-pH profile.     

A novel carbonic anhydrase from the mantle of the pearl oyster (Pinctada fucata)

A novel carbonic anhydrase (CA) has been purified from the mantle of the pearl oyster, Pinctada fucata, by ammonium sulfate precipitation and affinity chromatography. Its molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be approximately 38 kDa. Native-PAGE shows that the novel CA can bind a fluorescent probe, 5-dimethylamino-1-naphthalenesulfonamide (DNSA), known to specifically bind carbonic anhydrase. Compared to carbonic anhydrase I (CAI) from human erythrocytes, the novel CA migrates faster indicating that it is more acidic. The effect of an inhibitor on the enzyme activity was also examined. The CA from the mantle showed a weak resistance to acetazolamide (AZ), a specific inhibitor of CA. When DNSA was bound to CA, it caused the wavelength of emission maximum intensity to blue shift to 454 nm upon excitation at 326 nm. Histochemical data indicates that the enzyme is distributed widely throughout the mantle tissue, being concentrated at the edge of the mantle. The evidence presented indicates a function for CA in the process of pearl formation and biomineralization.     

The use of prontosil for the visualization of carbonic anhydrase bands in polyacrylamide gels. Application to the carbonic anhydrase isozymes of erythrocytes and white skeletal muscle of the rabbit

Prontosil, a carbonic anhydrase inhibitor of orange-red colour, is used to visualize carbonic anhydrase bands during isoelectric focusing in polyacrylamide gels. 5–60 ng of the sulfonamide Prontosil are added to the 100–200 μl samples before application to the gels. Bound Prontosil moves into the gel together with carbonic anhydrase and stains the enzyme bands formed there, while unbound Prontosil remains on top of the gels. The method is specific, no proteins other than carbonic anhydrase were observed to be stained, and it requires no special equippment. It was applied to chloroform/ethanol extracts of erythrolysates and while muscle homogenates from rabbits. Densitometric evaluation of the Prontosil-stained bands obtained with these extracts showed that rabbit red cells contain roughly equla amounts of carbonic anhydrase isoenzymes B and C while in rabbit white skeletal muscle isoenzyme C is predominant and little B enzyme occurs. These results confirm previous findings obtained by affinity chromatography of erythrolysates and muscle homogenates.     

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.     

Functional and molecular determination of carbonic anhydrase levels in bovine and cultured human chondrocytes

In this study, bovine articular and human chondrocytes from the C-20/A4 cell line were tested for the functional activity and molecular presence of the enzyme carbonic anhydrase. This enzyme is classically considered to be important in the maintenance of high cellular buffering capacity by catalysing the slow attainment of equilibrium between CO(2) and HCO(3)(-). The first functional assay measured the rate of pH equilibration after administration of a fixed dose of CO(2) solution to cell lysates. Compared to positive controls (human erythrocytes, murine M1 cells and purified carbonic anhydrase), chondrocyte lysates attained equilibrium at a significantly slower rate, similar to the rate obtained with a negative control (Xenopus oocytes). A second functional assay studied CO(2) hydration kinetics in intact C-20/A4 cells, using a pH-sensitive fluorescent dye, as the CO(2) content of the extracellular solution was changed. It was shown that C-20/A4 cells accelerate hydration only to a small degree. Hydration kinetics were reduced to the spontaneous rate in the presence of acetazolamide. Western immunoblotting with isoform-nonspecific antibodies to carbonic anhydrase demonstrated weak staining in both bovine and human chondrocytes.     

Light-induced stimulation of carbonic anhydrase activity in pea thylakoids

Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6-8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light-generated proton gradient across the thylakoid membrane thus facilitating the production of CO(2) from HCO(3)(-) and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier.     

Benzolamide is not a membrane-impermeant carbonic anhydrase inhibitor

Benzolamide, an orphan drug belonging to the pharmacological class of sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) is widely used in many physiological and pharmacological studies, together with the clinically employed classical drugs, acetazolamide, methazolamide, ethoxzolamide or dichlorophenamide, it being frequently stated that benzolamide is a membrane-impermeant inhibitor. We prove here that this is false: in fact benzolamide is rather similar to acetazolamide from the point of view of penetrability through blood red cell membranes. Unlike these neutral drugs, the cationic, positively-charged CAIs incorporating either tetraalkyl ammonium or pyridinium moieties, due to their salt-like character are indeed membrane-impermeant, being the only type of low molecular weight compound possessing such properties. Selective inhibition of membrane-associated CA isozymes is relevant indeed in many physiological studies and also pharmacologically, since the tumor-associated isozymes (CA IX and XII) are both membrane-bound.     

Characterisation of carbonic anhydrase in Plasmodium falciparum

Here we report the existence, purification and characterisation of carbonic anhydrase in Plasmodium falciparum. The infected red cells contained carbonic anhydrase approximately 2 times higher than those of normal red cells. The three developmental forms of the asexual stages, ring, trophozoite and schizont were isolated from their host red cells and found to have stage-dependent activity of the carbonic anhydrase. The enzyme was purified to homogeneity from the crude extract of P. falciparum using multiple steps of fast liquid chromatographic techniques. It had a Mr of 32 kDa and was active in a monomeric form. The human red cell enzyme was also purified for comparison with the parasite enzyme. The parasite enzyme activity was sensitive to well-known sulfonamide-based inhibitors of both bacterial and mammalian enzymes, sulfanilamide and acetazolamide. The kinetic properties and the amino terminal sequences of the purified enzymes from the parasite and host red cell were found to be different, indicating that the purified protein most likely exhibited the P. falciparum carbonic anhydrase activity. In addition, the enzyme inhibitors had antimalarial effect against in vitro growth of P. falciparum. Moreover, the vital contribution of the carbonic anhydrase to the parasite survival makes the enzyme an attractive target for therapeutic evaluation.     

Catalytic enhancement of human carbonic anhydrase III by replacement of phenylalanine-198 with leucine

Carbonic anhydrase III, a cytosolic enzyme found predominantly in skeletal muscle, has a turnover rate for CO2 hydration 500-fold lower and a KI for inhibition by acetazolamide 700-fold higher (at pH 7.2) than those of red cell carbonic anhydrase II. Mutants of human carbonic anhydrase III were made by replacing three residues near the active site with amino acids known to be at the corresponding positions in isozyme II (Lys-64----His, Arg-67----Asn, and Phe-198----Leu). Catalytic properties were measured by stopped-flow spectrophotometry and 18O exchange between CO2 and water using mass spectrometry. The triple mutant of isozyme III had a turnover rate for CO2 hydration 500-fold higher than wild-type carbonic anhydrase III. The binding constants, KI, for sulfonamide inhibitors of the mutants containing Leu-198 were comparable to those of carbonic anhydrase II. The mutations at residues 64, 67, and 198 were catalytically independent; the lowered energy barrier for the triple mutant was the sum of the energy changes for each of the single mutants. Moreover, the triple mutant of isozyme III catalyzed the hydrolysis of 4-nitrophenyl acetate with a specific activity and pH dependence similar to those of isozyme II. Phe-198 is thus a major contributor to the low CO2 hydration activity, the weak binding of acetazolamide, and the low pKa of the zinc-bound water in carbonic anhydrase III. Intramolecular proton transfer involving His-64 was necessary for maximal turnover.     

Purification and properties of cyclostome carbonic anhydrase from erythrocytes of hagfish

1. Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been purified from erythrocytes of hagfish (Myxine glutinosa). A single form with low specific CO2 hydration activity was isolated. The purified carbonic anhydrase appeared homogeneous judging from polyacrylamide gel electrophoresis and gel filtration experiments. The protein has a molecular weight of about 29 000, corresponding to about 260 amino acid residues. This molecular weight is in accordance with other vertebrate carbonic anhydrases with the exception of the elasmobranch enzymes, which have Mr 36 000--39 000. 2. The molecular weight obtained for hagfish carbonic anhydrase indicates that a carbonic anhydrase with Mr approx. 29 000 is the ancestral type of the vertebrate enzyme rather than, as in sharks, a heavier carbonic anhydrase molecule. 3. The circular dichroism spectrum may indicate a somewhat different structural arrangement of aromatic amino acid residues in this enzyme than in the mammalian carbonic anhydrases. 4. The enzyme is strongly inhibited by acetazolamide and also to a lesser extent by monovalent anions. 5. Zn2+, which is essential for activity, appears, contrary to other characterized carbonic anhydrases, less strongly bound in the active site of the enzyme.     

Sarcolemmal carbonic anhydrase in red and white rabbit skeletal muscle

Sarcolemmal vesicles of white and red skeletal muscles of the rabbit were prepared by consecutive density gradient centrifugations in sucrose and dextran according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13,862-13,871). White and red muscle membrane fractions enriched in sarcolemma were characterized by high ouabain-sensitive Na+, K(+)-ATPase, by high Mg2(+)-ATPase activity, and by a high cholesterol content. Ca2(+)-ATPase activity, a marker enzyme for sarcoplasmic reticulum, was not detectable in the highly purified white and red muscle sarcolemmal fractions. White and red muscle sarcolemmal fractions exhibited no significant differences with regard to Na+, K(+)-ATPase, Mg2(+)-ATPase, and cholesterol. Specific activity of carbonic anhydrase in white muscle sarcolemmal fractions was 38 U.ml/mg and was 17.6 U.ml/mg in red muscle sarcolemma. Inhibition properties of sarcolemmal carbonic anhydrase were analyzed for acetazolamide, chlorzolamide, and cyanate. White muscle sarcolemmal carbonic anhydrase is characterized by inhibition constants, KI, toward acetazolamide of 4.6 X 10(-8) M, toward chlorzolamide of 0.75 X 10(-8) M, and toward cyanate of 1.3 X 10(-4) M. Red muscle sarcolemmal carbonic anhydrase is characterized by KI values toward acetazolamide of 8.1 X 10(-8) M, toward chlorzolamide of 6.3 X 10(-8) M, and toward cyanate of 0.81 X 10(-4) M. In contrast to the high specific carbonic anhydrase activities in sarcolemma, carbonic anhydrase activity in sarcoplasmic reticulum from white muscle varied between values of only 0.7 and 3.3 U.ml/mg. Carbonic anhydrase of red muscle sarcoplasmic reticulum ranged from 2.4 to 3.7 U.ml/mg.     

Purification and characterization of human salivary carbonic anhydrase

A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.     

Effect of incorporating a thiophene tail in the scaffold of acetazolamide on the inhibition of human carbonic anhydrase isoforms I,II, IX and XII

The high resolution crystal structure of 5-(2-thienylacetamido)-1,3,4-thiadiazole-2-sulfonamide complexed to human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoform hCA II is reported. The compound binds in a similar manner with acetazolamide when the sulfamoyl–thiadiazolyl–acetamido fragment of the two compounds is considered, but the thienyl tail was positioned in the subpocket 2, rarely observed by other investigated CA inhibitors. This positioning allows interaction with amino acid residues (such as Asn67, Ile91, Gln92 and Val121 which are variable in other isoforms of medicinal chemistry interest, such as hCA I, IX and XII. Indeed, the investigated sulfonamide was a medium potency hCA I and II inhibitor but was highly effective as a hCA IX and XII inhibitor. This different behavior with respect to acetazolamide (a promiscuous inhibitor of all these isoforms) has been explained by resolving the crystal structure, and may be used to design more isoform-selective compounds.     

On the lack of specificity of the cobalt-bicarbonate method for carbonic anhydrase.

Data are presented that support a nonenzymic mechanism for the staining obtained with the cobalt-bicarbonate method. The biochemically inactive apocarbonic anhydrase and Cu+2 apocarbonic anhydrase stain positively and this stain is inhibited by acetazolamide. The staining of the acetazolamide resistant carbonic anhydrase of male rat liver is inhibited by 10-6 M acetazolamide, at which concentration no biochemical inhibition is observed. There is no correlation between the biochemical and histochemical inhibitory potencies of a number of sulfonamides. The nonsulfonamide inhibitor, KCNO, does not inhibit staining. When incubations are performed in media exposed to atmospheres of increasing CO2 content, staining is not abolished until the atmospheric pCO2 approaches that generated by the medium itself. This finding renders the carbonic anhydrase catalyzed dehydration of HCO3- an improbable reaction for the staining. Studies with modified media show differences in staining patterns and in sensitivity to acetazolamide inhibition which question the specificity of the method for carbonic anhydrase.