Human Carbonic Anhydrase Isoenzymes Research Articles

Pharmacological profiles of the potent carbonic anhydrase inhibitor dorzolamide hydrochloride, a topical antiglaucoma agent

Carbonic anhydrase (CA) plays an important role in the secretion of aqueous humor. The orally administered CA inhibitor acetazolamide lowers the intraocular pressure (IOP) of patients with glaucoma. However, approximately 50% of patients stop treatment with acetazolamide as a consequence of intolerable side effects due to the extraocular inhibition of the enzyme. This prompted attempts to develop a topically active CA inhibitor. Merck Research Laboratories focused on developing a water- and solvent-soluble compound to penetrate the cornea. Dorzolamide hydrochloride is a potent inhibitor of human CA isoenzyme II, with an IC50 value of 0.18 nM in vitro. In contrast, its inhibitory activity against human CA isozyme I is much weaker (IC50 value of 600 nM). Topically administered dorzolamide penetrated the ciliary body, inhibited its CA activity and had a hypotensive effect in rabbits; in contrast, topical administration of acetazolamide or methazolamide did not decrease IOP. In clinical trials, dorzolamide administered 3 times daily was effective in lowering IOP in patients with open-angle glaucoma or ocular hypertension. The hypotensive effect of dorzolamide 0.5% was similar to that of oral CA inhibitors or timolol (0.25%) twice daily. Dorzolamide did not induce the severe systemic adverse events associated with oral CA inhibitors. Dorzolamide was as effective as pilocarpine or dipivefrine as an adjunctive therapy in patients receiving beta-adrenergic antagonists. Dorzolamide also reduced IOP and accelerated retinal arteriovenous passage time in addition to improving visual function in patients with normal-tension glaucoma.

Enzyme histochemical and immunohistochemical localization of carbonic anhydrase as a marker of ductal differentiation in the developing rat parotid gland.

Carbonic anhydrase has been localized to the acini and ducts of mature rat parotid glands. This enzyme has been associated with ion transport, a prominent function of striated and excretory ducts in salivary glands, suggesting that it might be used as a marker of ductal differentiation. The purpose of this study was histochemically to document developmental changes in carbonic anhydrase in the ducts of the rat parotid gland. Parotid glands were excised from rats at representative developmental ages. Enzyme histochemistry was done on frozen sections fixed in acetone, and immunohistochemistry was performed with antibodies to human carbonic anhydrase isoenzymes I, II, and VI on paraffin sections of glands fixed in Helly's fluid. Carbonic anhydrase activity was weak until age 21 days after birth, when it had increased slightly in the acini and intercalated ducts and moderately in striated and excretory ducts. The adult pattern was attained by 28 days, in which reactions were moderate to strong in the striated and excretory ducts and modest in the acini and intercalated ducts. Immunohistochemical reactions were weak until 14 days, then increased rapidly, and by 28 days approached the adult pattern of virtually none in the acini and modest to moderately strong in the striated and excretory ducts. The order of reaction intensity of the antibodies was II > I > VI. Carbonic anhydrase is a useful marker of the functional differentiation of the striated and excretory ducts of the developing rat parotid gland.

The preclinical pharmacology of dorzolamide hydrochloride, a topical carbonic anhydrase inhibitor.

Dorzolamide hydrochloride (S,S-5,6-dihydro-4H-4-ethylamino-6-methylthieno [2,3-b]thiopyran-2-sulfonamide-7,7-dioxide HCl; MK-507; L-671,152) is a water-soluble, potent inhibitor of human carbonic anhydrase isoenzymes II and IV in vitro, the respective IC50 values being 0.18 nM and 6.9 nM. In contrast, it was found to be a much weaker inhibitor of human carbonic anhydrase isoenzyme I (IC50 value of 600 nM). The topical administration of one 50 microliters drop of 0.5%, 1% and 2% solutions of dorzolamide maximally lowered the intraocular pressure (IOP) of glaucomatous monkeys by 22%, 30% and 37%, respectively. Good ocular hypotensive activity was also observed in ocular normotensive and hypertensive rabbits. Its site of action was within the eye, and the reductions in IOP in both species was achieved via decreased aqueous humor inflow. The duration of action of 2% dorzolamide was shorter than that of 0.5% timolol in glaucomatous monkeys. The IOP lowering activity of timolol in this paradigm was enhanced by the concomitant instillation of dorzolamide. Both acutely and repeatedly administered 2% dorzolamide did not decrease regional ocular blood flow in the rabbit, and the topical instillation of the drug had no adverse effects on the eye of rabbits, dogs and monkeys. Dorzolamide has been approved in a number of countries for use in patients with ocular hypertension or open-angle glaucoma.

Carbonic Anhydrase Antibody in Sera from Patients with Endometriosis

Sera from 16 of 23 (69.6%) patients with endometriosis, a potential autoimmune disease, and 2 of 17 (11.8%) control individuals had autoantibodies against the bovine carbonic anhydrase (CA) molecular weight marker, as determined by the Western blot technique. The reactivity of these antibodies to purified human CA I, human CA II, and two preparations of bovine CA II were investigated. Of the 16 endometriosis patients who were reactive to the bovine CA molecular weight marker, 14 were reactive to at least one purified human CA isoenzyme tested, 8 were reactive to at least one purified bovine CA II, and 2 did not react with any of the CA isoenzymes tested. Variation in cross-reactivity between species and in the biochemical characteristics of various CA isoenzyme preparations may partially explain these findings. Autoantibodies to CA isoenzymes have recently been reported in other autoimmune diseases. Further investigation is required to determine the significance of CA autoantibody production in patients with endometriosis.

Immunohistochemistry of carbonic anhydrase in human placenta and fetal membranes

The localization of human carbonic anhydrase (CA) isoenzymes HCA I, HCA II, and rat CA II have been studied in human umbilical cord, chorion laeve including amnion and placenta from first and second trimester and also from term pregnancies. Detection techniques of immunofluorescence and immunoperoxidase were used in cryostat and paraffin sections. Both isoenzymes were found in the villous syncytiotrophoblast throughout pregnancy. HCA I staining patterns in the villous endothelium were highly variable whereas increasing immunoreactivity levels of endothelial HCA II were detected as pregnancy advances. The extravillous cytotrophoblast showed generally weaker levels of immunoreactivity. In amnionic epithelium of membranes, chorionic plate and umbilical cord, higher activities for HCA I, HCA II and rat CA II were found than in all other localizations. Our findings emphasize the importance of enzyme mediated bicarbonate/CO2 removal from the feto-placental unit as opposed to simple bicarbonate diffusion or carrier mediated transport. As effective transfer routes should be considered not only umbilical cord--placental villi--intervillous space, but also fetal kidney--amnionic fluid--amnion--uterine vessels.

Competitive time-resolved immunofluorometric assay for quantifying carbonic anhydrase VI in saliva

A competitive time-resolved immunofluorometric assay sensitive and robust enough for quantifying human salivary carbonic anhydrase isoenzyme VI (HCA VI) was developed. The solid-phase immunoassay is based on competition between Eu(3+)-labeled HCA VI and salivary HCA VI for polyclonal rabbit anti-HCA VI antibodies that are attached to microtiter plate wells precoated with sheep anti-rabbit IgG. The subsequent immunoassay including the separation of free and bound HCA VI requires only one incubation step, after which the Eu3+ of the bound labeled antigen is released into an enhancement solution. The highly fluorescent Eu chelates formed in this solution are then quantified by time-resolved fluorometry (Delfia). The time-resolution principle effectively obviates possible interferences from complex biological material such as saliva. The assay detection limit was 1.5 micrograms/L. Intra- and interassay imprecisions (CVs) were 5.1% and 5.3%, respectively. The mean analytical recovery was 93%. The mean +/- SD concentration of HCA VI in paraffin-stimulated saliva was 6.8 +/- 4.3 mg/L (n = 30) and the secretion rate was 10.2 +/- 7.9 micrograms/min. The method was useful for further investigations of the role of HCA VI in difficult matrices, e.g., saliva.

Carbonic anhydrase isoenzymes II and I are present in the zona glomerulosa cells of the human adrenal gland.

Human carbonic anhydrase isoenzymes I and II (HCA I and II) were purified from human erythrocytes by inhibitor affinity chromatography and ion-exchange chromatography. These isoenzymes were then located in the human adrenal gland using specific polyclonal antisera raised in rabbits and specific detection by immunohistochemical techniques. Both HCA II and I were located in the zona glomerulosa cells, although the staining for HCA I was faint. The cells of the zona fasciculata and the zona reticularis failed to stain with either antiserum. Control stainings with preimmune or anti-HCA VI sera were negative. The presence of HCA II and I in the zona glomerulosa cells may be linked to regulation of the biosynthesis or secretion of mineralocorticoids.

Inhibitory effect of synthetic aminophosphonic acid derivatives on human carbonic anhydrase

A series of phosphonic analogs of amino acids, peptides and their derivatives was synthesized in order to examine their ability to inhibit human carbonic anhydrase (HCA) isoenzymes (I and II). The aminophosphonic acids with amino protection possess much stronger inhibitory capacity ( K i = 10 −6-10 −5 M) than the free aminophosphonic acids ( K i = 10 −5-10 −3 M). The aminophosphonic acids with both N- and P-protection do not have an inhibitory effect on the esterase activity of HCA. In a comparison of the enantiomeric pairs of Cbz-Ala P(OH) 2, the d-conformer has several-fold stronger inhibitory potency. All aminoalkylphosphonates investigated possess one order of magnitude stronger inhibitory activity towards isoenzyme HCA-I than towards HCA-II. A computer-directed analysis was used to study the possible interactions in an aminoalkylphosphonate [Cbz- d-Ala P(OH) 2]-(HCA-II) complex.

Immunohistochemical localization of carbonic anhydrase isoenzymes in the human male reproductive tract

The distribution of human carbonic anhydrase (HCA) isoenzymes I, II and VI in the human male reproductive tract was studied using specific antisera against affinity purified isoenzymes in conjunction with the peroxidase-antiperoxidase complex method. HCA VI-specific staining could not be demonstrated in any of the tissues studied, and HCA I was observed only in red blood cells. Immunostaining denoted HCA II in the epithelia of the seminal vesicle, ampulla of the ductus deferens and distal ductus deferens. Some cells in the epithelium of the corpus and cauda epididymidis also stained for HCA II. The staining for HCA II in the epithelium of the reproductive tract declined from the strongly positive seminal vesicle to the proximal part of the ductus deferens, which stained negatively. There were also HCA II-positive particles derived from the apical protrusions of the epithelium in the lumina of the seminal vesicle, ampulla of the ductus deferens and ductus deferens. The physiological role of HCA II is linked to the secretion of bicarbonate into the seminal plasma and thereby to the regulation of sperm motility and pH in the seminal plasma.

A single-step solid phase radioimmunoassay for quantifying human carbonic anhydrase I and II in cerebrospinal fluid

A single-step solid phase radioimmunoassay was developed to detect human carbonic anhydrase (CA) isoenzymes I (CA I) and II (CA II) in cerebrospinal fluid (CSF). The assay is capable of routinely detecting both isoenzymes at ng levels compared to the μg levels of the traditional catalytic methods, which failed to demonstrate any CA activity in CSF. When the values of immunoreactive CA II in CSF were corrected for blood contamination (the CA I/CA II ratio of blood was about 7.9), the amount of brain tissue originated CA II could be calculated. The CA II values in CSF samples from 13 patients with multiple sclerosis were higher than those in CSF samples from 11 patients with various peripheral neurological disorders. Since CA II has been specifically localized to oligodendrocytes and myelin, our preliminary results suggest the possibility of CA II leakage from oligodendrocytes and myelin into CSF in demyelinating diseases.

The investigation of cobalt(II) substituted carbonic anhyrase and carboxypeptidase A

It is generally accepted that information obtained on cobalt(II) complexes can be transferred to the analogous zinc compounds. Therefore substitution of zinc(II) with cobalt(II) in zinc containing enzymes allows one to investigate these systems through spectroscopic techniques. The data reported here are concerned with carbonic anhydrase (CA) and carboxypeptidase A (CPA). 1H NMR data in D 2O on CoCa reveal a relatively sharp signal assigned to the β proton of the histidine bound to the metal. Its T −1 value is constant with pH for the bovine CA isoenzyme B (BCAB) whereas it s lower at low [H in the case of human CA isoenzyme (HCAB). These data parallel the pH dependence of the water 1H NMR data at every magnetic field [1, 2]. It is proposed that in the latter case (HCAB) a change in coordination chemistry occurs at low pH, and in particular that five coordinate species are obtained, whereas CoBCAB at every pH and CoHCAB at high pH are pseudotetrahedral. The general equilibrium is ▪ Model compounds show that nuclear longitudinal relaxation decreases from tetra- to five- to six-coordination in cobalt(II) complexes. The above equilibrium is consistent with the difference in the electronic spectra and in the pK a between the two isoenzymes. Water 1H NMR data, coordinated histidine 1H NMR data, electronic and EPR data are in our opinion consistent with five-coordination in CoCPA [3]. We believe that five coordination is reached through two water molecules. N 3 − is found to bind ▪ cobalt(II) providing a derivative the electronic spectrum of which can be interpreted as being due to tetracoordination. The affinity of N 3 − for the enzyme decreases with pH, the pK a being around 9. This behavior of N 3 − is analogous to that shown with CoHCAB both with respect to change in coordination number and pH dependence of the affinity constants.

Human carbonic anhydrase isoenzyme C

The effects of some alcohol and aldehyde containing fixatives on the antigenicity of human carbonic anhydrase isoenzyme C (HCA C) were tested in order to reveal the most suitable method for the immunohistochemical localization of this enzyme. The use of 2% and 4% paraformaldehyde or 2% glutaraldehyde solutions before immunoperoxidase (PAP) staining resulted in the loss of HCA C-specific reactivity in the surface epithelial cells of human appendicular and gastric mucosae, whereas the antigenic reactivity of HCA C was well retained in the parietal cells of gastric glands. In corresponding tissue sections fixed with one of the alcohol containing solutions (abs. methanol, methanol + chloroform 2:1 or Carnoy fluid) both the surface epithelial and parietal cells showed HCA C-specific immunostaining after the PAP procedure. In addition, the antigenicity of HCA C was found to be well preserved in some tubular cells of human kidney fixed in Carnoy fluid. The paraffin infiltration at relatively low temperature did not markedly affect the enzyme antigenicity. Fixation in Carnoy fluid coupled with paraffin embedding at 55-60 degrees C in vacuo was found to give the best preservation of the antigenicity of HCA C with good morphological integrity for light microscopical localization.

Carbonic anhydrase isoenzyme B in erythrocytes of subjects with chronic acidosis

A specific and quantitative immunological method for determination of human erythrocyte carbonic anhydrase isoenzyme B has been used to ascertain the contents of this enzyme in the erythrocytes of healthy persons and of subjects with chronic metabolic and respiratory acidosis. The investigations have shown significant increase of carbonic anhydrase type B in the erythrocytes of patients suffering from renal failure with chronic acidosis, and in patients with chronic obstructive lung disease and chronic respiratory acidosis. The erythrocytes of acidotic uremie patients have a significantly higher content of erythrocyte carbonic anhydrase isoenzyme B than do the erythrocytes of uremie subjects without chronic acidosis. In chronic obstructive lung disease, the content of this enzyme in erythrocytes was significantly higher in the hypercapnic patients than in the normocapnic ones. In renal failure, significant correlation was found between carbonic anhydrase isoenzyme B and standrad bicarbonate. In chronic obstructive lung disease no significant correlation was found between carbonic anhydrase and pCO 2.

Immunohistochemical localization of human carbonic anhydrase isoenzyme C.

Methods for immunohistochemical localization of human carbonic anhydrase isoenzyme C (HCA C) with indirect fluorescent antibody and immunoperoxidase techniques are described. Both methods revealed large amounts of this "high activity" isoenzyme in the mucosae of human stomach and appendix. With the indirect immunofluorescent method the presence of the enzyme in human erythrocyte cytoplasm was also demonstrated. Correlations of present findings with those obtained with the traditional histochemical methods for demonstration of carbonic anhydrase activity are discussed.

A radioimmunosorbent technique for the assay of B- and C-types of carbonic anhydrase in human tissues.

A radioimmunosorbent technique for the assay of the human carbonic anhydrase isoenzymes HCA B and HCA C in tissue fluids was developed. The sensitivity of the method was 0.2 ng/ml and the precision was 5% in duplicate determinations for both enzymes. The presence in a tissue of up to 20 times higher concentrations of one isoenzyme will not interfere with the assay of the other. Haemolysates contained (mean +/- SE, n = 11) 12.1 +/- 0.52 and 1.5 +/- 0.06 mg enzyme/g Hb, and serum 0.63 +/- 0.12 and 0.2 +/- 0.02 microgram/ml of HCA B and HCA C, respectively. Pilot experiments indicated that the isoenzymes can be determined also in tissues, i.e. urine, saliva and cerebrospinal fluid, where catalytic methods previously have indicated absence of or only weak carbonic anhydrase activity. N-terminals of both enzymes were not antigenic.

Carbonic anhydrase isoenzyme B in erythrocytes of anemic and non-anemic uremic subjects

A specific and quantitative immunological method for determination of human erythrocyte carbonic anhydrase isoenzyme B has been used to determine the contents of this enzyme in the erythrocytes from healthy persons and anemic and non-anemic subjects with renal insufficiency. The investigations have shown significant increase of carbonic anhydrase type B in the erythrocytes of patients with renal failure. The erythrocytes of anemic uremie patients have a significantly higher content of erythrocyte carbonic anhydrase isoenzyme B than the erythrocytes of non-anemic uremic subjects. Significant correlations were found between human erythrocyte carbonic anhydrase type B and hemoglobin and between it and plasma bicarbonate. No correlation was found between human erythrocyte carbonic anhydrase and plasma creatinine or creatinine clearance and serum zinc.

Carbonic anhydrase isoenzyme B in erythrocytes of subjects with different types of anemia

A specific and quantitative immunological method for determination of human erythrocyte carbonic anhydrase (HCA) isoenzyme B has been used to determine the contents of enzyme in the erythrocytes from healthy persons and from subjects with different types of anemia. The investigations have shown a statistically significant increase of HCA type B in the erythrocytes of subjects suffering from uremic anemia, cancer anemia, megaloblastic anemia while in sideropenic anemia the content was normal. In erythrocytes from patients with acute bleeding anemia the content of HCA type B was significantly decreased. An inverse relationship was found between B-hemoglobin and HCA type B in megaloblastic anemia but not in the other types of anemia.

Carbonic anhydrase isoenzymes in the erythrocytes of new-born premature and full-term infants

A specific and quantitative immunological method for the determination of human erythrocyte carbonic anhydrase isoenzymes B and C has been used to determine these enzymes in erythrocytes of umbilical cord blood. The investigations have shown a content of both isoenzymes less than 1 : 10 of that in adults. Premature infants have significantly lower values than full-term infants. The influence of carbonic anhydrase in the respiratory distress syndrome is discussed. No conclusions are drawn. The content of carbonic anhydrase in erythrocytes has been suggested as a laboratory parameter of maturity. In our investigations it does not seem to be useful for the evaluation of maturity.

Carbonic anhydrase isoenzyme B in erythrocytes of subjects with thyroid disorders

A specific and quantitative immunological method for determination of human erythrocyte carbonic anhydrase (H.C.A.) isoenzyme B has been used to determine the contents of enzyme in the erythrocytes from healthy persons and from subjects with thyroid disorders. The investigations have shown a statistically significant decrease of H.C.A. type B in the erythrocytes of hyperthyroid subjects and an increase of this isoenzyme in hypothyroid subjects. Correlation was found between H.C.A. type B and serum-thyroxin. The value of H.C.A. type B determination in the diagnosis of thyroid disorders is discussed.

Carbonic anhydrase isoenzyme B in the erythrocytes of uremic subjects

A specific and quantitative immunological method for determination of human erythrocyte carbonic anhydrase isoenzyme B has been used to determine the contents of enzyme in the erythrocytes from healthy persons and subjects with uremia. The investigations have shown a significant increase of carbonic anhydrase type B in the erythrocytes of patients with renal failure. Correlation was found between human erythrocyte carbonic anhydrase type B and Pt-creatinine-clearance but not between it and plasma standardbicarbonate.