Human Carbonic Anhydrase Isoenzymes Research Articles

Carbonic anhydrase inhibitory properties of phenolic sulfonamides derived from dopamine related compounds

The effects of some phenolic sulfonamides were determined on the cytosolic carbonic anhydrase isoenzyme I and II (hCA I and II). Both isoenzymes were purified separately from human erythrocytes, using the Sepharose-4B-l-tyrosine-sulfanilamide affinity column chromatography method. In continuation of the study, we identified the inhibitory effects of phenolic sulfonamides 1–4 on the esterase activity of hCA I, and II. The inhibitory effects of phenolic sulfonamides 1–4 were tested on human carbonic anhydrase isoenzymes hCA I, and II. Among the compounds 1–4, compound 1 was concluded to show the best inhibitory effects. According to our data, IC50 values of compound 1 were found as 3.55 and 2.94μM for hCA I, and hCA II, respectively. On the other hand, Ki values of this compound were found as 0.827 and 0.745μM for both isoenzymes, respectively.

Oxidation of cyanobenzocycloheptatrienes: Synthesis, photooxygenation reaction and carbonic anhydrase isoenzymes inhibition properties of some new benzotropone derivatives

The oxidation of some cyanocycloheptatrienes with CrO3 and pyridine was investigated and a few new nitrile functionalised benzotropone derivatives were obtained. Photooxygenation reaction of these products was also studied. The structures of the formed products were determined on the basis of NMR spectroscopy and the formation mechanism of unusual products was discussed. Human carbonic anhydrase isoenzymes I, and II (hCA I and hCA II) inhibition properties of nitrile functionalized new benzotropone derivatives were also studied. Both CA isozymes were inhibited in the low micromolar range by these nitrile functionalized benzotropone analogues. The newly synthesized benzotropone derivatives showed inhibition constants in the sub-micromolar range (2.51–4.06μM). The best hCA I inhibition was observed in 5H-benzocycloheptene-7-carbonitrile (Ki: 2.88±0.86μM). On the other hand, 5-oxo-5H-benzocycloheptatriene-7-carbonitrile showed the powerful inhibitory effect against hCA II (Ki: 2.51±0.34μM).

ChemInform Abstract: Novel Sulfamides and Sulfonamides Incorporating the Tetralin Scaffold as Carbonic Anhydrase and Acetylcholine Esterase Inhibitors.

AbstractNew compounds (V), (VII), (X) and (XI) inhibit human carbonic anhydrase isoenzymes I and II in the low micromolar and submicromolar range and show moderate inhibition capacity toward acetylcholine esterase inhibitors.

Carbonic anhydrase inhibitors. Inhibition of human cytosolic isoforms I and II with (reduced) Schiff’s bases incorporating sulfonamide, carboxylate and carboxymethyl moieties

A library of Schiff bases was synthesized by condensation of aromatic amines incorporating sulfonamide, carboxylic acid or carboxymethyl functionalities as Zn2+-binding groups, with aromatic aldehydes incorporating tert-butyl, hydroxy and/or methoxy groups. The corresponding amines were thereafter obtained by reduction of the imines. These compounds were assayed for the inhibition of two cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes, hCA I and II. The Ki values of the Schiff bases were in the range of 7.0–21,400nM against hCA II and of 52–8600nM against hCA I, respectively. The corresponding amines showed Ki values in the range of 8.6nM–5.3μM against hCA II, and of 18.7–251nM against hCA I, respectively. Unlike the imines, the reduced Schiff bases are stable to hydrolysis and several low-nanomolar inhibitors were detected, most of them incorporating sulfonamide groups. Some carboxylates also showed interesting CA inhibitory properties. Such hydrosoluble derivatives may show pharmacologic applications.

Facile, highly efficient, and clean one-pot synthesis of acridine sulfonamide derivatives at room temperature and their inhibition of human carbonic anhydrase isoenzymes

Reaction of dimedone, 4-amino-N-(diamino- methylene)benzenesulfonamide, and aromatic aldehydes was successfully realized using sulfuric acid as a cheap catalyst. Synthesis of novel acridine sulfonamide compounds was performed providing high yields in water as the solvent at room temperature. This method has several advantages such as use of a green solvent, high yields, and efficient one-pot procedure. In addition, human carbonic anhydrase isoenzymes (hCA I and hCA II) were purified from erythrocyte cells by affinity chromatography. The inhibi- tory effects of acetazolamide and the newly synthesized acridine sulfonamides on hydratase and esterase activities of these isoenzymes was studied in vitro. The esterase IC50 values of the new compounds are 47.2-230.1 lM for hCA I and 50.1-275.0 lM for hCA II.

Novel Sulphamides and Sulphonamides Incorporating the Tetralin Scaffold as Carbonic Anhydrase and Acetylcholine Esterase Inhibitors

Reactions of amino, aminomethyl tetralins and benzyl alcohol with chlorosulphonyl isocyanate (CSI) afforded sulphamoyl carbamates. The sulphamoyl carbamates were converted to sulphamides by palladium-catalysed hydrogenolysis. Sulphonamides were synthesized from the reactions of amines with MeSO2 Cl. Inhibition of human (h) carbonic anhydrase (CA) isoenzymes (hCA I, hCA II) and acetylcholine esterase (AChE) was investigated with the synthesized compounds. hCA I and hCA II were inhibited in the low micromolar or sub-micromolar range. The Ki values were in the range of 0.91-9.56 µM against hCA I and of 3.70-27.88 µM against hCA II. Sulphamides 11-13 and sulphonamides 14-16 had moderate inhibition capacity toward AChE. These findings suggest the novel sulphamides 11-13 and sulphonamides 14-16 as AChE and CA isoenzyme inhibitory agents.

Synthesis and characterization of a proton transfer salt between dipicolinic acid and 2-amino-6-methylbenzothiazole and its complexes, and their inhibition studies on carbonic anhydrase isoenzymes

A novel proton transfer compound (HMeABT)+(HDPC)−·H2O (1) obtained from 2-amino-6-methylbenzothiazole (MeABT) and dipicolinic acid (H2DPC) and its Fe(III), Fe(II), Co(II), Ni(II) and Cu(II) complexes (2–6) have been prepared and characterized by elemental, spectral (1H NMR, IR and UV–Vis) and thermal analyses, magnetic measurement and molar conductivity. Additionally, single crystal X-ray diffraction techniques were applied to all complexes.All compounds, including acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibition effects on human carbonic anhydrase isoenzymes (hCA I and hCA II) for their hydratase and esterase activities. Although there is no inhibition for hydratase activities, all compounds have inhibited the esterase activities of hCA I and II. The comparison of the inhibition studies of 1–6 to parent compounds, MeABT and H2DPC, indicates that 1–6 have more inhibitory effects. The inhibition effects of 2–6 are also compared to the simple metal complexes of MeABT and H2DPC, and the complexes from proton transfer salt (2–6) have shown better inhibition effects. Nevertheless, all the studied compounds have less inhibition effects than AAZ.

Facile synthesis and characterization of novel pyrazole-sulfonamides and their inhibition effects on human carbonic anhydrase isoenzymes

In the current study, a series of pyrazole-sulfonamide derivatives (2–14) were synthesized, characterized, and the inhibition effects of the derivatives on human carbonic anhydrases (hCA I and hCA II) were investigated as in vitro. Structures of these sulfonamides were confirmed by FT-IR, 1H NMR, 13C NMR and LC–MS analysis. 1H NMR and 13C NMR revealed the tautomeric structures. hCA I and hCA II isozymes were purified from human erythrocytes and inhibitory effects of newly synthesized sulfonamides on esterase activities of these isoenzymes have been studied. The Ki values of compounds were 0.062–1.278μM for hCA I and 0.012–0.379μM for hCA II. The inhibition effects of 7 for hCA I and 4 for hCA II isozymes were almost in nanomolar concentration range.

Synthesis and characterisation of novel Co(II) complexes of pyrazole carboxylate derivated of sulfonamide as carbonic anhydrase inhibitors

Two new metal complexes, diaquabis(4-benzoyl-1,5-diphenyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-carboxamide)cobalt(II) dihydrate (2) and diaquabis(ethyl-1-(3-nitrophenyl)-5-phenyl-3-(5-sulfamoyl-1,3,4-thiadiazol-2-ylcarbamoyl)-1H-pyrazole-4-carboxylate)cobalt(II) monohydrate (4), containing sulfonamide have been synthesized by the reaction of Co(II) with 4-benzoyl-1,5-diphenyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (1) and ethyl-1-(3-nitrophenyl)-5-phenyl-3-(5-sulfamoyl-1,3,4-thiadiazol-2-ylcarbamoyl)-1H-pyrazole-4-carboxylate (3), respectively. The structures of Co(II) complexes 2 and 4 have been characterised by spectroscopic methods and elemental analyses. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of ligands 3 and 4, acetazolamide as a control compound and the newly synthesized complexes on the activity of hydratase and esterase of these isoenzymes have been studied in vitro. The concentration of compounds 2 and 4 producing a 50% inhibition of hydratase activity (IC(50) values) were 0.473 ± 0.025 and 0.065 ± 0.002 μm for hCA-I and 0.213 ± 0.015 and 0.833 ± 0.021 μm for hCA-II, respectively. The IC(50) values of synthesized compounds 2 and 4 for esterase activity were, 0.058 ± 0.006 and 0.297 ± 0.015 μm for hCA-I and 0.110 ± 0.010 and 0.052 ± 0.002 μm for hCA-II, respectively. In relation to esterase activity, the inhibition equilibrium constants (K(i) ) were determined as 0.039 ± 0.004 and 0.247 ± 0.035 μm on hCA-I and 0.078 ± 0.002 and 0.363 ± 0.015 μm on hCA-II for 2 and 4, respectively. The synthesized compounds 2 and 4 had effective inhibitory activity (P < 0.0001) on hCA-I and hCA-II than the corresponding free ligands, 1 and 3, and acetazolamide. Compounds 2 and 4 might be considered as potential inhibitors.

Synthesis and characterization of metal complexes of heterocyclic sulfonamide as carbonic anhydrase inhibitors

Three novel metal complexes of N-[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl]-4-benzoyl-1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3-carboxamide which possess strong carbonic anhydrase (CA) inhibitory properties have been synthesised. The structure of these compounds has been investigated by elemental analysis, FT-IR, LC/MS, UV–vis spectrophotometric method and magnetic susceptibility. Human carbonic anhydrase isoenzymes hCA-I and hCA-II were purified from erythrocyte cells by the affinity chromatography. The inhibitory effects of newly synthesized complexes and acetazolamide (AAZ) as a control compound on hydratase and esterase activities of these isoenzymes have been studied in vitro by comparing IC50 and Ki values and it has been found that the newly synthesised complexes behave as very powerful inhibitors against hCA-I and hCA-II than parent ligand (1) and than AAZ.

Synthesis and characterization of novel dioxoacridine sulfonamide derivatives as new carbonic anhydrase inhibitors

Novel dioxoacridine sulfonamide compounds were synthesized from reaction of cyclic 1,3-diketones, sulfanilamide (4-amino benzene sulfonamide) and aromatic aldehydes. The structures of these compounds were confirmed by using spectral analysis (IR, H-NMR, 13C-NMR, and mass). Human carbonic anhydrase isoenzymes (hCA I and hCA II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of sulfanilamide, acetazolamide (AAZ), and newly synthesized sulfonamides on hydratase and esterase activities of these isoenzymes have been studied in vitro. The IC50 values of compounds for esterase activity are 0.71–0.11 µM for hCA I and 0.45–0.12 µM for hCA II, respectively. The Ki values of these inhibitors were determined as 0,38–0,008 µM for hCA I and 0,19–0,001 µM for hCA II, respectively.

Synthesis and characterization of a novel amino salicylato salt and its Cu(II) complex and their inhibition studies on carbonic anhydrase isoenzymes

A novel amino salicylato salt, 2-amino-6-methylpyridinium 2-hydroxy-5-sulfonatobenzoate ( 1), and its Cu(II) complex, 2-ammonio-6-methylpyridinium bis(μ 6-5-sulfonatosalicylatoaquacopper(II)) ( 2), have been synthesized from free ligands, namely 5-sulfosalicylic acid (H 3ssa) and 2-amino-6-methylpyridine (amp). Compounds 1 and 2 have been characterized by elemental, spectral ( 1H NMR, IR and UV–Vis) and thermal analyses. Additionally, magnetic measurements and the single crystal X-ray diffraction technique have been applied to compound 2. Compound 2 crystallizes in the triclinic P 1 ¯ space group. In the symmetric unit, the Cu(II) ion exhibits a distorted square planar configuration, coordinated by two carboxylate oxygen atoms (O2 and O2 i ), one phenolic oxygen atom (O1) of the ssa 3− anion and a water molecule (O1w). The free ligands H 3ssa and amp, and the products 1 and 2, and acetazolamide (AAZ) as a control compound have been also evaluated for their in vitro inhibitor effects on human carbonic anhydrase isoenzymes (hCA I and hCA II), purified from the erythrocyte cell by affinity chromatography for their hydratase and esterase activities. In relation to esterase activities, the inhibition equilibrium constants ( Ki) have also been determined. A comparison of the inhibition studies of the newly synthesized compounds 1 and 2 with the parent compounds H 3ssa and amp, and to AAZ, indicates that 1 and 2 have an effective inhibitory activity on hCA I and II, and can be used as potential inhibitors.

Synthesis and characterisation of two novel proton transfer compounds and their inhibition studies on carbonic anhydrase isoenzymes

Two novel proton transfer compounds were prepared between 2,4-dichloro-5-sulphamoylbenzoic acid (lasamide) (Hsba) and ethylenediamine (en), namely ethane-1,2-diaminium 2,4-dichloro-5-sulphamoylbenzoate (1), and also between Hsba and 2-amino-3-methylpyridine (2-amino-3-picoline) (amp), namely 2-amino-3-methylpyridinium 2,4-dichloro-5-sulphamoylbenzoate (2). All these were characterised by elemental, spectral (IR and UV-vis), thermal analyses, and single crystal X-ray diffraction studies. Compounds 1 and 2 crystallised in the P-1 and P21/c space groups, respectively. Intermolecular non-covalent interactions, such as ion pairing, hydrogen bonding, and π-π stacking were observed for these ionic compounds. The free ligands Hsba, en and amp, the products 1 and 2, and acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibitor effects on the human carbonic anhydrase isoenzymes (hCA I and hCA II) purified from erythrocyte cells by affinity chromatography for their hydratase and esterase activities. The half maximal inhibitory concentration (IC50) values for products 1 and 2 with respect to hydratase activity are 0.15 and 0.32 µM for hCA I and 0.06 and 0.15 µM for hCA II, respectively. The IC50 values of the same inhibitors for esterase activity are 0.13 and 0.8 µM for hCA I and 0.14 and 0.1 µM for hCA II, respectively. In relation to esterase activities, the inhibition equilibrium constants (Ki) were also determined and found to be 0.137 and 0.99 µM on hCA I and 0.157 and 0.075 µM on hCA II for 1 and 2, respectively. The comparison of the inhibition studies of the newly synthesised compounds 1 and 2 to the parent compounds Hsba and amp and also to AAZ indicated that 1 and 2 have an effective inhibitory activity on hCA I and II, and might be used as potential inhibitors.

Synthesis of 5-amino-1,3,4-thiadiazole-2-sulphonamide derivatives and their inhibition effects on human carbonic anhydrase isozymes

In this study, some novel inhibitors were synthesised from the further stage reactions of 4-benzoyl-1-(4-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride with 5-amino-1,3,4-thiadiazole-2-sulphonamide 1 (inhibitor 1). They were characterised by elemental and spectral (1H NMR, 13C NMR, IR) analyses. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of inhibitor 1, acetazolamide (2) and the 11 newly synthesised amides (8–18) on the hydratase and esterase activities of these isoenzymes (hCA-I and hCA-II) were studied in vitro. In relation to these activities, the inhibition equilibrium constants (Ki) were determined. The Ki values for the new compounds (8–18) were observed to be well below that of the parent compound inhibitor 1 and were also compared to 2 under the same experimental conditions. The comparison of the newly synthesised amides to inhibitor 1 and to 2 indicated that the new derivatives preferentially inhibited hCA-II and were more potent inhibitors of hCA-II than the parent inhibitor 1 and 2.

Synthesis and investigation of inhibition effect of fluorinated sulfonamide derivatives on carbonic anhydrase

Series of perfluoroalkanesulfonamides 1, sodium salt of perfluoroalkanesulfonamides 2 and polyfluoroalkanesulfonamides 3 derivatives were synthesized and characterized by 1H NMR, 13C NMR, 19F NMR, IR and HRMS. Inhibition effects of these compounds on bovine carbonic anhydrase (bCA) and human carbonic anhydrase isoenzyme II (hCA) have been investigated. Comparing IC 50 values of the synthesized molecules 1, 2 and 3, it has been found that compound 2b is a more potent inhibitor than acetazolamide on hCA. Moreover 2b does not present cellular toxicity on sheep red globules.

Synthesis, characterization and antiglaucoma activity of a novel proton transfer compound and a mixed-ligand Zn(II) complex

A novel proton transfer compound, pyridin-2-ylmethanaminium 2,4-dichloro-5-sulfamoylbenzoate (1), and a mixed-ligand Zn(II) complex, bis(2,4-dichloro-5-sulfamoylbenzoate)(2-aminomethylpyridine)aquazinc(II) monohydrate (2), have been synthesized from the same free ligands, which are 2,4-dichloro-5-sulfamoylbenzoic acid (Hsba) and 2-aminomethylpyridine (amp). They have been characterized by elemental, spectral ((1)H NMR, IR and UV-vis.) and thermal analyses. Additionally, magnetic measurement and single crystal X-ray diffraction technique were applied to compound 2. In the complex, Zn(II) ion exhibits a distorted octahedral configuration coordinated by O1 and O1(i) atoms of two mono dentante sba anions and N1, N2, N2(i) atoms of bidentante amp anion and a water molecule (O1w). The free ligands Hsba and amp, and the products 1 and 2, and acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibitor effects on human Carbonic Anhydrase isoenzymes (hCA I and hCA II) purified from erythrocyte cell by affinity chromatography for their hydratase and esterase activities. The IC(50) values of products 1 and 2 for hydratase activity are 0.26 and 0.13microM for hCA I and 0.30 and 0.15microM for hCA II, respectively. The IC(50) values of the same inhibitors for esterase activity are 0.32 and 0.045microM for hCA I and 0.29 and 0.23microM for hCA II, respectively. In relation to esterase activities, the inhibition equilibrium constants (K(i)) were also determined and found 0.25 and 0.058microM on hCA I and 0.22 and 0.24microM on hCA II for 1 and 2, respectively. The comparison of the inhibition studies of newly synthesized compounds 1 and 2 to parent compounds Hsba and amp and to AAZ indicated that 1 and 2 have effective inhibitory activity on hCA I and II, and might be used potential inhibitors.

Effects of new 5-amino-1,3,4-thiadiazole-2-sulfonamide derivatives on human carbonic anhydrase isozymes

Pyrazole carboxylic acid amides of 5-amino-1,3,4-thiadiazole-2-sulfonamide 1 (inhibitor 1) were synthesized from 4-benzoyl-1-(4-nitrophenyl)-5-phenyl-1 H-pyrazole-3-carbonyl chloride and 4-benzoyl-1-(3-nitrophenyl)-5-phenyl-1 H-pyrazole-3-carbonyl chloride compounds. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by the affinity chromatography. The inhibitory effects of inhibitor 1, acetazolamide ( AAZ), and of 16 newly synthesized amides ( 8–11, 12a– f, 13a– c, 14a– b, and 15) on hydratase and esterase activities of these isoenzymes have been studied in vitro. The average IC 50 values of the new compounds ( 8–11, 12a– f, 13a– c, 14a– b, and 15) for hydratase activity ranged from 3.25 to 4.75 μM for hCA-I and from 0.055 to 2.6 μM for hCA-II. The mean IC 50 values of the same inhibitors for esterase activity were in the range of 2.7–6.6 μM for hCA-I (with the exception of inhibitor 10, which did not inhibit the esterase activity of hCA-I) and of 0.013–4.2 μM for hCA-II. The K i values for new compounds ( 8–11, 12a– f, 13a– c, 14a– b, and 15) were observed well below that of the parent compound inhibitor 1 and were also comparable to that of AAZ under the same experimental conditions. The comparison of newly synthesized amides to inhibitor 1 and to AAZ indicated that the new derivatives preferentially inhibit hCA-II and are more potent inhibitors of hCA-II than the parent inhibitor 1 and AAZ.

Effects of Morphine and Hashish (Cannabis sativa) on Activities of Human Red Blood Cell Carbonic Anhydrase Isoenzymes

Carbonic anhydrase (CA) exists in all living organisms and converts CO2 (waste product) to H2CO3. Two different forms of CA are present in human erythrocytes. These forms are known as CA-I and CA-II. Whereas CA-I has a low activity, CA-II is highly active. In the current study, CA-I and CA-II were purified by affinity chromatography from human blood. Then the effects of hashish (Cannabis sativa L.) and morphine on the CA-I and CA-II activities were measured by following esterase activity, and the results are presented.

Immunohistochemical localization of carbonic anhydrases I, II, and VI in the developing rat sublingual and submandibular glands This article is a US Government work and, as such, is in the public domain in the United States of America.

Carbonic anhydrase has been localized to the acini and ducts of mature rat salivary 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 to immunohistochemically document developmental changes in carbonic anhydrase in the ducts of the rat sublingual and submandibular glands. Immunohistochemistry was performed with antibodies to human carbonic anhydrase isoenzymes I, II and VI on sections of sublingual and submandibular glands from rats at representative postnatal developmental ages. Reactions were weak in the ducts of both glands at 1 day, then progressively increased. By 42 days, reactions had the adult pattern of virtually none in the mucous or seromucous acini, moderate to strong in the striated and excretory ducts, and none to weak in the intercalated ducts. Weak to moderate reactions were observed in the granular convoluted tubules of the submandibular gland as they became recognizable at age 42 days. Reactions to carbonic anhydrase I and II antibodies also increased from none (1 day) to modest (42 days) in the demilunes of the sublingual gland. The order of reaction intensity of the antibodies was II > I > VI. When localized via these anti-human antibodies, carbonic anhydrase is a useful marker of the functional differentiation of the striated and excretory ducts of the developing rat sublingual and submandibular glands. Anat Rec 258:269–276, 2000. © 2000 Wiley-Liss, Inc.

Immunohistochemical localization of carbonic anhydrases I, II, and VI in the developing rat sublingual and submandibular glands.

Carbonic anhydrase has been localized to the acini and ducts of mature rat salivary 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 to immunohistochemically document developmental changes in carbonic anhydrase in the ducts of the rat sublingual and submandibular glands. Immunohistochemistry was performed with antibodies to human carbonic anhydrase isoenzymes I, II and VI on sections of sublingual and submandibular glands from rats at representative postnatal developmental ages. Reactions were weak in the ducts of both glands at 1 day, then progressively increased. By 42 days, reactions had the adult pattern of virtually none in the mucous or seromucous acini, moderate to strong in the striated and excretory ducts, and none to weak in the intercalated ducts. Weak to moderate reactions were observed in the granular convoluted tubules of the submandibular gland as they became recognizable at age 42 days. Reactions to carbonic anhydrase I and II antibodies also increased from none (1 day) to modest (42 days) in the demilunes of the sublingual gland. The order of reaction intensity of the antibodies was II > I > VI. When localized via these anti-human antibodies, carbonic anhydrase is a useful marker of the functional differentiation of the striated and excretory ducts of the developing rat sublingual and submandibular glands.