Immunoelectron microscopic localization of carbonic anhydrase III in rat skeletal muscle

Summary The subcellular distribution of carbonic anhydrase III in rat soleus and vastus lateralis muscles was studied using an immunogold technique. The enzyme protein was found to be distributed diffusely in the cytoplasm of skeletal muscle cells. Red skeletal muscle (mainly type I fibers) revealed very strong immunogold staining whereas in white muscle (mainly type II fibers) gold particles were almost completely absent. No immunoreaction was observed in mitochondria or in other intracellular organelles.     

Immunocytochemical localization of carbonic anhydrase isozyme III in equine thymus

Sections of equine thymus were examined for the presence of carbonic anhydrase (CA) isozymes by an immunohistochemical method. Carbonic anhydrase III, a major enzyme of skeletal muscle, was localized in some of the epithelial-reticular cells of the equine thymus. This finding suggests the presence of a new type of cell in the thymic cortex. The concentration of CA-III in the thymus was 17 micrograms/g wet tissue. CA-I and CA-II were not found in equine thymus.     

Immunocytochemical localization of carbonic anhydrase isozyme III in equine thymus

Summary Sections of equine thymus were examined for the presence of carbonic anhydrase (CA) isozymes by an immunohistochemical method. Carbonic anhydrase III, a major enzyme of skeletal muscle, was localized in some of the epithelial-reticular cells of the equine thymus. This finding suggests the presence of a new type of cell in the thymic cortex. The concentration of CA-III in the thymus was 17 g/g wet tissue. CA-I and CA-II were not found in equine thymus.     

Characterization of human carbonic anhydrase III from skeletal muscle

A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.     

Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues

Carbonic anhydrase (CA) III was demonstrated immunocytochemically in epithelium in some regions of salivary gland ducts, colon, bronchi, and male genital tract and in adipocytes, in addition to skeletal muscle and liver where the isozyme was previously localized. Basal cells beneath the submandibular gland's excretory ducts in guinea pig stained for CA III. Carbonic anhydrase III occurred alone in some and with CA II in other sites but was often absent from CA-II-containing types of cells. This was exemplified by CA III's abundance in CA-II-positive proximal colon and its sparsity in the CA-II-rich distal colon of the mouse. Striated ducts in guinea pig, but not mouse salivary glands, stained darker for CA and appeared accordingly to function more actively in ion transport compared with excretory ducts. Carbonic anhydrase content varied among genera in liver and pancreas and between mouse species and strains in salivary glands and kidney. Newly observed murine sites of CA II activity included Auerbach's plexus and a population of leukocytes infiltrating the lamina propria in small intestine, and several types of cells in the male genital tract. In immunoblot tests, antisera to CA III showed no cross reactivity with antisera to CA II, but those to CA II disclosed weak cross reactivity with CA III.     

Membrane-associated carbonic anhydrase IV in skeletal muscle: subcellular localization

 Carbonic anhydrase IV (CA IV) was examined by light microscopy and electron microscopy in rat soleus muscle. Semithin sections of aldehyde-fixed Epon-embedded muscle were stained with rabbit anti-rat lung CA IV and the avidin-biotin-peroxidase complex. With this technique, capillaries and sarcolemma showed positive CA IV staining. For electron microscopy, rat soleus specimens were aldehyde-fixed, with or without subsequent osmication, and embedded in Epon. Ultrathin sections were immunostained with anti-rat lung CA IV/immunogold. Omitting osmium allowed ample antigen-antibody reactions but could not prevent the release of glycosylphosphatidylinositol-anchored CA IV from the membranes, which led to apparent background staining. Postosmication significantly reduced tissue antigenicity but kept the antigen bound to the membranes and thus allowed a very precise localization of CA IV. By electron microscopy, membrane-bound CA IV is found to be associated with capillary endothelium, sarcolemma, and sarcoplasmic reticulum (SR). Conceivably, the presence of SR staining in ultrathin sections and its absence in semithin sections reflect a problem of accessibility of the antigenic sites. Accepted: 17 May 1996     

Comparative immunohistolocalization of carbonic anhydrase isozymes I, II and III in the equine and bovine digestive tract

Summary Immunohistochemical localizations of carbonic anhydrase isozymes (CA-I, CA-II and CA-III) in equine and bovine digestive tracts were studied. In the horse, epithelial cells in both the oesophagus and non-glandular part of the stomach lacked all three isozymes. In contrast, surface epithelial and parietal cells in the glandular region of the stomach showed reactivity for CA-II. In the small intestine, absorptive columnar cells covering the villi in the duodenum were positive for CA-II. The epithelium of the jejunum and ileum lacked all three isozymes. In the large intestine, CA-II was detected in the columnar cells in the upper part of the crypt. In cattle, epithelial cells of the oesophagus showed reactions for CA-I and CA-III but not for CA-II. Although the absorptive epithelial cells of the small intestine lacked CA-I, CA-II and CA-III, those of the upper part of large intestine crypts were heavily stained for all three isozymes.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I,II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K_i values of the molecules 3–6 were in the range of 41.12–363 μM against hCA I, of 0.381–470 μM against hCA II and of 0.578–1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K_A values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Rat lung carbonic anhydrase: activity, localization, and isozymes

Carbonic anhydrase activity in rat lungs perfused free of blood was localized by homogenization of the tissue followed by differential centrifugation. Four fractions were obtained from the homogenate, a cell debris pellet with a mitochondrial pellet and a microsomal pellet with a clear cytosol supernatant. The last named fraction contained 67% of the total enzyme activity; the cell debris contained 18%, and the mitochondrial and microsomal contained 8 and 7%, respectively. Of the 33% of enzyme activity associated with the pellet fraction, 25% could be experimentally defined as membrane associated by its solubilization with 0.3 M tris-(hydroxymethyl) aminoethane sulfate buffer. The remainder was defined as membrane bound. Purification of the soluble carbonic anhydrase from the lung yielded two isozymes with electrophoretic and inhibitor sensitivities apparently identical with the blood isozymes. Hemoglobin analysis showed that the lung isozymes could not have included more than 0.03% enzyme from blood contamination. The carbonic anhydrase activity present in the whole rat lung would give an average acceleration of the CO2 hydration reaction under physiological conditions over the uncatalyzed rate of 122, sufficient to maintain equilibration between CO2 and plasma HCO3- during blood transit of the lung. If the membrane-associated activity is mostly on the plasma membrane of the endothelial cells and available to the capillary blood, it would be sufficient to give this acceleration. We suggest that the possible source of this membrane-associated activity might be adsorption from the blood of carbonic anhydrase liberated by erythrocyte lysis.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Expression of carbonic anhydrase isozymes II and III in developing bovine parotid gland

Two cytosolic carbonic anhydrase isozymes (CA-II and CA-III) were studied by immunohistochemistry in bovine parotid glands during fetal development. In a 3-month-old fetus of crown-rump length (CRL) 17 cm, the expression of CA-II in undifferentiated epithelial cells was observed, whereas immunostaining for CA-III remained negative. At 26 cm CRL (4–5 months old), weak expression of CA-III in large ductal epithelial cells was noted. The accumulation of secreted granules in primary acinar cells was initially observed at this stage. In a newborn calf, anti-CA-II reactivity almost disappeared from most duct segments. The time-dependent expression and distribution of the isozymes in parotid glands may reflect different biological functions of these structurally closely related isozymes. Bovine parotid acinar cells of fetuses would thus appear to possess all the cellular structures and immunohistochemical properties at 4 and 5 months of gestation. CA-II subsequently disappeared from duct segments and nearly all acinar cells in adults were present at or just after birth.     

Novel carbonic anhydrase isozymes I, II and IV activators incorporating sulfonyl-histamino moieties.

Sulfonylamido(ureido) derivatives of histamine were synthesized by an original procedure in order to obtain tight-binding activators of the zinc enzyme carbonic anhydrase (CA), exploiting the binding energy of the alkyl/arylsulfonyl moieties with amino acid residues at the entrance of the active site. In contrast to the lead molecule, histamine, the new derivatives possessed higher affinity for three different CA isozymes, as evidenced by compairing the affinity constants of these compounds for isozyme CA II.     

Immunohistochemical localization of carbonic anhydrase isoenzymes II and III in quail kidney

The immunohistochemical localization of carbonic anhydrase isoenzymes has never been investigated in avian renal tissue previously. Enzyme activity has largely been documented by histochemical and physiological reports. In this investigation, specific antisera were used to study the distribution of the cytosolic carbonic anhydrase II and III isoenzymes in the quail kidney. Comparison between the present findings and the corresponding histochemical patterns, previously obtained in the same species by a cobalt phosphate precipitation method, resulted in the bulk of renal carbonic anhydrase activity being attributed to the carbonic anhydrase II isoenzyme. Conversely, moderate carbonic anhydrase III immunostaining appeared to be confined to the smooth muscle cells of ureteral and arteriolar walls. Indirect evidence of the occurrence, in the quail kidney, of a membrane-associated carbonic anhydrase form, antigenically distinct from the II and III isoforms, was inferred.     

Immunocytochemical localization of carbonic anhydrase in myelinated fibers in peripheral nerves of rat and mouse

Rat sciatic nerve, spinal root, and cranial nerve were immunostained with an antibody against rat brain carbonic anhydrase II (ca), to determine the localization of ca in the rat peripheral nervous system (PNS). Similar methods were applied to mouse nerves to see if that antigen could be detected in the PNS of this species. In rat nerves, intense immunostaining was observed in the axoplasm of many of the myelinated fibers, whereas others were stained less intensely or were negative. A heterogeneous pattern of immunostaining was also found in neuronal perikarya within the ganglia, and in some regions of the ganglia ca immunostaining was found in putative satellite cells and their processes. Ca in rat PNS therefore appears to occur at both neuronal and glial sites, whereas it is exclusively glial in the CNS. In longitudinal sections of some fibers within rat nerves, ca immunostaining could be detected at the inner boundaries of the myelin sheaths. In mouse nerves, axoplasmic staining was observed but it was fainter than in rat nerves. Interspecies differences were most obvious in the dorsal columns of the spinal cord. In rat, intensely stained axons proceeded through the roots into the gracilis or cuneate and often into the gray matter. In mouse, there was much less immunostaining of axons but more intense ca immunostaining in CNS myelin than in the CNS myelin in the rat cord. The implications concerning putative functions of ca in the rodent nervous system are discussed.     

Characterisation of cDNA clones for rat muscle carbonic anhydrase III

cDNA clones for rat muscle carbonic anhydrase III have been isolated from a gt-11 library and sequenced. Comparison with human CAIII cDNA showed about 90% homology to rat. The rat clones were used to estimate mRNA from liver and muscle on Northern blots and showed that the sexual dimorphism of CAIII in rat liver relates to a difference in mRNA levels.     

Novel purification of carbonic anhydrase III from human, rat and baboon muscle

A new method of inhibitor elution from DEAE cellulose is described for carbonic anhydrase III. Highly purified fractions free of other isozymes were obtained after one column elution.     

Immunoelectron microscopy of carbonic anhydrase isozyme VI in rat submandibular gland: comparison with isozymes I and II.

Carbonic anhydrase (CA) was purified from the saliva of pilocarpine-treated rats by inhibitor-affinity chromatography, and its localization in the rat submandibular gland was studied by the indirect immunoperoxidase technique using a monoclonal antibody (MAb) raised against the enzyme. SDS-polyacrylamide gel electrophoresis of the CA VI gave three bands of 33, 39, and 42 KD. Enzyme digestion experiment showed that the 42 KD molecule was degraded into the 39 KD molecule and the 39 KD molecule into the 33 KD molecule. The cleavage of the 42 KD molecule was independent and that of the 39 KD molecule was dependent on endo-beta-N-acetylglucosaminidase F. The 42 KD molecule was detected in the CA purified from the pilocarpine-treated but not the untreated salivary gland. The MAb recognized all the three components of the enzyme. Immunostaining for CA VI was seen in the cytosol and secretory granules of serous acinar cells and in the duct luminal contents. Staining specific for erythrocyte CA (CA I and CA II) was observed in the cytosol of the epithelial cells of granular, striated, and excretory ducts. Among these duct cells, the agranular varieties in the granular and excretory ducts were essentially devoid of the immunoreactivity.     

Synthesis of rat muscle carbonic anhydrase III in a cell-free translation system

Carbonic anhydrase III (CA III) was identified in the products of rat soleus muscle RNA translation in vitro by both a radioimmunoassay and a specific immunoprecipitation technique followed by SDS--polyacrylamide gel electrophoresis analysis of the precipitated antigen. The primary translation product has the same Mr-value as the native isoenzyme. CA III mRNA was found to represent approximately 0.55% of the total mRNA present in rat soleus muscle.