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.     

Human carbonic anhydrase isoenzyme C

Summary 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 21 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° C in vacuo was found to give the best preservation of the antigenicity of HCA C with good morphological integrity for light microscopical localization.Grant support from the Finnish Culture Foundation     

Immunohistochemical demonstration of human carbonic anhydrase isoenzyme II in brain tumours

Summary Carbonic anhydrase (CA) is a functionally important enzyme in the central nervous system (CNS), where it is involved in the control of the acid-base balance and regulates the production of cerebrospinal fluid (CSF). Isoenzyme II (CA II) is the most widely distributed CA in the CNS, being present in at least myelin, oligodendrocytes, astrocytes and the choroid plexus. This study was undertaken to examine the presence of CA II in different brain tumours from 31 patients. Specific antibodies recognizing CA II were used in immunoperoxidase staining of tumour specimens. Anti-CA I and VI sera and normal rabbit serum were used as controls. CA II-positive staining was observed in all the astrocytic tumours (n = 9), oligodendrogliomas (n = 3) and medulloblastomas (n = 3). The most malignant tumours exhibited the strongest staining. In addition, four acoustic neurinomas, one plexiform neurofibroma, one choroid plexus papilloma, one ependymoblastoma and one subependymoma expressed the enzyme. Meningiomas (n = 4) and neuronal tumours (N = 4), including one dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos), were negative. Anti-CA I, VI and normal rabbit sera showed no specific staining in tumour cells. The presence of CA II in the astrocytomas was confirmed by Western blotting, which revealed a distinct 29 kDa polypeptide band corresponding the CA II. Anti-CA I serum showed similarly a single 29 kDa band, recognizing the enzyme which is abundantly present in the erythrocytes. The present results demonstrate that despite the malignant transformation of the cells, the expression of CA II is sustained in astrocytic tumours, oligodendrogliomas, ependymal and choroid plexus tumours and tumours of nerve sheath cell origin. Our results suggest that some tumours contain abundant CA II, which might leak into the CSF.     

Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme

Summary Rabbits immunized with low-activity ruminal carbonic anhydrase (RCA) isoenzyme, extracted from ruminal epithelial cells isolated by digestion with trupsin, yielded anti-RCA sera which reacted specifically with bovine RCA in double agar gel diffusion and immunoelectrophoretic tests, but failed to cross-react with bovine erythrocyte CA. The localization of RCA was identified in histological sections and isolated ruminal epithelial cell preparations by indirect immunofluorescence and immunoperoxidase tests as the basal, spinosum and granulosum layers of ruminal mucous epithelium.     

Immunohistochemical demonstration of carbonic anhydrase isoenzyme C in the epithelium of the human ciliary processes

Extravascular location of two main carbonic anhydrase isoenzymes was immunohistochemically investigated in human ciliary processes. The high-activity carbonic anhydrase isoenzyme C was clearly demonstrated in the ciliary epithelium, but was absent from the ciliary stroma. The low-activity isoenzyme B was evident neither in the epithelium nor in the stroma.     

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

Summary 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 vescle, 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.     

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.     

Purification and localization of human carbonic anhydrase

Summary Three different isoenzymes of human carbonic anhydrase are now well characterized. Carbonic anhydrase I and II have been known for several years and are located in high amounts in red blood cells as well as in many other tissues.Carbonic anhydrase III, a protein showing CO₂ hydratase and p-nitrophenylphosphatase activity was isolated from skeletal muscle some years ago. Earlier observations based on enzyme activity and radioimmunoassay studies have suggested that this protein is present in greater quantities in red skeletal muscles than in white ones. We have purified CA III from human soleus muscle and using obtained monospecific polyclonal antibody localized this protein in the same muscle fibers which show acid resistant ATPase activity. Using this protein as a marker for type I muscle fibers, fiber classification into type I and II could now be done also from paraffin embedded sections.This study is supported by the Research Council of Physical Education and Sport, Ministry of Education, Finland     

Immunohistochemical demonstration of carbonic anhydrase.

Rabbits were immunized using human erythroxyte carbonic anhydrase B (HCA B) purified by the modified methods of Armstrong et al. (1966) and Bernstein and Schraer (1972). The globulin fraction was isolated by ammonium sulphate precipitation. The anti-HCA B globulin was specific, when judged using the double diffusion technique of Ouchterlony and immunoelectrophoresis. No cross reaction with human erythrocyte carbonic anhydrase C was found, but cross reactions with erythrocyte carbonic anhydrase from rat, mouse and guinea pig were observed. Flurorescein isothiocyanate conjugated goat anti-rabbit globulin was used for the localization of HCA B in tissue sections and erythrocytes on slides.     

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.     

Ultrastructural immunocytochemical localization of carbonic anhydrase

Summary Specific antibodies against human erythrocyte carbonic anhydrase isozyme C were used to determine the ultrastructural localization of this enzyme in the collecting ducts of rat kidney. Using a pre-embedding labeling technique, carbonic anhydrase C was found in the cytoplasm of intercalated cells, whereas the principal cells were negative.     

Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands

Human salivary carbonic anhydrase (HCA VI) was purified by inhibitor affinity chromatography and its location in the human parotid and submandibular glands identified, using a polyclonal antiserum raised against the purified enzyme in rabbits in conjunction with the peroxidase-antiperoxidase complex method. The antibodies raised against the purified enzyme in rabbits did not crossreact with the HCA II or I. However, they slightly recognized human IgA; the antiserum was therefore absorbed with human IgA before immunohistochemical use. HCA VI-specific staining was detected in the cytoplasm and particularly in the secretory granules of the serous acinar cells of both parotid and submandibular glands, the staining of the secretory granules being most distinct in paraformaldehyde-fixed tissues. Some epithelial cells and the luminal content of the striated ducts also gave a specific HCA VI staining. Staining specific for HCA II was also found in the granules of the serous acinar cells, particularly in the submandibular gland when Carnoy fluid fixation was used. Slight HCA II-specific staining was also detected in the striated ductal cells in the Carnoy fluid-fixed specimens. No staining specific for HCA I was detected. The results indicate that the serous acinar cells in human parotid and submandibular glands contain abundant HCA II and HCA VI. Interestingly, only HCA VI is secreted into the saliva, although both enzymes appear to be located in structures resembling the secretory granules in the acinar cells. The enzymes probably form a mutually complementary system regulating the salivary buffer capacity.     

The catalytic mechanism of carbonic anhydrase. Hydrogen-isotope effects on the kinetic parameters of the human C isoenzyme.

1. The steady-state kinetics of the interconversion of CO2 and HCO3 catalyzed by human carbonic anhydrase C was studied using 1H2O and 2H2O as solvents. The pH-independent parts of the parameters k(cat) and Km are 3-4 times larger in 1H2O than in 2H2O for both directions of the reaction, while the ratios k(cat)/Km show much smaller isotope effects. With either CO2 or HCO3 as substrate the major pH dependence is observed in k(cat), while Km appears independent of pH. The pKa value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2H2O than in 1H2O. 2. The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhudrase C is approximately 35% faster in 2H2O than in 1H2O. In both solvents the pKa values of the pH-rate profiles are similar to those observed for the CO2-HCO3 interconversion. 3. It is tentatively proposed that the rate-limiting step at saturating concentrations of CO2 or HCO3 is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO2 and HCO3 involves a proton trnasfer or not.     

Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat.

The distribution of carbonic anhydrase isozyme II (CA II)-like immunoreactivity (-LI) in the gustatory epithelium was examined in the adult rat. In the circumvallate and foliate papillae, CA II-LI was observed in the cytoplasm of the spindle-shaped taste bud cells, with weak immunoreaction in the surface of the gustatory epithelium. No neuronal elements displayed CA II-LI in these papillae. There was no apparent difference in the distribution pattern between the anterior and posterior portions of the foliate papillae. In immunoelectron microscopy, immunoreaction products for CA II were diffusely distributed in the entire cytoplasm of the taste bud cells having dense round granules at the periphery of the cells. No taste bud cells displaying CA II-LI were detected in the fungiform papillae, but a few thick nerve fibers displayed CA II-LI. In the taste buds of the palatal epithelium, neither taste bud cells nor neuronal elements exhibited CA II-LI. The present results indicate that CA II was localized in the type I cells designated as supporting cells in the taste buds located in the posterior lingual papillae of the adult animal.     

Immunohistochemical demonstration of carbonic anhydrase isoenzyme VI (CA VI) expression in rat lower airways and lung.

Carbonic anhydrase isoenzyme VI (CA VI), which is transported in high concentrations in saliva and milk into the alimentary tract, is an important element of mucosal protection in the upper alimentary tract. Like alimentary tract mucosa, the respiratory tract mucosa is also exposed to heavy microbial, physical, and chemical stress. The protective and renewal-promoting factors present in the surface mucus of the respiratory tract are mainly produced by the seromucous tracheobronchial glands. Here we studied the secretion of CA VI by these glands in adult and developing rats using immunohistochemical techniques. The serous acinar and duct cells of the tracheobronchial glands stained for CA VI. The presence of the enzyme also in the duct content indicates its active secretion into the surface mucus. CA VI was also visible in the secretory cells and at the base of the ciliated cells of the tracheobronchial surface epithelium. Moreover, the Clara cells of the bronchiolar surface epithelium stained for CA VI. These findings are consistent with the hypothesis that CA VI has a mucosa-protective role not only in the gastrointestinal tract but also in the respiratory tract, where CA VI may act as a pivotal pH neutralizer and growth factor.     

Immunolabeling of carbonic anhydrase isoenzyme C and glial fibrillary acidic protein in paraffin-embedded tissue sections of human brain and retina

The specificities of carbonic anhydrase isoenzyme C (CA C) and glial fibrillary acidic (GFA) protein as immunocytochemical markers for different glial cell populations in human brain and retina were studied using indirect immunofluorescence and peroxidase-antiperoxidase complex methods. With antibodies against CA C, only those cerebral cells that were morphologically oligodendrocytes and Müller cells of the retina showed positive immunostaining reaction, whereas antibodies against GFA protein selectively labeled cerebral astrocytes and a part of the glial cells and fibers in the inner layers of the retina. In double labeling, when both glial cell markers were successively localized in the same cerebral tissue sections, GFA protein immunofluorescence was never found in the immunoperoxidase-stained CA C-positive cells, which further supports the oligodendrocyte-specificity of CA C in human brain.     

Immunohistochemical study of carbonic anhydrase III in the extraocular muscles of human embryos.

The differentiation of extraocular muscles was studied immunohistochemically in externally normal human embryos (Carnegie stages 13-23), using antibodies to carbonic anhydrase (CA) III and beta-enolase as the markers of type 1 and type 2 muscle fibers, respectively. At stage 18, some myoblasts were immunoreactive to beta-enolase antibodies, however, CA-III immunoreactivity was not observed around the optic vesicle. At stage 20, CA-III immunoreactivity appeared in some muscle fibers of extraocular muscles. From stage 21 to stage 23, CA-III-immunoreactive fibers increased and almost equalled the number of beta-enolase-immunoreactive fibers. These findings suggest that CA-III-immunoreactive type 1 fibers appear in the late stage of myogenesis compared with beta-enolase-immunoreactive type 2 fibers.