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.     

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 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.     

Immunohistochemical localization of human carbonic anhydrase isoenzyme C

Summary 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.     

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 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.     

The distribution of carbonic anhydrase II in human, pig and rat oesophageal epithelium

Carbonic anhydrase II (CA II) is present in human oesophageal epithelial cells and probably involved in protecting the mucosa against acidic gastric refluxate. If this is the case, then it is likely that the enzyme will be more concentrated at or near the gastro-oesophageal junction. To answer this question, and determine whether CA II is present and similarly distributed in other species, we also examined the oesophageal epithelium of the rat and pig. In the rat, CA II was largely absent from the oesophageal epithelium, but present in the stratified squamous epithelium of the gastric forestomach as an approximately 2 mm-long collar around the entrance to the corpus, a site that roughly corresponds to the gastro-oesophageal junction in other animals. The enzyme was present mainly in basal and prickle cells. In upper and middle pig oesophagus, CA II was largely confined to basal cells and isolated groups of stratified superficial prickle cells. CA II-containing epithelial cells were highly concentrated in the thickened epithelium at the gastro-oesophageal junction (about four-times thicker than upper or middle). Reactive cells were present throughout the depth of the epithelium, but noticeably more concentrated in the basal and superficial prickle cell layers. CA II was also prominent in the most superficial cell layers in islands of the oesophageal mucosa within the gastric cardia. In man, CA II was confined largely to the basal half of the epithelium in the upper and middle regions of oesophagus. The distribution of CA II at the gastro-oesophageal junction took different forms. In general, there were more CA II-reactive cells at or closer to the lumen. The superficial prickle cell layers tended to exhibit more CA II than the deeper layers, with basal and epibasal cells containing little or no enzyme. In other regions of the same specimens, CA II-containing cells were present from the basal to the most luminal layers. If CA II in oesophageal epithelial cells in the region of the gastro-oesophageal junction (or in the case of the rat the forestomach/corpus junction) is important in the defence against refluxate, then it is in a vulnerable site, since bile salts are potent inhibitors of the enzyme. The action of bile salts on CA II may be an important factor in the initiation of oesophageal disease.     

Immunohistochemical demonstration of carbonic anhydrase

Summary 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. Fluorescein isothiocyanate conjugated goat anti-rabbit globulin was used for the localization of HCA B in tissue sections and erythrocytes on slides.     

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.     

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 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.     

Decreased total carbonic anhydrase esterase activity and decreased levels of carbonic anhydrase 1 isozyme in erythrocytes of type II diabetic patients

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pH_i for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes. Jehan Ornasir—these studies were undertaken as a partial requirement of her M.S. Degree, Graduate School, Howard University, Washington, DC, USA     

Immunohistochemical demonstration of the carbonic anhydrase isoenzymes I and II in pancreatic tumours

Summary The location of carbonic anhydrase (CA) isoenzymes I, II and VI in normal and neoplastic pancreatic tissue was studied using polyclonal antisera and the immunoperoxidase technique. Samples were obtained from patients with well-differentiated (n = 4), moderately differentiated (n = 1) and poorly differentiated (n = 4) ductal adenocarcinomas, cystadenocarcinoma (n = 2), adenosquamous carcinoma (n = 1), acinar adenocarcinoma (n = 1), gastrinoma (n = 3), insulinoma (n = 3) and glucagonoma (n = 1). The control specimens were from a patient with traumatic laceration of the pancreas. The normal and malignant endocrine tissue showed intense positive staining for CA I localized in the cells expressing glucagon. In the exocrine pancreatic tissue, CA II was detected in the normal and neoplastic ductal epithelium. No specific staining was detected with anti-CA VI serum in either normal or malignant tissue.     

Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone,cartilage and giant cell tumor

Summary Carbonic anhydrase isoenzymes I and II have been localized in human bone and cartilage. Osteoclasts are strongly positive for carbonic anhydrase II but very little if any reaction is observed for carbonic anhydrase I. In tendon giant cell tumor osteoclastlike-giant cells contained high amounts of carbonic anhydrase II suggesting the close relation of these cells to normal osteoclasts. In growth plate cartilage strong staining was obtained in late proliferative and hypertrophic chondroxytes as well as in extracellular matrix of hypertrophic zone also only with anti-human carbonic anhydrase II.     

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.     

Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis

Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80-90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50-60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification.