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.
With use of specific antibodies against human and rat erythrocyte carbonic anhydrase C and human carbonic anhydrase B, only the isozyme C could be detected by immunofluorescence in rat kidney epithelial cells. In the postnatal kidney a few cells were positive after 2 days, but the number of fluorescent cells increased during the first few weeks of life to reach the final adult levels after 3 wk in the cortex and 5 wk in the medulla. In the postnatal and adult kidney a characteristic mosaic pattern of fluorescence was seen in the late distal tubule, the connecting segment, and the collecting tubule, where the mitochondria-rich dark cells were brightly fluorescent. In addition, in later postnatal stages and in the adult, the entire epithelium of the initial portion of descending thin limbs of Henle (long loops) was labeled. Some kidney regions that had previously been shown to contain carbonic anhydrase activity by biochemical and histochemical techniques stained only weakly with the immunocytochemical method. This suggests either that the enzyme in these regions does not cross-react strongly with our antibodies or that these regions contain only low amounts of carbonic anhydrase, at the limit of the detection threshold of our techniques.
The regional and cellular distribution of the high activity carbonic anhydrase isoenzyme (CA C or CA II) in the mouse nervous system was investigated by an indirect immunoperoxidase (peroxidase-antiperoxidase) method using cross-reactive antibodies prepared against human CA C. In the mature brain an overall strong CA C specific reactivity was revealed in the heavily myelinated nerve tracts, the main immunostaining originating from small, intensively reacting cells interpreted as oligodendrocytes and from the myelin sheaths. An obvious staining was also revealed in the choroid plexus cells, especially in their free borders, and in the erythrocytes ofthe blood vessels, while Literature Cited
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