Toshiho Nishita

Distribution of Carbonic Anhydrase Isozyme VI in the Developing Bovine Parotid Gland

The distribution of bovine carbonic anhydrase isozyme VI (CA-VI), purified from bovine saliva, was studied immunohistochemically using antiserum against bovine CA-VI in bovine parotid glands during fetal and postnatal development. A weak expression of CA-VI in undifferentiated epithelial cells and ductal cells was observed in a 4- to 5-month-old fetus with a 26-cm crown-rump length. The reaction in both acinar and ductal cells subsequently persisted during late gestation and birth. Although anti-CA-VI reactivity was still seen in both regions immediately following birth, the reactivity had almost completely disappeared from most duct segments by 1 month following birth. Changes in the localization and time-dependent expression of the isozyme in parotid glands may reflect changes in the biological function of structurally closely related isozymes.

Comparative immunochemical studies of carbonic anhydrase III in horses and other mammalian species

1. Carbonic anhydrase III (CA-III) from different mammalian species (horse, cow, dog, cat, rat and rabbit) has been analyzed by the immunodiffusion technique with anti-equine CA-III serum. 2. Immunodiffusion demonstrated the absence of cross-reactivity between isozyme CA-I, CA-II, and CA-III. 3. Cross-reactions were observed between the CA-III from all the species examined except the rabbit. 4. Molecular weights and isoelectric points of CA-III from different species were determined by Western blotting.

Expression and localization of carbonic anhydrase in bovine mammary gland and secretion in milk

Little attention has been paid to carbonic anhydrase VI (CA VI), a secretory type isozyme, in the bovine mammary gland, although the gland is an important exocrine gland and CA VI is known to localize in exocrine glands such as salivary and lacrimal glands in various animal species. In the present study mRNA expression and protein localization of CA VI in isolated gland tissues and in cloned epithelial cells from the mammary gland of Holstein cows (Bos taurus) were observed by reverse transcript polymerase chain reaction and immunocytochemistry. Also, changes of CA VI concentrations in milk were measured for 2 months postpartum by an enzyme-linked immunosorbent assay. CA VI gene expression was detected in the gland tissues and epithelial cells, and CA VI protein was localized in the cytoplasm of the epithelial cells. Colostrum contained the highest concentration of CA VI protein (100 ng/ml), decreasing in an exponential manner (P<0.001). We conclude that bovine mammary epithelial cells synthesize and secrete CA VI in colostrum at higher concentration than in normal milk, implying its role to compensate for low CA VI secretion in neonatal calves.

Immunohistochemical localization of carbonic anhydrase isozymes in the rat carotid body

The rat carotid body was immunohistochemically stained for carbonic anhydrase I, II and III (CA‐I, CA‐II and CA‐III). Immunoreactivity for CA‐I was distributed in type I cells, type II cells and nerve bundles. Smooth muscle cells and endothelial cells of blood vessels were also strongly stained for CA‐I. CA‐II immunoreactivity was distinctly positive in type I cells and nerve bundles. Vascular smooth muscle cells were weakly positive, and type II cells were negative for CA‐II. CA‐III immunoreactivity was identified in type I cells and vascular smooth muscle cells. Our results suggest that carbonic anhydrase isozymes in type I cells play an important role in chemoreception for hypercapnia. Immunoreactivities for CA‐I and CA‐II in the nerve fibres may participate in the synergic action of carotid sinus nerve between hypoxic and hypercapnic stimuli.

Immunohistochemistry of carbonic anhydrase isozymes (CA‐I, II and III) in canine salivary glands: a distributional and comparative assessment

The immunohistolocalization of carbonic anhydrase isozymes (CA‐I, II, III) in canine salivary glands was studied using antiserum against CA‐I, II, III. In parotid glands, immunostaining intensely localized cytosolic CA‐II antiserum throughout the cytoplasm of acinar secretory cells and ductal epithelial cells, especially in the striated duct region. CA‐III reactivity in the glands was only seen selectively at the intercalated ductal cells. In contrast, no immunoreaction localized CA‐I in the gland. In the submandibular and sublingual glands, CA‐I, II, and III were all observed in the ductal segments of the glands, whereas serous demilune appeared devoid of all three cytosolic CA isozymes. In contrast, in zygomatic glands (i.e. dorsal buccal glands) all CA isozymes were observed in both serous demilune and ductal segments. In all of the salivary glands examined, no mucous acinar cells were found to be reactive for any CA.

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

SummaryImmunohistochemical 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.

Immunohistolocalization and Gene Expression of the Secretory Carbonic Anhydrase Isozymes (CA-VI) in Canine Oral Mucosa, Salivary Glands and Oesophagus

The immunohistolocalization of secretory carbonic anhydrase isoenzymes (CA‐VI) in canine salivary glands, parotid, submandibular, sublingual and zygomatic glands, oral and oesophageal mucosa was studied using a specific antiserum against a canine CA‐VI. In addition, the gene expression of CA‐VI from the same tissue was studied using a real‐time reverse‐transcriptase polymerase chain reaction. In all salivary glands and oesophageal gland, immunostaining intensely localized CA‐VI antiserum throughout the cytoplasm of serous acinar cells, including serous demilune and ductal epithelial cells. In contrast, no immunoreaction localized CA‐VI in the mucous acinar cells of the gland. CA‐VI gene transcripts were also detected in the same areas. The physiological significance of secretory CA‐VI in the oral and oesophageal cavity is thought to play a highly specialized role in the maintenance of bicarbonate level in saliva and to protect mucosa from acid injury. It is shown that the major sites of the CA‐VI secretion in dogs were in serous (demilune) secretory cells in all four major salivary glands and oesophageal glands in particular.

Immunohistochemical studies of the carbonic anhydrase isozymes in the bovine placenta

Placentae from 11 cows, ranging from about 80 to 270 days gestation, were studied for immunohistochemical localization of carbonic anhydrase isozymes. CA isozymes were localized using the avidin-biotin-peroxidase complex (ABC) method in the bovine placenta. CA-II was found in the fetal trophoblastic cells with single nuclei as well as in erythrocytes of maternal and fetal blood. The percentage of positive cells for anti-CA-II in the trophoblastic cells did not change during development of gestation in the fetal bovine. CA-I and CA-III were not detected in the bovine placenta.

Measurement of Carbonic Anhydrase Isozyme VI (CA-VI) in Bovine Sera, Saliva, Milk and Tissues

Concentrations of bovine carbonic anhydrase isozyme VI (CA-IV) in bovine serum, saliva, normal milk, colostrum, submandibular gland, liver, and mammary gland were determined. CA-VI was purified from bovine saliva and an antibody to CA-VI was generated. The concentrations of CA-VI in the saliva (7.8 ± 7.9 μg/ml), serum (2.1± 5.7 ng/ml), milk (7.9 ± 12.1 ng/ml), submandibular gland (284.7 μg/g protein), liver (921.0 ± 180.7 ng/g protein) and mammary gland (399.6 ± 191.2 ng/g protein) were determined by ELISA. No seasonal change in CA-VI levels was observed in normal milk. The concentration of CA-VI in colostrum (day 1 post partum) was 119 ng/ml and decreased rapidly by 1 month following birth. Mammary gland contained much smaller amounts than the submandibular gland. CA-VI mRNA was detected in the liver and mammary gland of cow by RT-PCR. The ELISA used in this study proved to be a precise and sensitive method for determining CA-VI concentrations in saliva, serum, milk and tissue specimens from cows. The ELISA may enable the study of changes in CA-VI associated with hereditary or metabolic disorders of the salivary gland, mammary gland and liver using small samples of saliva, serum or milk.

Nucleotide sequence and expression of a cDNA encoding canine carbonic anhydrase VI (CA-VI).

A full-length cDNA clone of a canine carbonic anhydrase VI (CA-VI) was generated from the canine parotid gland by using a reverse transcription-polymerase chain reaction (RT-PCR) technique with degenerate primers designed from conserved regions of the same locus in humans and bovines employing RACE (rapid amplification of cDNA ends) techniques. The cDNA sequence was 1351 base pairs (bp) long and was predicted to encode a 320-amino-acid polypeptide containing a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI showed the highest similarity of 74% to that of human CA-VI. RT-PCR analysis with primers specific to the canine CA-VI demonstrated strong signals in the major salivary glands and weak signals in the minor salivary glands and esophagus of a healthy dog. No CA-VI mRNA was detected in the pancreas, liver or the digestive tract except the esophagus.