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.
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.
ABSTRACT. Equine carbonic anhydrase isozymes (CA-I and CA-II) were purified from erythrocytes by several column chromatography. Polyclonal anti-CA-I and anti-CA-II sera were produced in rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISA) were established to determine the developmental changes in CA-I and CA-II levels in equine erythrocytes. Concentrations of CA-I and CA-II in erythrocytes from 150 clinically normal thoroughbreds (123 racehorses and 27 riding horses) were determined by ELISA. Mean (± SD) concentrations of CA-I and CA-II in racehorses were 1.70 ± 0.48 and 0.94 ± 0.13 mg/g hemoglobin (Hb), respectively. Mean concentrations of CA-I and CA-II in riding horses were 2.34 ± 0.52 and 0.76 ± 0.08 mg/g Hb, respectively. When the CA levels in racehorses and riding horses were compared, the CA-I level in riding horses was higher than that in racehorses (p=0.01). The CA-II level in racehorses was higher than that in riding horses (p=0.02). These data suggest that the levels of CA isozymes in erythrocytes of racehorses were influenced by chronic physical stress. The CA-I concentration in erythrocytes of 2-month-old horses was approximately 0.25 mg/g Hb. The CA-I level noticeably increased during the first year of life and approached normal adult levels by 2 years. The CA-II level decreased slightly with age, indicating different regulation of CA-I and CA-II expression during development. KEY WORDS: carbonic anhydrase, equine, ELISA, erythrocyte.
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 microg/ml), serum (2.1+/- 5.7 ng/ml), milk (7.9 +/- 12.1 ng/ml), submandibular gland (284.7 microg/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.
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.
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