Immunohistolocalization of carbonic anhydrase isozyme (CA-VI) in bovine mammary glands

The localization of bovine carbonic anhydrase isozyme VI (CA-VI) was examined immunohistochemically in bovine mammary glands during early lactation period (after 2-3 days of postpartum) and dry period (at about 2 months preparturition in adults), and young calves (at 30 and 150 days after birth) using specific CA-VI antiserum. The immunoreaction for anti-CA-VI antiserum was very weak in the mammary glands in young (prepubescent) calves. In dry period, CA-VI was also weakly expressed in secretory epithelial (acinar) and ductal cells. In contrast, the reaction was intense in mammary gland cells in early lactation period. Dot blotting analysis indicated that anti-CA-VI reacted positively to beastings and mature saliva, but weakly or not at all to milk during the dry period or calf saliva, respectively. The intense expression of CA-VI in the mammary glands in early lactation period might compensate for low levels of secretion from functionally and structurally immature salivary glands in young calves.     

Measurement of carbonic anhydrase isozyme VI (CA‐VI) in swine sera,colostrums, saliva,bile, seminal plasma and tissues

Swine secretory carbonic anhydrase VI (CA‐VI) was purified from swine saliva and an antibody to CA‐VI was generated. A specific and sensitive enzyme‐linked immunosorbent assay (ELISA) has been developed for the measurement of swine CA‐VI. The assay can detect as little as 5 ng/mL of swine CA‐VI. Typical standard curves were determined for a range of CA‐VI solutions (7.8 to 500 ng/mL). The coefficients of variation for these solutions were less than 5%. When 500, 250 or 100 ng/mL of swine CA‐VI was added to swine sera, the recoveries were 102.0%, 109.7% and 100.2%, respectively. The concentrations of CA‐VI in the saliva (26.2 ± 30.4 µg/mL), sera (3.3 ± 4.9 ng/mL), bile (153.0 ± 114.0 ng/mL), seminal plasma (124.0 ± 39.0 ng/mL) and parotid gland (441.3 ± 90.0 µg/g wet tissue), submaxillary gland (88.1 ± 124.4 µg/g wet tissue), sublingual gland (58.6 ± 24.6 µg/g wet tissue) and gallbladder (2.4 ± 1.3 µg/1g wet tissue) were determined by ELISA. The concentration of CA‐VI in colostrum was 163.3 ± 101.4 ng/mL and did not decrease within 10 days following parturition. An immunohistochemical reaction to anti‐CA‐VI antiserum was observed in the columnar epithelial cells lining the gallbladder. These data suggest that secretory CA‐VI plays various roles in pH regulation and the maintenance of ion and fluid balance.     

Immunohistochemistry of the bovine secretory carbonic anhydrase isozyme (CA-VI) in bovine alimentary canal and major salivary glands

In the present study, we firstly demonstrated immunohistochemical expressions of secretory carbonic anhydrase (CA-VI) isozyme in bovine forestomach, large intestine and major salivary glands. CA-VI was detected in basal layer epithelial cells of esophageal and forestomach stratified epithelium, in mucous cells of upper glandular region of large intestine, in serous acinar cells of the parotid gland, in serous demilune cells and some ductal liner cells of mandibular, monostomatic sublingual and esophageal glands. These immunohistolocalizations suggested that bovine CA-VI plays various roles in pH regulation, maintenance of ion and fluid balance, and cell proliferation.     

Immunocytochemical localisation of carbonic anhydrase isozyme III in equine skeletal muscle

The location of carbonic anhydrase III (CA-III) in frozen sections of biopsies of Thoroughbred horse skeletal muscle was studied. Fibre types were determined by ATP-ase and succinate dehydrogenase staining. CA-III isozyme was detected using a peroxidase conjugated anti-CA-III antibody. CA-III was found to be localised in slow twitch oxidative fibres (ST), but was also present in fast twitch oxidative (FTH) fibres in small amounts. Fast twitch glycolytic (FT) fibres were stained lightly compared with control sections. The concentrations of CA-III in muscle and liver were 70 micrograms/mg protein and 4 micrograms/mg protein, respectively. CA-I and CA-II were not found in muscle extracts by the double immunodiffusion method.     

Purification of swine carbonic anhydrase isoenzyme III and measurement of its levels in tissues and plasma

The changes in the levels of carbonic anhydrase isozyme III (CA‐III) in swine plasma and urine have not been previously determined or reported. CA‐III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA‐III from swine muscle tissues and determined CA‐III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA‐III in the tissues of female swine (age, 3 months) and plasma of young swine (age, 1–5 months) and adult female pigs (age, 2–3 years) were determined using the ELISA system for swine CA‐III. The mean (± SD) levels of CA‐III in the skeletal muscles were 3.8 ± 3.2 mg/g (wet tissue), and in the plasma, 230 ± 193 ng/ml at 1 month, 189 ± 208 ng/ml at 2 months, 141 ± 148 ng/ml at 3 months, 78 ± 142 ng/ml at 4 months and 53 ± 99 ng/ml at 5 months. The mean level of CA‐III in the plasma samples from 2‐ to 3‐year‐old pigs was 18 ± 60 ng/ml. CA‐III in the plasma samples was found to decrease from 1 month until 3 years of age (p < 0.01). We performed far‐western blotting to clarify the cause of the observed decrease in CA‐III in plasma. Our results demonstrated that CA‐III is bound to the transferrin and albumin. In addition, we determined that the levels of CA‐III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.     

Purification of chicken carbonic anhydrase isozyme-III (CA-III) and its measurement in White Leghorn chickens

Background

The developmental profile of chicken carbonic anhydrase-III (CA-III) blood levels has not been previously determined or reported. We isolated CA-III from chicken muscle and investigated age-related changes in the levels of CA-III in blood.

Methods

CA-III was purified from chicken muscle. The levels of CA-III in plasma and erythrocytes from 278 female chickens (aged 1-93 weeks) and 68 male chickens (aged 3-59 weeks) were determined by ELISA.

Results

The mean level of CA-III in female chicken erythrocytes (1 week old) was 4.6 μg/g of Hb, and the CA-III level did not change until 16 weeks of age. The level then increased until 63 weeks of age (11.8 μg/g of Hb), decreased to 4.7 μg/g of Hb at 73 weeks of age, and increased again until 93 weeks of age (8.6 μg/g of Hb). The mean level of CA-III in erythrocytes from male chickens (3 weeks old) was 2.4 μg/g of Hb, and this level remained steady until 59 weeks of age. The mean plasma level of CA-III in 1-week-old female chickens was 60 ng/mL, and this level was increased at 3 weeks of age (141 ng/mL) and then remained steady until 80 weeks of age (122 ng/mL). The mean plasma level of CA-III in 3-week-old male chickens was 58 ng/mL, and this level remained steady until 59 weeks of age.

Conclusion

We observed both developmental changes and sex differences in CA-III concentrations in White Leghorn (WL) chicken erythrocytes and plasma. Simple linear regression analysis showed a significant association between the erythrocyte CA-III level and egg-laying rate in WL-chickens 16-63 weeks of age (p < 0.01).     

Distribution of carbonic anhydrase in digestive tract of pond loach (Misgurnus anguilicaudatus)

Carbonic anhydrase (CA) family is discovered in various species including mammals, avians and even plants, but it is rarely reported in fishes. Ten Pond Loaches were used in this study. The results indicated that no evidence of distribution of CA in esophagus. Nonetheless CA was found in the mucosal epithelium of stomach and in the villous epithelium of intestine demonstrated histochemically by the numerous black sedimentation of cobaltous sulfide (CoS) in these areas. In addition, black sedimentations of CoS were also found in all the vascular endothelium examined and red blood cells of digestive tract. The distribution of CA in Pond loach was more closely resemble amphibians than to other species, suggesting evolutional adaptation for Pond loach in aquatic environments.     

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.     

Evaluation of a topically administered carbonic anhydrase inhibitor (MK-927) in normotensive and glaucomatous beagles.

A new topically administered anhydrase inhibitor, MK-927, was evaluated for its ocular hypotensive activity in normotensive and glaucomatous Beagles. Single- and multiple-dose studies were performed. Six concentrations of the drug were evaluated in the single-dose study and the 2% solution was used for multiple-dose evaluation. The decrease in intraocular pressure (IOP) was greater in glaucomatous Beagles at the higher concentrations of the drug. The 2 and 4% solutions of MK-927 significantly lowered IOP (mean, 5 mm of Hg; SEM +/- 1.6 and SEM +/- 1.2, respectively) in normotensive and glaucomatous Beagles. In the multiple-dose study, IOP was significantly decreased in the normotensive (mean, 4 mm of Hg; SEM +/- 0.74) and glaucomatous Beagles (mean, 9 mm of Hg; SEM +/- 1.2). The maximal effect was observed by day 4. A contralateral effect was found in glaucomatous Beagles, with the maximal effect on day 4.     

Biochemical and developmental characterization of carbonic anhydrase II from chicken erythrocytes

Background

Carbonic anhydrase (CA) of the chicken has attracted attention for a long time because it has an important role in the eggshell formation. The developmental profile of CA-II isozyme levels in chicken erythrocytes has not been determined or reported. Furthermore, the relations with CA-II in erythrocyte and egg production are not discussed. In the present study, we isolated CA-II from erythrocytes of chickens and determined age-related changes of CA-II levels in erythrocytes.

Methods

Chicken CA-II was purified by a combination of column chromatography. The levels of CA-II in the hemolysate of the chicken were determined using the ELISA system in blood samples from 279 female chickens, ages 1 to 93 weeks, 69 male chickens, ages 3 to 59 weeks and 52 weeks female Araucana-chickens.

Results

The mean concentration of CA-II in hemolysate from 1-week-old female was 50.8 ± 11.9 mg/g of Hb. The mean levels of CA-II in 25-week-old (188.1 ± 82.6 mg/g of Hb), 31-week-old (193.6 ± 69.7 mg/g of Hb) and 49-week-old (203.8 ± 123.5 mg/g of Hb) female-chickens showed the highest level of CA-II. The levels of CA-II in female WL-chickens significantly decreased at 63 week (139.0 ± 19.3 mg/g of Hb). The levels of CA-II in female WL-chicken did not change from week 63 until week 93.The mean level of CA-II in hemolysate of 3-week-old male WL-chickens was 78.3 ± 20.7 mg/g of Hb. The levels of CA-II in male WL-chickens did not show changes in the week 3 to week 59 timeframe. The mean level of CA-II in 53-week-old female Araucana-chickens was 23.4 ± 1.78 mg/g of Hb. These levels of CA-II were about 11% of those of 49-week-old female WL-chickens. Simple linear regression analysis showed significant associations between the level of CA-II and egg laying rate from 16 week-old at 63 week-old WL-chicken (p < 0.01).

Conclusions

Developmental changes and sexual differences of CA-II concentration in WL-chicken erythrocytes were observed. The concentration of CA-II in the erythrocyte of WL-chicken was much higher than that in Araucana-chicken (p < 0.01).     

Immunohistolocalization of carbonic anhydrase isoenzymes (CA-I, -II and -III ) in canine epididymis

The immunolocalization of the efferent duct and the epididymis in canine was firstly examined using an the immunohistochemical method with the canine carbonic anhydrase (CA) -I, CA-II and CA-III antisera. The efferent duct was immunonegative for all present canine CA antisera. However, some slender shaped epithelial cells in the head and body segments of the epididymal duct were intensely reacted to the CA-II antiserum. These results suggested that the CA-II might be controlled in the luminal environment in the head and body segments of the canine epididymis by the proton and bicarbonate balance for the maintenance of the spermatozoal stability and movement.     

Measurement of erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) in racehorses and riding horses

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.     

Comparison of the distribution of carbonic anhydrase isozymes (CA-I, CA-II, CA-III) in the rat gastrointestinal tract.

The present paper described the immunohistochemical distributions of carbonic anhydrase (CA) isozymes. CA-I, CA-II and CA-III, in the epithelium lining the rat gastrointestinal tract, with rabbit antibodies to equine CA-I, CA-II and CA-III. Prior to the immunohistochemical examinations, the crossreactivities of these antibodies to the rat-antigens were confirmed in this study. In the stomach, surface epithelial cells and parietal cells of the glandular region showed an immunoreactivity only to CA-II. In the large intestine, each immunoreactivity to CA isozyme (CA-I, CA-II and CA-III) was localized in the upper portion of intestinal glands, and decreased toward the distal digestive tract, but absent in the small intestine. The present histological findings suggested that the CA isozymes might play a role in the ion-transportation during the water absorption in the rat large intestine.