Localization of carbonic anhydrase activity in the developing boar testis

Embryonic and fetal pig gonads were obtained immediately after the sow's slaughter at 18, 21, 25, 28, 30, 36, 55, 63, 80 or 108 days of pregnancy. Semithin plastic sections were incubated for localization of carbonic anhydrase (CA) activity using a cobalt precipitation technique. In the embryonic gonad, CA activity was only present in the coelomic epithelium and in the endothelium of scattered blood capillaries. In the early testes (30-36 days) the CA activity was also localized in the cytoplasm of the sustentacular cells. Both spermatogonia and the developing interstitial cells were negative. At later stages, the testes presented a clear CA cytoplasmic activity in the Sertoli cells and a membrane-bound activity in the peritubular capillaries, resembling the enzymatic localization in the adult. The epithelium of the rete testis had a clear membrane-bound CA activity. CA histochemistry is useful as a marker for topographical studies of Sertoli cells during the prenatal development in the pig.     

Localization of carbonic anhydrase in the rat lung

Summary The localization of carbonic anhydrase in the rat lung has been demonstrated, at light and electron microscopic levels, by the cobalt bicarbonate histochemical method of Hansson. Focal deposits of the cobalt sulfide reaction product were found not only in the capillary endothelium of the alveolar walls, but also in the small and large alveolar cells. The histochemical reaction was abolished by two potent inhibitors, acetazolamide (10^–5 to 10^–6 M) and KCNO (5×10^–3 to 10×10^–3 M). Physiological assay with Maren's method indicated that values for carbonic anhydrase activity in rat lung are 4.4±0.8 UA/mg of protein, 25.0±5.5 UA/mg of nitrogen, and 369±86 UA/g of wet weight. In addition, it was calculated that after fixation in glutaraldehyde-formaldehyde-picric acid about 9% activity is retained.     

Pulmonary carbonic anhydrase in vertebrate gas exchange organs

Carbonic anhydrase (CA) catalyzes the interconversion of CO(2) and HCO(3)(-). Intracellular (extravascular) and intravascular (extracellular) CA has been identified and localized in the lungs of reptiles and mammals. Less information is known, however, on the presence of intravascular CA in the lungs of amphibians and avians. In the present study, perfusion studies were used to compare the catalytic activity of pulmonary intravascular CA in reptiles and mammals. In addition, SDS-resistant CA activity was examined in microsomal fractions prepared from gill/lung tissue from representative animals in each vertebrate class. Finally, the CNO(-) sensitivity of the microsomal CA activity was compared. No SDS-resistant CA activity was found in gill microsomal fractions of several fish species. In contrast, the data suggest that SDS-resistant, intravascular pulmonary CA activity is present in air-breathing vertebrates with vastly differing lung morphologies and that the kinetics of inhibition is remarkably comparable between the vertebrate classes.     

Localization of carbonic anhydrase in legume nodules

Extracts of the central infected zone and the surrounding cortex of nodules from Lupinus angustifolius L., Vigna unguiculata L. (Walp), Pisum sativum L., Phaseolus vulgaris L., Vicia faba L. and Medicago sativa L. contained significant activities of carbonic anhydrase (CA). Immunoassay of extracts using antisera to a putative nodule CA (Msca1) cloned from M. sativa also indicated expression in both tissue types. Quantitative confocal microscopy using laser scanning imaging and a fluorescent CA‐specific probe (5‐dimethylaminonaphthalene‐1‐sulfonamide [DNSA]) localized expression to the infected cells in the central zone tissue and a narrow band of 2–3 files of cells in the cortical tissue that corresponded to the inner cortex. In the infected cells, the enzyme activity was distributed evenly in the cytosol, but in the inner cortical cells, it was restricted to the periphery – possibly to the plasma membrane or cell wall. The functions of CA in these two tissues are considered in relation to the carbon metabolism of nodules and the participation of the inner cortex in the regulation of gaseous diffusive resistance.     

Localization and quantification of carbonic anhydrase activity in the symbiotic Scyphozoan Cassiopea xamachana

The relationship between density and location of zooxanthellae and levels of carbonic anhydrase (CA) activity was examined in Cassiopea xamachana. In freshly collected symbiotic animals, high densities of zooxanthellae corresponded with high levels of CA activity in host bell and oral arm tissues. Bleaching resulted in a significant loss of zooxanthellae and CA activity. Recolonization resulted in full restoration of zooxanthellar densities but only partial restoration of CA activity. High levels of CA activity were also seen in structures with inherently higher zooxanthellar densities, such as oral arm tissues. Similarly, the oral epidermal layer of bell tissue had significantly higher zooxanthellar densities and levels of CA activity than did aboral bell tissues. Fluorescent labeling, using 5-dimethylaminonapthalene-1-sulfonamide (DNSA) also reflected this tight-knit relationship between the presence and density of zooxanthellae, as DNSA-CA fluorescence intensity was greatest in host oral epithelial cells directly overlying zooxanthellae. However, the presence and density of zooxanthellae did not always correspond with enzyme activity levels. A transect of bell tissue from the margin to the manubrium revealed a gradient of CA activity, with the highest values at the bell margin and the lowest at the manubrium, despite an even distribution of zooxanthellae. Thus, abiotic factors may also influence the distribution of CA and the levels of CA activity.     

Histochemical demonstration of carbonic anhydrase activity

Summary Freeze-dried frozen sections are floated on the surface of the freshly prepared incubation mixture (CoSO₄ 1.75 × 10^–3 M, H₂SO₄ 5.3 × 10^–2 M, NaHCO₃ 1.57 × 10^–2 M and KH₂PO₄ 1.17 to 11.7 × 10^–3 M; demonstration of weak activity requires high phosphate). A compound containing cobalt and phosphorous precipitates at carbonic anhydrase sites and is converted to CoS. Adequate staining requires only 2–10 minutes of incubation. Actazolamide inhibits the staining reaction in specific concentrations. Actazolamidein vivo, 20 mg/kgi.v. to mice 30 minutes before sacrifice also inhibited the staining. The proportion phosphorous in the specific precipitate increases with KH₂PO₄ of the medium (shown by the addition of⁶⁰Co and³²P). An explanation of the reaction mechanism is given, based on the catalyzed loss of CO₂ in the surface layer. The inclusion of phosphate in the medium makes this modification ofHäusler's method so sensitive that it shows carbonic anhydrase activity in for instance stratum spinosum of the skin.This investigation was supported by grants from the Medical Faculty, University of Uppsala and from the U.S. National Institutes of Health (Grant NB 3060 to E.Bárány).     

Localization of carbonic anhydrase in guinea pig Bowman's glands.

We examined the histochemical localization of carbonic anhydrase (CA) in Bowman's glands by light and electron microscopy. Neither CAI nor CAII was detected immunohistochemically in the duct cells. However, by enzyme histochemistry the duct cells revealed electron-dense precipitates demonstrative of CA in the microvilli and intercellular digitations. The reaction product was also noted in small vesicles in the cytoplasm of duct cells. In cells of the acini, the well-developed short microvilli, basolateral cell membrane, and mitochondria along the basolateral membrane showed strong deposits indicating CA activity. Dense reaction product of CA was also detected in a small core within the electron-lucent granules of the secretory cells, although CAI and CAII were not detected by immunostaining in the secretory granules. Although the functional significance of CA in Bowman's glands is obscure, the enzyme may play a role in regulation of pH and ion balance in the mucous layer covering the olfactory epithelium. The presence of CA activity in the ducts suggests that these structures are not simple tubes serving as a conduit for secretory substances but participate in modifying the luminal content by secreting CA. (J Histochem Cytochem 47:1525-1531, 1999)     

Role of tubular secretion and carbonic anhydrase in vertebrate renal sulfate excretion

The renal proximal tubule of vertebrates performs an essential role in controlling plasma SO(4)(2-) concentration ([SO(4)(2-)]). Although net tubular SO(4)(2-) reabsorption is the predominate control process in terrestrial vertebrates, a facilitated secretory flux is also present. In contrast, marine teleosts obtain excess SO(4)(2-) from drinking, and increased plasma [SO(4)(2-)] is prevented predominately through net tubular secretion. Tubular SO(4)(2-) secretion is accomplished by at least two electroneutral anion exchange processes in series. Movement of SO(4)(2-) into the cell across the basolateral membrane is pH dependent, suggesting SO(4)(2-)/OH(-) exchange. Luminal HCO(3)(-) and Cl(-) can facilitate SO(4)(2-) movement out of the cell across the brush-border membrane. The molecular identities of the anion exchangers are unknown but are probably homologues of SO(4)(2-) transporters in the mammalian SLC26 gene family. In all species tested, glucocorticoids increase renal SO(4)(2-) excretion. Whereas glucocorticoids downregulate SO(4)(2-) reabsorptive mechanisms in terrestrial vertebrates, they may also stimulate a mediated secretory flux. In the marine teleost, cortisol increases the level of SO(4)(2-)/HCO(3)(-) exchange at the brush-border membrane, tubular carbonic anhydrase (CA) activity, CAII protein, and a proportion of tubular SO(4)(2-) secretion that is CA dependent. CA activity is required for about one-half of this net SO(4)(2-) secretion but is also required for about one-half of the net reabsorption in bird proximal epithelium. A CA-SO(4)(2-)/anion exchanger metabolon arrangement is proposed that may speed both the secretory and reabsorptive processes.     

Histochemical demonstration of carbonic anhydrase activity in the uterus

Summary Positive histochemioal reaction for carbonic anhydrase (CAH) activity was observed in the glandular and lining epithelium of human and rat uteri. The CAH activity in the rat endometrium showed cyclic changes during the estrous cycle. Only one main component of CAH activity was revealed by electrophoresis, under conditions when the erythrocytes showed several isozymes.     

Carbonic anhydrase activity of intact carbonic anhydrase II-deficient human erythrocytes

Intact erythrocytes from subjects with deficiency of blood carbonic anhydrase (CA) II and from normal subjects were assayed for enzyme activity by use of an 18O exchange technique in a solution containing 25 mM (CO2 + NaHCO3) plus 125 mM NaCl. At 25 degrees C and pH 7.4, the catalyzed reaction velocity was 0.32 +/- 0.04 M/s for the CA II-deficient and 1.60 +/- 0.12 M/s for the normal cells, a ratio of 1:5. Under the same conditions at 37 degrees C the relative difference between the CA II-deficient and normal cells was much less: the velocity for the CA II-deficient cells was 0.84 +/- 0.07 M/s and for the normal cells 1.60 +/- 0.32 M/s, a ratio of 1:1.9. Results were comparable for the hemolysates with the NaHCO3 reduced to 85 mM (the corresponding intracellular concentration): at 25 degrees C CA II-deficient cells had a velocity of 0.36 +/- 0.01 M/s compared with 1.12 +/- 0.04 M/s for the normal cells, a ratio of 1:3.1. At 37 degrees C again the relative difference between hemolysates from CA II normal and deficient cells was much less: the CA II-deficient cells had a reaction velocity of 1.17 +/- 0.22 M/s vs. 2.60 +/- 0.36 M/s for the normal cells, a ratio of 1:2.2. The greater fractional reduction of enzyme velocity of CA II-deficient cells at 25 degrees C compared with 37 degrees C appears to be explained by a greater chloride inhibition of the presumed CA I at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical demonstration of carbonic anhydrase activity in the alimentary canal

Summary Carbonic anhydrase (CAH) activity was histochemically demonstrated in various parts of the alimentary canal of rat and in the stomach of man using the method of Waldeyer and Häusler (1959). The most intense histochemical reaction was observed in the parietal cells of the rat stomach, and reactions of decreasing intensity in the epithelial cells of the colon, appendix, jejunoileum, duodenum and oesophagus in the order mentioned. An intense reaction was also observed in the parietal cells of the stomach of man and a weak activity in the pyloric glands. After electrophoresis on cellulose acetate film the CAH activity in human and rat stomach mucosa showed one band with the same migration rate as the fastest moving band of the erythrocyte CAH.     

Localization of carbonic anhydrase in living osteoclasts with bodipy 558/568-modified acetazolamide, a thiadiazole carbonic anhydrase inhibitor.

We describe the synthesis of Bodipy 558/568-modified acetazolamide, a fluorescent inhibitor of carbonic anhydrase and its use to localize the enzyme in living cells. The modified acetazolamide, with its specific sulfonamide group intact, labeled cells at concentrations as low as 10(-9) M, with a minimal loading time of 5 min. The staining was decreased by 57.4% by preincubating cells with unaltered acetazolamide (1:100) or with trifluoromethane sulfonamide, 6-ethoxyzolamide, and 5-(3-hydroxybenzoyl)-thiophene-2-sulfonamide. The efficacy of the inhibitor was unchanged by the fluorescent label, as determined by an acridine orange assay that detects acidification of osteoclasts, the cell model used in this study. This compound should prove to be useful for studying carbonic anhydrase in many organisms because of the high degree of conservation of the active site of this enzyme. (J Histochem Cytochem 47:545-550, 1999)     

Localization of carbonic anhydrase in the sperm-storing regions of the turkey and quail oviduct

The localization of carbonic anhydrase in the sperm storage regions of turkey and quail was investigated using a histochemical method showing the activity of all the isozymes present. Intense carbonic anhydrase activity was found in the turkey sperm storage tubules and infundibular storage glands, whereas no activity could be detected in the quail at these sites. Both species did, however, show strong membrane-bound and cytoplasmic activity in the non-ciliated cells of the utero-vaginal surface epithelium and scattered cells of the vaginal epithelium. The enzyme catalyses the reaction , and the presence of carbonic anhydrase in these regions makes rapid changes in pH possible. It is suggested that increasing pH and/or the addition of bicarbonate stimulates sperm motility needed during transfer of the oviducal lumen. A lowering of the pH would keep the sperm qui escent during storage. The duration of sperm storage is considerably longer in the turkey than in the quail. The high quantity of carbonic anhydrase in the turkey sperm storage tubules may, thus, play a role in the duration of sperm storage.     

Light-induced stimulation of carbonic anhydrase activity in pea thylakoids

Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6-8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light-generated proton gradient across the thylakoid membrane thus facilitating the production of CO(2) from HCO(3)(-) and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier.     

Localization of carbonic anhydrase in the salivary glands of the cockroach,Periplaneta americana

Carbonic anhydrase (CA) activity was localized in the salivery glands of the cockroach, Periplaneta americana, by (1) Hansson's histochemical technique, and (2) the use of the fluorescent sulphonamide, 5-dimethyl-amino-naphthalene-1-sulphonamide (DNSA). Both techniques reveal the same distribution pattern of CA in the four morphologically different cell types of the glands: peripheral cells, central cells, inner acinar duct cells, and distal duct cells. Positive reactions with Hansson's cobalt/phosphate technique were found in the apical regions of the peripheral cells and the distal duct cells, and were inhibited by 10^–5 M acetazolamide in control experiments. No staining could be detected in the central cells and the inner acinar duct cells. The fluorescent CA inhibitor DNSA (10^–4 M) specifically stained the peripheral cells and the distal duct cells in methanolfixed cryostat sections, whereas the central cells and the inner acinar duct cells remained unstained. The role of CA in the peripheral cells is not clear. CA activity in the distal duct cells may provide the protons needed to run the vacuolar-type H⁺-ATPase on the apical infoldings of the cells. This ATPase may be involved in modification of the primary saliva.