Localization of carbonic anhydrase activity in the vertebrate nephron

Synopsis The localization of carbonic anhydrase activity in the vertebrate nephron has been examined with particular reference to the proximal tubule and collecting duct. In all species studied, activity was present in the proximal tubular epithelium. In the pigeon and turtle, distinctive and similar patterns of staining were observed in the glomerulus and first portion of the proximal tubule. In the rat and rhesus monkey, the entire proximal tubule exhibited activity; in these species it has been shown previously with micropuncture techniques that there is a high absorptive capacity of this nephron segment for bicarbonate. In contrast, large portions of the dog proximal tubule were inactive; similar studies in this animal have shown tubular concentrations of bicarbonate only slightly lower than plasma levels. In the rat and dog, the entire length of the collecting duct was diffusely and intensely active; in contrast, pigeon collecting duct showed no activity. An alternating pattern of inactive and intensely active cells was observed in the collecting ducts of the toad, turtle, rabbit and monkey. A similar pattern has been described in the turtle and toad bladder, tissues utilized forin vitro studies of ion transport and H⁺ secretion.     

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.     

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.     

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).     

Spasm behavior and the diffuse nerve-net in Cassiopea xamachana (Scyphozoa: Coelenterata)

A quick train of diffuse nerve-net (DNN) impulses is required to initiate a patterned flurry of strong contractions, the ‘spasm,’ of the striated muscles of the rhizostome scyphomedusan Cassiopea xamachana. A number of spasms are illustrated. Using this response as a tool, the connectivity of the DNN has been established. The occurrence and variability of the spasm is illustrated with an analysis of their occurrence in a typical, extended experiment. It is proposed that the spasm is an integral and essential component of the behavior of scyphomedusae, and that its control by the DNN exemplifies how complex behavior is generated.     

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)     

Toxinological studies of the venom from Cassiopea xamachana nematocysts isolated by flow cytometry

The tentacle epithelial tissue of Cassiopea xamachana contains nematocysts and symbiotic algal particles. These two structures were dissociated, analyzed and sorted by flow cytometry. A simple separating method was developed utilizing the algal chlorophyll autofluorescence and the nematocysts' fluorescence after the uptake of fluorescent stains. A five-fold increase in mouse lethality; significantly more potent hemolytic and cytosensing activities; as well as a cleanup in the capillary electropherogram and SDS gel profiles for the crude nematocyst venom preparations prepared by fluorescence activated cell sorting (FACS), was observed relative to alternative methods. Because the hemolytic potency of pre-sorting nematocyst venom was minimal and the post-sorting counterpart was significantly positive, the possibility that algae inhibited the venom's toxinological activity was considered.     

A systematic quantification of carbonic anhydrase transcripts in the mouse digestive system

Background  

Carbonic anhydrases (CAs) are physiologically important enzymes which participate in many gastrointestinal processes such as acid and bicarbonate secretion and metabolic pathways including gluconeogenesis and ureagenesis. The genomic data suggests that there are thirteen enzymatically active members of the mammalian CA isozyme family. In the present study, we systematically examined the mRNA expression levels of all known CA isozymes by quantitative real-time PCR in eight tissues of the digestive system of male and female mice.     

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)     

Electrophoretic and histochemical studies of carbonic anhydrase activity

Summary A simple method for separation of carbonic anhydrase activity into components by electrophoresis on cellulose acetate strips is described. With this method, using barbiturate buffer systems at various pH values, two main components of CAH in rat erythrocytes, and the splitting of each of these into two minor components were revealed. Two components were also observed in the CAH activity in kidney and lens homogenates, and one component in brain homogenate. A modification of Häusler's histochemical method for CAH was adapted for visualization of the electrophoretically separated bands. This rendered the evalution of the results easier than with the quantitative measurements alone. The quantitative measurement of CAH activity in electrophoretic strips corresponded with the degree of staining by the histochemical method. This among other facts supports the view of the specificity of the histochemical method used. Some examples of the histochemical staining pattern of the CAH activity in rat tissues are given.     

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.     

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)