Summary
Transmission electron microscopy and immunohistochemistry, the latter employing the avidin—biotin—peroxidase (ABC complex) technique, were utilized to localize copper—zinc superoxide dismutase (CuZn—SOD) enzyme activity in the epithelial cells of the toad urinary bladder mucosa. This ‘scavenger’ enzyme catalyses the dismutation (reduction—oxidation) of the superoxide anion (O2 −), a toxic free radical generated during normal cellular respiration. In unstimulated epithelial cells, enzyme activity was seen in the cytosol of granular, mitochondrial-rich and goblet cells. The basal cells were generally devoid of enzyme activity. In addition to the cytosol, SOD activity was also seen in association with the apical plasma membrane of the epithelial cells. In the presence of the steroid hormone aldosterone (10−7 m, 30 min—6 h), CuZn—SOD activity was markedly increased along the luminal mucosal membrane of granular, mitochondrial-rich and goblet cells. This increase was seen as early as 30 min after the addition of hormone, and as long as 6 h after treatment. The cytosolic reaction was usually decreased or absent under these conditions. From the data presented, it appears that CuZn—SOD is involved in electrolyte (sodium) transport in the epithelial cells of the toad urinary bladder. The latter may involve hormone-induced alterations in luminal cell membrane structure and chemistry.
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ALLEN, J. E., GOODMAN, D. B. P., BESARAB, A. & RASMUSSEN, H. (1983) Studies on the biochemical basis of oxygen toxicity.Biochim. biophys. Acta 320, 708–28.
BEAUCHAMP, C. & FRIDOVICH, I. (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels.Anal. Biochem. 44, 276–87.
BOUCHER, R. C., NAVARTE, J., COTTON, C., STUTTS, M. J., KNOWLES, R. R., FINN, A. L. & GATZY, J. T. (1982) Sodium absorption in mammalian airways. InFluid and Electrolyte Abnormalities in Exocrine Glands in Cystic Fibrosis (edited by QUINTON, P. M., MARTINEZ, J. R. & HOPFER, U.), pp. 271–8. San Francisco: San Francisco Press.
BRADFORD, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–54.
CRABBE, J. (1961) Stimulation of active sodium transport by the isolated toad bladder with aldosteronein vitro.J. Clin. Invest. 40, 2103–10.
CRAPO, J. D. & MCCORD, J. M. (1976) Oxygen-induced changes in pulmonary superoxide dismutase assayed by antibody titrations.Amer. J. Physiol. 231, 1196–203.
CROUCH, R. K., GANDY, S. E., KIMSEY, G., GALBRAITH, R. A., GALBRAITH, G. M. P. & BUSE, M. G. (1981) The inhibition of islet superoxide dismutase by diabetogenic drugs.Diabetes 30, 235–41.
DAVIS, W. L., GOODMAN, D. B. P., SHIBATA, K. & KIPNIS, M. (1988) The immunolocalization of copper—zinc superoxide dismutase in the epithelial cells of the toad urinary bladder.Anat. Rec. 220, 29A.
EDELMAN, I. S., BOGOROCH, R. & PORTER, G. A. (1963) On the mechanism of action of aldosterone on sodium transport: the role of protein synthesis.Proc. Natl. Acad. Sci. USA 50, 1169–77.
EDELMAN, I. S. & FIMOGNARI, G. M. (1968) I. Modes of hormone action. On the biochemical mechanism of action of aldosterone.Recent Progr. Horm. Res. 24, 1–44.
FRANKEL, E. N. (1980) Lipid oxidation.Progr. Lipid Res. 19, 1–14.
FREEMAN, B. A. & CRAPO, J. D. (1982) Biology of disease: Free radical and tissue injury.Lab. Invest. 47, 412–26.
FRIDOVICH, I. (1978) The biology of oxygen radicals: The superoxide radical is an agent of oxygen toxicity — superoxide dismutases provide an important defense.Science 201, 875–80.
GOODMAN, D. B. P., ALLEN, J. E. & RASMUSSEN, H. (1971) Studies on the mechanism of action of aldosterone: Hormone-induced changes in lipid metabolism.Biochemistry 10, 3825–31.
GOODMAN, D. B. P., WONG, M. & RASMUSSEN, H. (1975) Aldosterone-induced membrane fatty acid metabolism in the toad bladder.Biochemistry 14, 2803–9.
HOCHSTEIN, P. & JAIN, S. K. (1981) Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging.Fed. Proc. 40, 183–8.
HSU, S-M., RAINE, L. & FANGER, H. (1981) The use of avidin—biotin—peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures.J. Histochem. Cytochem. 29, 577–80.
LIEN, E. L., GOODMAN, D. B. P. & RASMUSSEN, H. (1975) Effects of an acetyl-coenzyme A carboxylase inhibitor and a sodium sparing diuretic on aldosterone-stimulated sodium transport, lipid synthesis, and phospholipid fatty acid composition in the toad urinary bladder.Biochemistry 14, 2749–54.
LIEN, E. L., GOODMAN, D. B. P. & RASMUSSEN, H. (1976) Effects of inhibitors of protein and RNA synthesis on aldosterone-stimulated changes in phospholipid fatty acid metabolism in the toad urinary bladder.Biochim. biophys. Acta 421, 210–17.
LING, G. L. (1962)Physical Theory of the Living State: The Association-Induction Hypothesis, with Consideration of the Mechanisms Involved in Ionic Specificity. Appendix H. New York: Blaisdell.
MARKLUND, S. L., HOLM, E. & HELLNER, L. (1982) Superoxide dismutase in extracellular fluids.Clin. Chim. Acta 126, 41–9.
MAURER, H. R. (1971)Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, pp. 44–5. New York: Degruyter.
MILLER, J. H. & MENDOZA, S. A. (1978) Lack of protection against oxygen toxicity byin vivo protective agents in isolated toad bladder.Aviation Space Environ. Med. 49, 665–70.
OLVER, R. E., DAVIS, B., MARIN, M. G. & NADEL, J. A. (1975) Active transport of Na and Cl across canine tracheal epitheliumin vitro.Annu. Rev. Respir. Dis. 112, 811–22.
REDMOND, T. M., DUKE, E. J., COLES, W. H., JIMSON, J.A. V. & CROUCH, R. K. (1984) Localization of corneal superoxide dismutase by biochemical and histochemical techniques.Exp. Eye Res. 38, 369–78.
SHARP, G. W. & LEAF, A. (1966) Mechanism of action of aldosterone.Physiol. Rev. 46, 593–633.
SWITZER, R. C., MERRIL, C. R. & SHIFRIN, S. (1979) A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.Anal. Biochem. 89, 231–7.
THAETE, L. G., CROUCH, R. K., SCHULTE, B. A. & SPICER, S. S. (1983) The immunolocalization of CuZn—superoxide dismutase in canine tissues.J. Histochem. Cytochem. 31, 1399–406.
THAETE, L. G., CROUCH, R. K. & SPICER, S. S. (1985) Immunolocalization of copper—zinc superoxide dismutase. II. Rat.J. Histochem. Cytochem. 33, 803–8.
UBELS, J. L. & HOFFERT, J. R. (1981) Ocular oxygen toxicity: the effects of hyperbaric oxygen on retinal Na+,K+ ATPase.Exp. Eye Res. 32, 77–84.
VARMA, S. D., KUMAR, S. & RICHARDS, R. D. (1979) Light induced damage to ocular lens cation pump. Prevention by vitamin C.Proc. Natl. Acad. Sci. USA 76, 3504–6.
VLADIMIROV, Y. A., OLENOV, V. I., SUSLOVA, T. B. & CHEREMISINA, Z. P. (1980) Lipid peroxidation in mitochondrial membrane.Adv. Lipid Res. 17, 173–81.
WEBER, K. & OSBORN, M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.J. Biol. Chem. 244, 4406–12.
WELSH, M. J., SMITH, P. L., FROMM, M. & FRIZZELL, R. A. (1982) Crypts are the site of intestinal fluid and electrolyte secretion.Science 218, 1219–21.
WIDDICOMB, J. H. & WELSH, M. J. (1980) Ion transport by dog tracheal epithelium.Fed. Proc. 39, 3062–6.
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Davis, W.L., Kipnis, M., Shibata, K. et al. The ultrastructural immunocytochemical localization of superoxide dismutase in the amphibian urinary bladder: Effect of aldosterone. Histochem J 21, 203–209 (1989). https://doi.org/10.1007/BF01747521
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DOI: https://doi.org/10.1007/BF01747521