Membrane-bound carbonic anhydrase in human retinal pigment epithelium

T. J. Wolfensberger, I. Mahieu, J. Jarvis-Evans, M. Boulton, N. D. Carter, A. Nogradi, E. Hollande, A. C. Bird

Research output: Contribution to journalArticle

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Abstract

Purpose. Inhibition of carbonic anhydrase (CA) by acetazolamide causes a decrease in the standing potential of the retinal pigment epithelium (RPE) and an increase in the rate of subretinal fluid absorption, and it may improve cystoid macular edema. These effects are thought to be mediated by the RPE. Given the solubility coefficient of acetazolamide, the drug is most likely to act by direct inhibition of membrane-bound CA (CA IV). To identify a substrate for acetazolamide in the RPE, the distribution of CA activity and the isoform of CA in the RPE membrane were investigated. Methods. Carbonic anhydrase activity was determined by Hansson's technique in fresh human eyes from donors of both sexes and different ages. The presence of the membrane- bound isoform CA IV was investigated immunohistochemically at the light and electron microscopic level, as well as by Western blotting in fresh RPE, and in adult and fetal RPE cultures. Results. Hansson's histochemical method demonstrated CA activity on the apical and basolateral cell membrane of the RPE. Using the γ-globulin fraction of a polyclonal antibody against pure CA IV, immunocytochemistry showed labeling for CA IV on the apical RPE membrane of morphologically polarized human adult and fetal RPE cultures. Gel electrophoresis and Western blotting demonstrated a major immunoreactive band at 55 kDa in homogenates, which was consistently reduced to approximately 35 kDa by incorporation of 0.1% Triton X-100 detergent. Conclusions. These results suggest that the clinical effects of carbonic anhydrase inhibitors on RPE function may be mediated via membrane-bound carbonic anhydrase activity in RPE and that CA IV is responsible for activity on the apical surface.

Original languageEnglish (US)
Pages (from-to)3401-3407
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume35
Issue number9
StatePublished - 1994
Externally publishedYes

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Carbonic Anhydrases
Retinal Pigment Epithelium
Carbonic Anhydrase IV
Membranes
Acetazolamide
Protein Isoforms
Western Blotting
Subretinal Fluid
Carbonic Anhydrase Inhibitors
Macular Edema
Globulins
Octoxynol
Detergents
Solubility
Electrophoresis
Gels
Immunohistochemistry
Cell Membrane
Electrons

Keywords

  • acetazolamide
  • carbonic anhydrase
  • cystoid macular edema
  • membrane-bound isoenzymes
  • retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Wolfensberger, T. J., Mahieu, I., Jarvis-Evans, J., Boulton, M., Carter, N. D., Nogradi, A., ... Bird, A. C. (1994). Membrane-bound carbonic anhydrase in human retinal pigment epithelium. Investigative Ophthalmology and Visual Science, 35(9), 3401-3407.

Membrane-bound carbonic anhydrase in human retinal pigment epithelium. / Wolfensberger, T. J.; Mahieu, I.; Jarvis-Evans, J.; Boulton, M.; Carter, N. D.; Nogradi, A.; Hollande, E.; Bird, A. C.

In: Investigative Ophthalmology and Visual Science, Vol. 35, No. 9, 1994, p. 3401-3407.

Research output: Contribution to journalArticle

Wolfensberger, TJ, Mahieu, I, Jarvis-Evans, J, Boulton, M, Carter, ND, Nogradi, A, Hollande, E & Bird, AC 1994, 'Membrane-bound carbonic anhydrase in human retinal pigment epithelium', Investigative Ophthalmology and Visual Science, vol. 35, no. 9, pp. 3401-3407.
Wolfensberger TJ, Mahieu I, Jarvis-Evans J, Boulton M, Carter ND, Nogradi A et al. Membrane-bound carbonic anhydrase in human retinal pigment epithelium. Investigative Ophthalmology and Visual Science. 1994;35(9):3401-3407.
Wolfensberger, T. J. ; Mahieu, I. ; Jarvis-Evans, J. ; Boulton, M. ; Carter, N. D. ; Nogradi, A. ; Hollande, E. ; Bird, A. C. / Membrane-bound carbonic anhydrase in human retinal pigment epithelium. In: Investigative Ophthalmology and Visual Science. 1994 ; Vol. 35, No. 9. pp. 3401-3407.
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AU - Mahieu, I.

AU - Jarvis-Evans, J.

AU - Boulton, M.

AU - Carter, N. D.

AU - Nogradi, A.

AU - Hollande, E.

AU - Bird, A. C.

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N2 - Purpose. Inhibition of carbonic anhydrase (CA) by acetazolamide causes a decrease in the standing potential of the retinal pigment epithelium (RPE) and an increase in the rate of subretinal fluid absorption, and it may improve cystoid macular edema. These effects are thought to be mediated by the RPE. Given the solubility coefficient of acetazolamide, the drug is most likely to act by direct inhibition of membrane-bound CA (CA IV). To identify a substrate for acetazolamide in the RPE, the distribution of CA activity and the isoform of CA in the RPE membrane were investigated. Methods. Carbonic anhydrase activity was determined by Hansson's technique in fresh human eyes from donors of both sexes and different ages. The presence of the membrane- bound isoform CA IV was investigated immunohistochemically at the light and electron microscopic level, as well as by Western blotting in fresh RPE, and in adult and fetal RPE cultures. Results. Hansson's histochemical method demonstrated CA activity on the apical and basolateral cell membrane of the RPE. Using the γ-globulin fraction of a polyclonal antibody against pure CA IV, immunocytochemistry showed labeling for CA IV on the apical RPE membrane of morphologically polarized human adult and fetal RPE cultures. Gel electrophoresis and Western blotting demonstrated a major immunoreactive band at 55 kDa in homogenates, which was consistently reduced to approximately 35 kDa by incorporation of 0.1% Triton X-100 detergent. Conclusions. These results suggest that the clinical effects of carbonic anhydrase inhibitors on RPE function may be mediated via membrane-bound carbonic anhydrase activity in RPE and that CA IV is responsible for activity on the apical surface.

AB - Purpose. Inhibition of carbonic anhydrase (CA) by acetazolamide causes a decrease in the standing potential of the retinal pigment epithelium (RPE) and an increase in the rate of subretinal fluid absorption, and it may improve cystoid macular edema. These effects are thought to be mediated by the RPE. Given the solubility coefficient of acetazolamide, the drug is most likely to act by direct inhibition of membrane-bound CA (CA IV). To identify a substrate for acetazolamide in the RPE, the distribution of CA activity and the isoform of CA in the RPE membrane were investigated. Methods. Carbonic anhydrase activity was determined by Hansson's technique in fresh human eyes from donors of both sexes and different ages. The presence of the membrane- bound isoform CA IV was investigated immunohistochemically at the light and electron microscopic level, as well as by Western blotting in fresh RPE, and in adult and fetal RPE cultures. Results. Hansson's histochemical method demonstrated CA activity on the apical and basolateral cell membrane of the RPE. Using the γ-globulin fraction of a polyclonal antibody against pure CA IV, immunocytochemistry showed labeling for CA IV on the apical RPE membrane of morphologically polarized human adult and fetal RPE cultures. Gel electrophoresis and Western blotting demonstrated a major immunoreactive band at 55 kDa in homogenates, which was consistently reduced to approximately 35 kDa by incorporation of 0.1% Triton X-100 detergent. Conclusions. These results suggest that the clinical effects of carbonic anhydrase inhibitors on RPE function may be mediated via membrane-bound carbonic anhydrase activity in RPE and that CA IV is responsible for activity on the apical surface.

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