Membrane-associated carbonic anhydrase in cultured rabbit nonpigmented ciliary epithelium

Qiang Wu, Nicholas A Delamere, William Pierce

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Purpose. To measure the activity of membrane-associated carbonic anhydrase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, determine its identity and its sensitivity to extra-cellular trypsin, and compare the ability of acetazolamide and a cell-impermeant dextran-bound CA inhibitor to change cytoplasmic pH. Methods. Studies were conducted using a cell line derived from rabbit NPE. The cells were lysed and separated into soluble and insoluble fractions by differential centrifugation. CA activity in these fractions was determined using a CO2 hydration assay. In studies with intact cells, a membrane-impermeable high-molecular-weight dextran- bound inhibitor (DBI) was synthesized and used to selectively bind and inhibit the extracellular-facing membrane-bound CA. Measurements of CA activity in intact red blood cells were conducted to confirm DBI remains extracellular. Acetazolamide, a membrane-permeable CA inhibitor, was used to inhibit total CA activity. Intracellular pH was determined using the pH- dependent absorbance of the fluorescent dye BCECF-AM. Results. A low-speed pellet enriched with plasma membrane material accounted for 22.3 ± 6.1% (n = 18) of the total CA activity in the cultured NPE. When intact cells were exposed to trypsin-EDTA, a 28% reduction of membrane-associated CA activity was observed; DBI inhibited this CA activity loss. Cytosolic CA activity was inhibited by 0.2% sodium dodecyl sulfate (SDS). In contrast, membrane- associated CA was SDS resistant, a characteristic of the CA-IV isozyme. By Western blot, CA-IV immunoreactive polypeptide was detected in the cultured cells and also in native rabbit and porcine ciliary epithelium. Inhibition of total CA activity with acetazolamide and inhibition of extracellular-facing membrane-associated CA with DBI caused an identical intracellular pH decrease in intact NPE cells. Conclusions. Expression of the CA-IV isozyme could account for the significant fraction of CA activity in the cultured NPE, which is membrane associated and SDS resistant. Sensitivity to tryptic hydrolysis suggests the membrane-associated CA partially faces extracellularly. As judged by responses to an extracellular CA inhibitor, the membrane-associated CA has a functional role in maintaining cytoplasmic pH.

Original languageEnglish (US)
Pages (from-to)2093-2102
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number10
StatePublished - Sep 1997
Externally publishedYes

Fingerprint

Carbonic Anhydrases
Epithelium
Rabbits
Membranes
Carbonic Anhydrase Inhibitors
Dextrans
Carbonic Anhydrase IV
Acetazolamide
Sodium Dodecyl Sulfate
Trypsin
Epithelial Cells
Cell Membrane
Centrifugation
Fluorescent Dyes
Edetic Acid
Cultured Cells

Keywords

  • Ciliary epithelium
  • Dextran-bound carbonic anhydrase inhibitor
  • Eye
  • Intracellular pH
  • Membrane-bound carbonic anhydrase

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Membrane-associated carbonic anhydrase in cultured rabbit nonpigmented ciliary epithelium. / Wu, Qiang; Delamere, Nicholas A; Pierce, William.

In: Investigative Ophthalmology and Visual Science, Vol. 38, No. 10, 09.1997, p. 2093-2102.

Research output: Contribution to journalArticle

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title = "Membrane-associated carbonic anhydrase in cultured rabbit nonpigmented ciliary epithelium",
abstract = "Purpose. To measure the activity of membrane-associated carbonic anhydrase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, determine its identity and its sensitivity to extra-cellular trypsin, and compare the ability of acetazolamide and a cell-impermeant dextran-bound CA inhibitor to change cytoplasmic pH. Methods. Studies were conducted using a cell line derived from rabbit NPE. The cells were lysed and separated into soluble and insoluble fractions by differential centrifugation. CA activity in these fractions was determined using a CO2 hydration assay. In studies with intact cells, a membrane-impermeable high-molecular-weight dextran- bound inhibitor (DBI) was synthesized and used to selectively bind and inhibit the extracellular-facing membrane-bound CA. Measurements of CA activity in intact red blood cells were conducted to confirm DBI remains extracellular. Acetazolamide, a membrane-permeable CA inhibitor, was used to inhibit total CA activity. Intracellular pH was determined using the pH- dependent absorbance of the fluorescent dye BCECF-AM. Results. A low-speed pellet enriched with plasma membrane material accounted for 22.3 ± 6.1{\%} (n = 18) of the total CA activity in the cultured NPE. When intact cells were exposed to trypsin-EDTA, a 28{\%} reduction of membrane-associated CA activity was observed; DBI inhibited this CA activity loss. Cytosolic CA activity was inhibited by 0.2{\%} sodium dodecyl sulfate (SDS). In contrast, membrane- associated CA was SDS resistant, a characteristic of the CA-IV isozyme. By Western blot, CA-IV immunoreactive polypeptide was detected in the cultured cells and also in native rabbit and porcine ciliary epithelium. Inhibition of total CA activity with acetazolamide and inhibition of extracellular-facing membrane-associated CA with DBI caused an identical intracellular pH decrease in intact NPE cells. Conclusions. Expression of the CA-IV isozyme could account for the significant fraction of CA activity in the cultured NPE, which is membrane associated and SDS resistant. Sensitivity to tryptic hydrolysis suggests the membrane-associated CA partially faces extracellularly. As judged by responses to an extracellular CA inhibitor, the membrane-associated CA has a functional role in maintaining cytoplasmic pH.",
keywords = "Ciliary epithelium, Dextran-bound carbonic anhydrase inhibitor, Eye, Intracellular pH, Membrane-bound carbonic anhydrase",
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T1 - Membrane-associated carbonic anhydrase in cultured rabbit nonpigmented ciliary epithelium

AU - Wu, Qiang

AU - Delamere, Nicholas A

AU - Pierce, William

PY - 1997/9

Y1 - 1997/9

N2 - Purpose. To measure the activity of membrane-associated carbonic anhydrase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, determine its identity and its sensitivity to extra-cellular trypsin, and compare the ability of acetazolamide and a cell-impermeant dextran-bound CA inhibitor to change cytoplasmic pH. Methods. Studies were conducted using a cell line derived from rabbit NPE. The cells were lysed and separated into soluble and insoluble fractions by differential centrifugation. CA activity in these fractions was determined using a CO2 hydration assay. In studies with intact cells, a membrane-impermeable high-molecular-weight dextran- bound inhibitor (DBI) was synthesized and used to selectively bind and inhibit the extracellular-facing membrane-bound CA. Measurements of CA activity in intact red blood cells were conducted to confirm DBI remains extracellular. Acetazolamide, a membrane-permeable CA inhibitor, was used to inhibit total CA activity. Intracellular pH was determined using the pH- dependent absorbance of the fluorescent dye BCECF-AM. Results. A low-speed pellet enriched with plasma membrane material accounted for 22.3 ± 6.1% (n = 18) of the total CA activity in the cultured NPE. When intact cells were exposed to trypsin-EDTA, a 28% reduction of membrane-associated CA activity was observed; DBI inhibited this CA activity loss. Cytosolic CA activity was inhibited by 0.2% sodium dodecyl sulfate (SDS). In contrast, membrane- associated CA was SDS resistant, a characteristic of the CA-IV isozyme. By Western blot, CA-IV immunoreactive polypeptide was detected in the cultured cells and also in native rabbit and porcine ciliary epithelium. Inhibition of total CA activity with acetazolamide and inhibition of extracellular-facing membrane-associated CA with DBI caused an identical intracellular pH decrease in intact NPE cells. Conclusions. Expression of the CA-IV isozyme could account for the significant fraction of CA activity in the cultured NPE, which is membrane associated and SDS resistant. Sensitivity to tryptic hydrolysis suggests the membrane-associated CA partially faces extracellularly. As judged by responses to an extracellular CA inhibitor, the membrane-associated CA has a functional role in maintaining cytoplasmic pH.

AB - Purpose. To measure the activity of membrane-associated carbonic anhydrase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, determine its identity and its sensitivity to extra-cellular trypsin, and compare the ability of acetazolamide and a cell-impermeant dextran-bound CA inhibitor to change cytoplasmic pH. Methods. Studies were conducted using a cell line derived from rabbit NPE. The cells were lysed and separated into soluble and insoluble fractions by differential centrifugation. CA activity in these fractions was determined using a CO2 hydration assay. In studies with intact cells, a membrane-impermeable high-molecular-weight dextran- bound inhibitor (DBI) was synthesized and used to selectively bind and inhibit the extracellular-facing membrane-bound CA. Measurements of CA activity in intact red blood cells were conducted to confirm DBI remains extracellular. Acetazolamide, a membrane-permeable CA inhibitor, was used to inhibit total CA activity. Intracellular pH was determined using the pH- dependent absorbance of the fluorescent dye BCECF-AM. Results. A low-speed pellet enriched with plasma membrane material accounted for 22.3 ± 6.1% (n = 18) of the total CA activity in the cultured NPE. When intact cells were exposed to trypsin-EDTA, a 28% reduction of membrane-associated CA activity was observed; DBI inhibited this CA activity loss. Cytosolic CA activity was inhibited by 0.2% sodium dodecyl sulfate (SDS). In contrast, membrane- associated CA was SDS resistant, a characteristic of the CA-IV isozyme. By Western blot, CA-IV immunoreactive polypeptide was detected in the cultured cells and also in native rabbit and porcine ciliary epithelium. Inhibition of total CA activity with acetazolamide and inhibition of extracellular-facing membrane-associated CA with DBI caused an identical intracellular pH decrease in intact NPE cells. Conclusions. Expression of the CA-IV isozyme could account for the significant fraction of CA activity in the cultured NPE, which is membrane associated and SDS resistant. Sensitivity to tryptic hydrolysis suggests the membrane-associated CA partially faces extracellularly. As judged by responses to an extracellular CA inhibitor, the membrane-associated CA has a functional role in maintaining cytoplasmic pH.

KW - Ciliary epithelium

KW - Dextran-bound carbonic anhydrase inhibitor

KW - Eye

KW - Intracellular pH

KW - Membrane-bound carbonic anhydrase

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