Studies on bicarbonate transporters and carbonic anhydrase in porcine nonpigmented ciliary epithelium

Mohammad - Shahidullah, Chi Ho To, Ryan M. Pelis, Nicholas A Delamere

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Purpose: Bicarbonate transport plays a role in aqueous humor (AH) secretion. The authors examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine nonpigmented ciliary epithelium (NPE). Methods: Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. Results: Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO2/ HCO3- -free HEPES buffer, exposure to a CO2/HCO3- -containing buffer caused rapid acidification followed by a gradual increase in pHi. Subsequent removal of CO2/HCO3- with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pHi. The rate of gradual alkalinization after the addition of HCO3- /CO2 was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethylamiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO3- /CO2 was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pHi. In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44%, respectively. Conclusions: The results suggest the NPE uses a Na+ -HCO-3 cotransporter to import bicarbonate and a Cl -/HCO3- exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.

Original languageEnglish (US)
Pages (from-to)1791-1800
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume50
Issue number4
DOIs
StatePublished - Apr 2009

Fingerprint

Carbonic Anhydrases
Bicarbonates
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
Swine
Epithelium
Aqueous Humor
Buffers
Acetazolamide
Methazolamide
Chloride-Bicarbonate Antiporters
HEPES
Chlorides
Sodium-Bicarbonate Symporters
Carbonic Anhydrase Inhibitors
Indicator Dilution Techniques
Ciliary Body
Fluorescein
Anions
Sodium
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Studies on bicarbonate transporters and carbonic anhydrase in porcine nonpigmented ciliary epithelium. / Shahidullah, Mohammad -; To, Chi Ho; Pelis, Ryan M.; Delamere, Nicholas A.

In: Investigative Ophthalmology and Visual Science, Vol. 50, No. 4, 04.2009, p. 1791-1800.

Research output: Contribution to journalArticle

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abstract = "Purpose: Bicarbonate transport plays a role in aqueous humor (AH) secretion. The authors examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine nonpigmented ciliary epithelium (NPE). Methods: Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. Results: Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO2/ HCO3- -free HEPES buffer, exposure to a CO2/HCO3- -containing buffer caused rapid acidification followed by a gradual increase in pHi. Subsequent removal of CO2/HCO3- with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pHi. The rate of gradual alkalinization after the addition of HCO3- /CO2 was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethylamiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO3- /CO2 was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pHi. In the intact eye, DIDS and acetazolamide reduced AH secretion by 25{\%} and 44{\%}, respectively. Conclusions: The results suggest the NPE uses a Na+ -HCO-3 cotransporter to import bicarbonate and a Cl -/HCO3- exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.",
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AU - To, Chi Ho

AU - Pelis, Ryan M.

AU - Delamere, Nicholas A

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N2 - Purpose: Bicarbonate transport plays a role in aqueous humor (AH) secretion. The authors examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine nonpigmented ciliary epithelium (NPE). Methods: Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. Results: Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO2/ HCO3- -free HEPES buffer, exposure to a CO2/HCO3- -containing buffer caused rapid acidification followed by a gradual increase in pHi. Subsequent removal of CO2/HCO3- with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pHi. The rate of gradual alkalinization after the addition of HCO3- /CO2 was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethylamiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO3- /CO2 was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pHi. In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44%, respectively. Conclusions: The results suggest the NPE uses a Na+ -HCO-3 cotransporter to import bicarbonate and a Cl -/HCO3- exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.

AB - Purpose: Bicarbonate transport plays a role in aqueous humor (AH) secretion. The authors examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine nonpigmented ciliary epithelium (NPE). Methods: Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. Results: Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO2/ HCO3- -free HEPES buffer, exposure to a CO2/HCO3- -containing buffer caused rapid acidification followed by a gradual increase in pHi. Subsequent removal of CO2/HCO3- with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pHi. The rate of gradual alkalinization after the addition of HCO3- /CO2 was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethylamiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO3- /CO2 was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pHi. In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44%, respectively. Conclusions: The results suggest the NPE uses a Na+ -HCO-3 cotransporter to import bicarbonate and a Cl -/HCO3- exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.

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