Responses of sodium-hydrogen exchange to nitric oxide in porcine cultured nonpigmented ciliary epithelium

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Abstract

Purpose. To better understand how nitric oxide (NO) alters the function of the nonpigmented ciliary epithelium (NPE), studies were performed to determine the influence of NO on sodium-hydrogen exchanger (NHE) activity. Methods. Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF (2',7'-bis(2-carboxyl)-5(6)- carboxyfluorescein-acetoxyethyl ester). Na-H exchanger (NHE) was examined by immunolocalization. Results. In cells acidified by 5 minutes of exposure to 20 mM ammonium chloride, pHi recovery was partially inhibited by sodium nitroprusside (SNP), an NO donor, and L-arginine, the endogenous substrate for NO synthase. SNP and dimethyl amiloride (DMA), an NHE inhibitor, inhibited pHi recovery to a similar degree. In bicarbonate-free buffer SNP+DMA elicited no additional change in pHi recovery beyond that elicited by DMA alone. This suggests that SNP causes NHE inhibition. the SNP's effect on pHi recovery was mimicked by 8-pCPT-cGMP but suppressed by ODQ and H-8. Ouabain alone reduced pHi recovery, but SNP+ouabain caused significant further reduction. Immunolocalization studies revealed NHE1 and -4 in native and cultured NPE. Conclusions. NHE1 and -4 are expressed at the NPE basolateral margin. The findings suggest the NHE is inhibited by NO which acts via a cGMP and protein kinase G signaling pathway. The NHE response does not appear to be the consequence of NO-induced Na,K-ATPase inhibition. Because NO synthases are expressed in porcine NPE, NO could act as an autocrine regulator of NHE activity. Although NHE inhibitors are known to lower intraocular pressure (IOP), further studies are needed to understand whether changes in NHE activity contribute to the IOP-lowering effect of NO donors.

Original languageEnglish (US)
Pages (from-to)5851-5858
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume50
Issue number12
DOIs
StatePublished - Dec 2009

Fingerprint

Sodium-Hydrogen Antiporter
Hydrogen
Nitric Oxide
Swine
Epithelium
Sodium
Nitroprusside
Amiloride
Nitric Oxide Donors
Ouabain
Intraocular Pressure
Nitric Oxide Synthase
Cyclic GMP-Dependent Protein Kinases
Ammonium Chloride
Bicarbonates
Single Nucleotide Polymorphism
Arginine
Buffers
Esters

ASJC Scopus subject areas

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

Cite this

@article{425ae7f276144af7ad182da6ab82c539,
title = "Responses of sodium-hydrogen exchange to nitric oxide in porcine cultured nonpigmented ciliary epithelium",
abstract = "Purpose. To better understand how nitric oxide (NO) alters the function of the nonpigmented ciliary epithelium (NPE), studies were performed to determine the influence of NO on sodium-hydrogen exchanger (NHE) activity. Methods. Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF (2',7'-bis(2-carboxyl)-5(6)- carboxyfluorescein-acetoxyethyl ester). Na-H exchanger (NHE) was examined by immunolocalization. Results. In cells acidified by 5 minutes of exposure to 20 mM ammonium chloride, pHi recovery was partially inhibited by sodium nitroprusside (SNP), an NO donor, and L-arginine, the endogenous substrate for NO synthase. SNP and dimethyl amiloride (DMA), an NHE inhibitor, inhibited pHi recovery to a similar degree. In bicarbonate-free buffer SNP+DMA elicited no additional change in pHi recovery beyond that elicited by DMA alone. This suggests that SNP causes NHE inhibition. the SNP's effect on pHi recovery was mimicked by 8-pCPT-cGMP but suppressed by ODQ and H-8. Ouabain alone reduced pHi recovery, but SNP+ouabain caused significant further reduction. Immunolocalization studies revealed NHE1 and -4 in native and cultured NPE. Conclusions. NHE1 and -4 are expressed at the NPE basolateral margin. The findings suggest the NHE is inhibited by NO which acts via a cGMP and protein kinase G signaling pathway. The NHE response does not appear to be the consequence of NO-induced Na,K-ATPase inhibition. Because NO synthases are expressed in porcine NPE, NO could act as an autocrine regulator of NHE activity. Although NHE inhibitors are known to lower intraocular pressure (IOP), further studies are needed to understand whether changes in NHE activity contribute to the IOP-lowering effect of NO donors.",
author = "Shahidullah, {Mohammad -} and Amritlal Mandal and Delamere, {Nicholas A}",
year = "2009",
month = "12",
doi = "10.1167/iovs.09-3453",
language = "English (US)",
volume = "50",
pages = "5851--5858",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "12",

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TY - JOUR

T1 - Responses of sodium-hydrogen exchange to nitric oxide in porcine cultured nonpigmented ciliary epithelium

AU - Shahidullah, Mohammad -

AU - Mandal, Amritlal

AU - Delamere, Nicholas A

PY - 2009/12

Y1 - 2009/12

N2 - Purpose. To better understand how nitric oxide (NO) alters the function of the nonpigmented ciliary epithelium (NPE), studies were performed to determine the influence of NO on sodium-hydrogen exchanger (NHE) activity. Methods. Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF (2',7'-bis(2-carboxyl)-5(6)- carboxyfluorescein-acetoxyethyl ester). Na-H exchanger (NHE) was examined by immunolocalization. Results. In cells acidified by 5 minutes of exposure to 20 mM ammonium chloride, pHi recovery was partially inhibited by sodium nitroprusside (SNP), an NO donor, and L-arginine, the endogenous substrate for NO synthase. SNP and dimethyl amiloride (DMA), an NHE inhibitor, inhibited pHi recovery to a similar degree. In bicarbonate-free buffer SNP+DMA elicited no additional change in pHi recovery beyond that elicited by DMA alone. This suggests that SNP causes NHE inhibition. the SNP's effect on pHi recovery was mimicked by 8-pCPT-cGMP but suppressed by ODQ and H-8. Ouabain alone reduced pHi recovery, but SNP+ouabain caused significant further reduction. Immunolocalization studies revealed NHE1 and -4 in native and cultured NPE. Conclusions. NHE1 and -4 are expressed at the NPE basolateral margin. The findings suggest the NHE is inhibited by NO which acts via a cGMP and protein kinase G signaling pathway. The NHE response does not appear to be the consequence of NO-induced Na,K-ATPase inhibition. Because NO synthases are expressed in porcine NPE, NO could act as an autocrine regulator of NHE activity. Although NHE inhibitors are known to lower intraocular pressure (IOP), further studies are needed to understand whether changes in NHE activity contribute to the IOP-lowering effect of NO donors.

AB - Purpose. To better understand how nitric oxide (NO) alters the function of the nonpigmented ciliary epithelium (NPE), studies were performed to determine the influence of NO on sodium-hydrogen exchanger (NHE) activity. Methods. Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF (2',7'-bis(2-carboxyl)-5(6)- carboxyfluorescein-acetoxyethyl ester). Na-H exchanger (NHE) was examined by immunolocalization. Results. In cells acidified by 5 minutes of exposure to 20 mM ammonium chloride, pHi recovery was partially inhibited by sodium nitroprusside (SNP), an NO donor, and L-arginine, the endogenous substrate for NO synthase. SNP and dimethyl amiloride (DMA), an NHE inhibitor, inhibited pHi recovery to a similar degree. In bicarbonate-free buffer SNP+DMA elicited no additional change in pHi recovery beyond that elicited by DMA alone. This suggests that SNP causes NHE inhibition. the SNP's effect on pHi recovery was mimicked by 8-pCPT-cGMP but suppressed by ODQ and H-8. Ouabain alone reduced pHi recovery, but SNP+ouabain caused significant further reduction. Immunolocalization studies revealed NHE1 and -4 in native and cultured NPE. Conclusions. NHE1 and -4 are expressed at the NPE basolateral margin. The findings suggest the NHE is inhibited by NO which acts via a cGMP and protein kinase G signaling pathway. The NHE response does not appear to be the consequence of NO-induced Na,K-ATPase inhibition. Because NO synthases are expressed in porcine NPE, NO could act as an autocrine regulator of NHE activity. Although NHE inhibitors are known to lower intraocular pressure (IOP), further studies are needed to understand whether changes in NHE activity contribute to the IOP-lowering effect of NO donors.

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U2 - 10.1167/iovs.09-3453

DO - 10.1167/iovs.09-3453

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SP - 5851

EP - 5858

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

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