Responses of sodium-hydrogen exchange to nitric oxide in porcine cultured nonpigmented ciliary epithelium.
OBJECTIVE
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 (pH(i)) 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, pH(i) 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 pH(i) recovery to a similar degree. In bicarbonate-free buffer SNP+DMA elicited no additional change in pH(i) recovery beyond that elicited by DMA alone. This suggests that SNP causes NHE inhibition. the SNP's effect on pH(i) recovery was mimicked by 8-pCPT-cGMP but suppressed by ODQ and H-8. Ouabain alone reduced pH(i) 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.
Shahidullah
Mohammad
M
Department of Physiology, University of Arizona, Tucson, Arizona 85724, USA. shahidua@email.arizona.edu
Mandal
Amritlal
A
Delamere
Nicholas A
NA
D000818:Animals; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D004848:Epithelium; D005452:Fluoresceins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013552:Swine
D005452:Fluoresceins; D005456:Fluorescent Dyes; D020030:Nitric Oxide Donors; D017923:Sodium-Hydrogen Exchangers; D009599:Nitroprusside; D009569:Nitric Oxide; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein