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M. Shahidullah, A. Mandal, N. A. Delamere; Sodium Nitroprusside Inhibits Intracellular pH Recovery in Cultured Porcine Nonpigmented Ciliary Epithelium. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3997.
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Previously we reported that nitric oxide (NO) donor, sodium nitroprusside (SNP), reduces aqueous humor secretion and intraocular pressure in the porcine eye1 . Here we examine whether NO donors alters intracellular pH (pHi) in cultured porcine NPE. Manipulation of intracellular pH by inhibition of sodium hydrogen exchange (NHE) has been shown to lower IOP in mouse2.
BCECF fluorescence imaging was used to study the effect of drugs on pHi recovery after an acid load. Expression of NHE isoforms in native and cultured NPE was detected by immunohistochemistry and immunoblot.
Both native and cultured porcine NPE expressed NHE1 and NHE4 isoforms. Treatment of cultured NPE with SNP inhibited pHi recovery concentration dependently (10 µM - 1 mM). L-arginine, the endogenous substrate for NO synthase also inhibited pHi recovery. Dimethyl amiloride (DMA), a potent inhibitor of NHE, produced a substantial inhibition of pHi recovery at100 µM. In cells exposed to SNP and DMA simultaneously, the extent of inhibition of pHi recovery was similar to that elicitedby SNP alone. This suggests that the effect of SNP on pHi recovery is mediated through inhibition of NHE. Exogenously applied cGMP, 8-pCPT-cGMP (10µM) mimicked the SNP effect on pHi recovery. The inhibitory effect of SNP on pHi recovery was blocked by 1µM of H-8, an inhibitor of cGMP-dependent protein kinase (PKG).
Primary cultures of porcine NPE continue to express NHE isoforms found in native NPE. NO donors inhibit pHi recovery through inhibition of NHE. The inhibitory effect of NO donors on NHE is mediated through cGMP-PKG signal transduction pathway.References1 Shahidullah M, Yap MK and To CH. cGMP, sodiun nitroprusside and sodium azide reduce aqueous humour formation in the isolated arterially perfused pig eye. British Journal of Pharmacology 2005; 144: 1-9.2 Avila MY, Seidler RW, Stone RA, et al. Inhibitors of NHE-1 Na+/H+ exchange reduce mouse intraocular pressure. Investigative Ophthalmology & Visual Science 2002; 43: 1897-1902.
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