Src Family Kinase Links Insulin Signaling to Short Term Regulation of Na,K-ATPase in Nonpigmented Ciliary Epithelium

The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na+/H+ exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pHi) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1–100 nM), the rate of pHi recovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na+-dependent pHi recovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 μM) completely abolished the pHi recovery by NHE. 18α-Glycyrrhetinic acid (18α-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pHi recovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined.

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Influence of bafilomycin A1on pHi responses in cultured rabbit nonpigmented ciliary epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.5.c1700 ◽

1997 ◽

Vol 273 (5) ◽

pp. C1700-C1706 ◽

Cited By ~ 5

Author(s):

Qiang Wu ◽

Nicholas A. Delamere

Keyword(s):

Plasma Membrane ◽

Disulfonic Acid ◽

Ciliary Epithelium ◽

Free Medium ◽

Acid Base Balance ◽

Carbonyl Cyanide ◽

Base Balance ◽

Rich Solution ◽

Cytoplasmic Acidification ◽

Nonpigmented Ciliary Epithelium

Aqueous humor secretion is in part linked to [Formula: see text]transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pHi). Because a recent report suggests that NPE cells have a plasma membrane-localized vacuolar H+-ATPase, the present study was conducted to examine whether vacuolar H+-ATPase contributes to pHi regulation in a rabbit NPE cell line. Western blot confirmed vacuolar H+-ATPase expression as judged by H+-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native ciliary epithelium. pHi was measured using 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Exposing cultured NPE to K+-rich solution caused a pHi increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl2. Bafilomycin A1 (0.1 μM; an inhibitor of vacuolar H+-ATPase) inhibited the pHi increase caused by high K+. The pHi increase was also inhibited by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chlorophenylhydazone but not by ZnCl2, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), omeprazole, low-Cl−medium, [Formula: see text]-free medium, or Na+-free medium. Bafilomycin A1 slowed the pHi increase after an NH4Cl (10 mM) prepulse. However, no detectable pHi change was observed in cells exposed to bafilomycin A1 under control conditions. These studies suggest that vacuolar H+-ATPase is activated by cytoplasmic acidification and by reduction of the proton electrochemical gradient across the plasma membrane. We speculate that the mechanism might contribute to maintenance of acid-base balance in NPE.

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