AJP - Cell Physiology, Vol 261, Issue 1 C132-C142, Copyright © 1991 by American Physiological Society

ARTICLES

pHi regulation in frog retinal pigment epithelium: two apical membrane mechanisms

H. Lin and S. S. Miller
School of Optometry, University of California, Berkeley 94720.

This study demonstrates that the apical membrane of frog retinal pigment epithelium (RPE) contains two intracellular pH (pHi) regulatory mechanisms, an electrogenic Na-HCO3 cotransporter blocked by DIDS and an amiloride-inhibitable Na-H antiporter. pHi was studied using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In these cells resting pHi equals 7.26 +/- 0.09 (n = 58). After an acid load (NH4Cl prepulse), pHi recovery required apical extracellular Na concentration ([Na]o) in HCO3 or HCO3-free Ringer. In HCO3 Ringer recovery was completely blocked by 1 mM apical DIDS (n = 5) but was not affected by absence of Cl. In HCO3-free Ringer, recovery was completely blocked by 1 mM apical amiloride (n = 3). At resting pHi, the intrinsic pH-buffering capacity of the cell is approximately 7.1 mM/pH and rises monotonically as pHi decreases. In HCO3 Ringer, the initial rate of acidification caused by apical Na removal, 0.39 +/- 0.03 pH/min (n = 26), was 80-90% inhibited by apical DIDS (n = 5) and 16% inhibited by 1 mM apical amiloride (n = 7), but not affected by absence of Cl. In HCO3 Ringer, initial rates of acidification induced by apical DIDS or amiloride were 0.11 +/- 0.06 (n = 5) and 0.03 +/- 0.02 pH/min (n = 7), respectively. These results indicate that the Na-HCO3 cotransporter accounts for 80-90% of the acid extrusion from frog RPE cells. Increasing apical [K]o from 2 to 5 mM approximates the in vivo apical [K]o changes during a light-dark transition and alkalinizes the cells. [K]o-induced alkalinization had an initial rate of 0.11 +/- 0.02 pH/min (n = 16), which was approximately 75% inhibited by apical DIDS (to 0.04 +/- 0.01 pH/min, n = 7) and completely blocked by HCO3/CO2 removal from both bathing solutions. [K]o-induced pHi changes alter RPE transport mechanisms and may affect RPE-photoreceptor interactions.

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