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Bicarbonate transport mechanisms in rabbit ciliary body epithelium

1991; Elsevier BV; Volume: 52; Issue: 4 Linguagem: Inglês

10.1016/0014-4835(91)90035-d

1096-0007

J. Mario Wolosin, Joseph A. Bonanno, David K. Hanzel, Terry E. Machen,

Metabolism and Genetic Disorders

Sections of whole ciliary body dissected from Dutch belted rabbits were incubated with the cell entrappable pH probe BCECEF-AM. This led to a highly specific localization of epifluorescence emission at the exposed, non-pigmented cell layer (npe) of the dual layered epithelium that covers this organ. The BCECF-loaded tissue sections were superfused in a flow-through chamber and the intracellular pH (pHi) of small groups (10–20) of cells was derived from the ratio of the emission intensities derived from excitations at 490 and 440 nm. In CO2/HCO3− Ringer's, npe pHi = 7·09±0·11. Replacement of CO2/HCO3− by Hepes increased pHi by 0·22±0·02, indicating alkali secretory activity under the bicarbonate-rich conditions. Replacement of Cl− by gluconate elicited a rapid, 0·6-U increase in pHi. This effect exhibited little dependence on Na+ and was inhibited by 0·5 mm dihydro-4,4′-diisothiocyanatostilbene -2,2′-disulfonate (H2DIDS). These results indicate the presence of an electroneutral Cl−/base exchange activity. Elevation of [K+] (by partial replacement of Na+) also elicited increases in pHi. In Cl−-free media pHi reached 7·8–8·0, a condition under which intracellular [HCO3−] is at least twice as high as its extracellular value. This effect did not occur in the absence of Na+. The Na+-dependent high [K+]-induced pHi increase was inhibited by H2DIDS. The effects of Ba2+ on pHi, alone and in combination with high [K+], as well as that of full K+ removal, suggested that the link between high [K+] and pHi increase was mainly due to the effect of cell depolarization on an electronegative Na+ dependent HCO3− transporter. Under normal physiological conditions, the two acid/base transport systems are the main determinants of npe pHi.