Reevaluation of Cl-/HCO3- exchange in cultured bovine corneal endothelial cells.
OBJECTIVE
To determine the apical versus basolateral polarity of the putative anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (Em) changes on HCO3- transport.


METHODS
BCECs grown on permeable supports were used for independent perfusion of apical and basolateral surfaces. Intracellular pH (pHi) was measured using the fluorescent dye BCECF. Relative changes in Em were measured using the fluorescent dye bis-oxonol. Western blot analysis was used to detect immunoreactivity against the anion exchanger (AE1 or AE2).


RESULTS
Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH units; both sides, 0.20 pH units). Application of 100 microM H2-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, on the apical side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidification (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkalinization (0.02 pH units), whereas basolateral exposure had no effect. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DPC), and niflumic acid (50-200 microM), known Cl- channel blockers, produced cellular acidification in control Ringer. Niflumic acid hyperpolarized Em and inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response.


CONCLUSIONS
Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cellular alkalinization after Cl- removal is caused primarily by depolarization of Em, which drives HCO3- influx through the basolateral electrogenic Na+:nHCO3- cotransporter. In contrast with cultured BCECs, AE2 may be present in fresh cells.
Bonanno
J A
JA
University of California, Morton D. Sarver Center for Cornea and Contact Lens Research, School of Optometry, Berkeley, USA.
Yi
G
G
Kang
X J
XJ
Srinivas
S P
SP
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000187:drug effects; D009544:Niflumic Acid / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D017920:Antiporters; D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D005456:Fluorescent Dyes; D009579:Nitrobenzoates; D062367:ortho-Aminobenzoates; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D009544:Niflumic Acid; C018564:dihydro-DIDS; C001209:fenamic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid