Identification and localization of acid-base transporters in the conjunctival epithelium

Acid-base transporters of rabbit and porcine conjunctival epithelia were identified and localized with immunoblotting and immunohistochemical techniques using specific antibodies against carriers commonly found in epithelia, i.e. the Cl(-)/HCO3(-)exchanger (AE2), Na(+)/H(+) exchanger (NHE-1, -2, -3) and the electrogenic Na(+)-(n)HCO3(-) cotransporter (NBC). Western blot analysis demonstrated that anti-AE2 reacted with an approximate 170 kDa protein in both rabbit and pig cell membranes prepared from separately isolated bulbar and palpebral conjunctivae. NHE1 was similarly identified in these distinct conjunctival regions but results with anti-NBC were ambiguous. Histochemical examinations indicated that the AE2 and NHE1 proteins reside on the basolateral surfaces of the plasma membrane throughout the multilayered tissue. The immunostaining of porcine cryosections for AE2 and rabbit sections for NHE1 was specific, because of its abolishment following either pre-absorption with the corresponding peptide or omission of the primary antibody. Screening with anti-NBC produced weak staining of the sections that appeared to be non-specific. For confirmation of these results, the acid-base transporters present in rabbit cell cultures of conjunctival epithelia were ascertained from the changes in intracellular pH (pH(i)) evoked upon sequential superfusion with media of altered composition. This approach readily obtained Na(+)- and Cl(-)-dependent pH(i)effects consistent with the existence of Cl(-)/HCO3(-) and Na(+)/H(+)exchange activities. Evidence for the presence of NBC could not be acquired, thereby substantiating the observations from the immunodetection techniques. The identity and location of the antiporters that were found suggested that these elements could contribute to transcellular Cl(-)transport in the basolateral-to-apical direction. To test this possibility, the effects of AE and/or NHE inhibition were determined on the bumetanide-insensitive Cl(-)-dependent short-circuit current across rabbit conjunctivae freshly isolated in Ussing-type chambers. These experiments revealed that such current is indeed sustained by the antiporters. Results with acetazolamide further suggested that the contribution of the acid-base transporters towards transepithelial Cl(-)secretion is variable and dependent upon individual rates of metabolic CO(2)production. Overall, the present study provides an initial identification of the acid-base transporters present in the conjunctiva. Besides their likely role in intracellular pH regulation, the parallel, basolateral expression of AE2 and NHE1 indicates that these elements do not directly contribute to the pH of the tear film but may complement the Na(+)-2Cl(-)-K(+)cotransporter in effectuating Cl(-)secretion.