Immunolocalization of Na-K-ATPase, Na-K-Cl and Na-glucose cotransporters in the conjunctival epithelium

Purpose. To investigate possible regional expression of transport systems i n the conjunctival epithelium given distinct differences in morphological a ppearance between the bulbar and palpebral epithelia as well as variations found in the proportions of Na absorptive versus Cl secretory activities in electrophysiological studies. Methods. Mouse monoclonal antibodies against the alpha1-subunit of Na-K-ATP ase and Na-K-Cl cotransporter (NKCC1) and a rabbit polyclonal antibody agai nst the Na- glucose cotransporter (SGLT1) were used in immunoblotting and i mmunofluorescent labeling of frozen fixed sections isolated from either the bulbar and palpebral regions of the conjunctiva. Results. Western blot analysis clearly demonstrated the presence of Na-K-AT Pase, NKCC1 and SGLT1 proteins in both bulbar and palpebral conjunctiva. In direct immunofluorescence studies on bulbar and palpebral conjunctival port ions revealed intense staining by the anti-NKCC1 and anti-alpha1-Na-K-ATPas e antibodies exclusively at the basolateral surfaces, whereas the anti-SGLT 1 antibody was used with porcine conjunctiva to elicit strong and unambiguo us staining along the apical plasma membrane. Conclusions. Proteins that mediate the transport activities of the Na-K-ATP ase and Na-K-Cl cotransporter are uniformly distributed throughout the palp ebral and bulbar regions of the rabbit conjunctival epithelium. Although th e Na- glucose cotransporter could be detected in immunoblots of the rabbit, this cotransporter appears to be uniformly distributed as well, based upon immunohistochemical sections of the pig conjunctiva. Thus, it appears like ly that mechanisms for Cl secretion and Na absorption reside in both bulbar and palpebral segments of the conjunctival epithelium.