Ultrastructural localization of Na+, K(+)-ATPase in the exorbital lacrimal gland of rat.

Ultrastructural localization of Na+, K(+)-ATPase in the exorbital lacrimal gland of rat was investigated quantitatively by protein A-gold technique. Na+, K(+)-ATPase was purified from the rat kidney, and anti-holo Na+, K(+)-ATPase antibody was obtained from the rabbit by injecting the purified enzyme. A specific antibody against the alpha-subunit of Na+, K(+)-ATPase was affinity purified. Immunoblot analysis revealed that the antibody bound specifically to the alpha-subunit of Na+, K(+)-ATPase of the lacrimal gland. Rats were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde, and the lacrimal glands were embedded in LR White resin. Ultrathin sections were incubated with affinity purified antibody against the alpha-subunit of Na+, K(+)-ATPase, and then with protein A-gold complex. The sections were observed under an electron microscope. Light microscopy with silver enhancement procedure revealed that Na+, K(+)-ATPase was located mainly on the basal region of the cells of intralobular and interlobular ducts. Quantitative immunoelectron microscopic analysis showed that gold particles were found on the basolateral surfaces of the interlobular and intralobular ducts cells and on the basolateral surface of the acinar cells, whereas no significant binding was observed on any part of the apical surfaces of these cells. Labeling density of gold particles was highest on the basolateral surface of the interlobular duct cells, secondarily highest on the basolateral surface of the intralobular duct cells, and lowest on the basolateral surface of the acinar cells. The distribution pattern of Na+, K(+)-ATPase in the acinar cells and the duct cells suggest that this enzyme may play an important role in primary secretion and in determining the composition of electrolytes in the final secretion, respectively.