Elasmobranch rectal gland cell: autoradiographic localization of [3H]ouabain-sensitive Na, K-ATPase in rectal gland of dogfish, Squalus acanthias.

Abstract

Specific binding of radiolabeled inhibitor was employed to localize the Na-pump sites, (Na,K-ATPase) in rectal gland epithelium, a NaCl-secreting osmoregulatory tissue which is particularly rich in pump sites. Slices of gland tissue from spiny dogfish were incubated in suitable [3H]ouabain-containing media and then prepared for Na,K-ATPase assay, measurement of radiolabel binding or quantitative freeze-dry autoradiography at the light microscope level. Gross freezing or drying artifacts were excluded by comparison with additional aldehyde-fixed slices. Characterization experiments demonstrated high-affinity binding which correlated with Na,K-ATPase inhibition and half-saturated at .apprx. 5 .mu.M [3H]ouabain. The normal half-loading time was .apprx. 1 h and low-affinity binding to nonspecific sites was negligible. Autoradiographs from both 1 and 4 h incubated slices showed .apprx. 85% of the bound [3H]ouabain to be localized within a 1-.mu.m wide boundary region where the highly infolded basal-lateral cell membranes are closest to the mitochondria. Most of the enormous Na,K-ATPase activity associated with rectal gland epithelium is in the basal-lateral cell membrane facing interstitial fluid and not in the luminal membrane facing secreted fluid. Distribution along the basal-lateral membrane appears to be nonuniform with a higher density of enzyme sites close to mitochondria.