ELASMOBRANCH RECTAL GLAND CELL - AUTORADIOGRAPHIC LOCALIZATION OF [OUABAIN-H-3-SENSITIVE NA, K-ATPASE IN RECTAL GLAND OF DOGFISH, SQUALUS-ACANTHIAS

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 ~ 5 μM [ 3H]ouabain. At this concentration, the normal half-loading time was ~ 1 h and low-affinity binding to nonspecific sites was negligible. Autoradiographs from both 1- and 4-h incubated slices showed ~ 85% of the bound [H]ouabain to be localized within a 1-μm wide boundary region where the highly infolded basal-lateral cell membranes are closest to the mitochondria. These results establish that 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. Moreover, distribution along the basal-lateral membrane appears to be nonuniform with a higher density of enzyme sites close to mitochondria.