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 approximately 5 microM [3H]ouabain. At this concentration, the normal half-loading time was approximately 1 h and low-affinity binding to nonspecific sites was negligible. Autoradiographs from both 1- and 4-h incubated slices showed approximately 85% of the bound [3H]ouabain to be localized within a 1-micrometer wide boundary region where the highly infolded basal-lateral cell membrane 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.
Elasmobranch rectal gland cell: autoradiographic localization of [3H]ouabain-sensitive Na, K-ATPase in rectal gland of dogfish, Squalus acanthias.
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J Eveloff, K J Karnaky, P Silva, F H Epstein, W B Kinter; Elasmobranch rectal gland cell: autoradiographic localization of [3H]ouabain-sensitive Na, K-ATPase in rectal gland of dogfish, Squalus acanthias.. J Cell Biol 1 October 1979; 83 (1): 16–32. doi: https://doi.org/10.1083/jcb.83.1.16
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