Differential covalent labeling of apical and basal-lateral membranes of the epithelium of the toad bladder

Summary

The apical (luminal) plasma membrane of toad bladder epithelial cells has been labeled with (¹²⁵I) diazo-diiodo sulfanilic acid (¹²⁵I-DDISA) as demonstrated by electron-microscopic autoradiography. The silver grains (¹²⁵I) were localized exclusively to the apical surface. At concentrations of DDISA of 10⁻³ m or less, binding to the apical membrane had no significant effect on the fine structure of the epithelium. At concentrations of DDISA of 10⁻⁶ m or less, the baseline short-circuit current (SCC), and the response to cyclic 3′,5′-adenosine monophosphate (cAMP) plus theophylline were unimpaired. At 10⁻⁵ m, baseline SCC was unchanged and the response to cyclic AMP plus theophylline was enhanced. At concentrations of 10⁻⁴ m and greater baseline SCC was depressed and the response to the nucleotide inhibited.

The basal-lateral epithelial plasma membranes were labeled by exposing the serosal side to pyridoxal phosphate and reducing the resultant Schiff base with sodium borotritide (³H-NaBH₄). In electron-microscopic autoradiographs, the silver grains (³H) were found over the basal and lateral surfaces of the epithelium. At concentrations of pyridoxal phosphate of 10⁻⁴ m and³H-NaBH₄ of 10⁻³ m, there were no significant changes in the fine structure of the epithelium. Addition of pyridoxal phosphate (10⁻⁴ m) and NaBH₄ (10⁻³ m) to the serosal side decreased the baseline SCC significantly but not the response to vasopressin.

Covalent attachment of the¹²⁵I and the³H was indicated by resistance to elution in the preparation of the sections for electron-microscopy and the reagent requirements for binding.