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

Abstract

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-NaBH1). 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 and 3H-NaBH1 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 the 125I and the 3H was indicated by resistance to elution in the preparation of the sections for electron-microscopy and the reagent requirements for binding.

Radio-iodination of plasma membranes of toad bladder epithelium

“…Ekblad et al [6] provided evidence of covalent labeling of the basal-lateral membranes of toad bladder epithelium with pyridoxal phosphate and 3H-NaBH4, as indicated by electron microscopic autoradiography. For the purpose of biochemical analysis, however, the yield of radioactive labeling of these membranes was low.…”

“…In these, as well as the studies with enzymatic radio-iodination, labeling of the surfaces did not perturb the basal or vasopressin-stimulated short-circuit current (SCC) of the toad bladder [6,40]. The radioactive yields of the labeled membranes, however, were too low for biochemical characterization of the labeled product and limited the utility of these procedures for isolation of the separated surfaces.…”

“…In addition to radio-iodination, the apical and basal-lateral surfaces of toad bladder epithelium have been differentially covalently labeled with ~25I-diazodiiodosulfanilic acid and pyridoxal phosphate + 3H-NaBH, [6]. In these, as well as the studies with enzymatic radio-iodination, labeling of the surfaces did not perturb the basal or vasopressin-stimulated short-circuit current (SCC) of the toad bladder [6,40].…”

Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone

“…3 and Table 2. The distribution of mitochondrial (cytochrome oxidase), lysosomal (fl-hexosaminidase) and exposed apical membrane proteins from unstimulated bladders (125I-labeling of apical membrane with diazotized iodosulfanilic acid) [8] were assayed in fractions from differential and density gradient centrifugation. The intermediate pellet fraction is enriched for 125I-labeled apical membrane, mitochondria and lysosomes as well as HRP ( Table 2).…”

“…Eighty microliters of 50 mM NaPO4, pH = 7.5, was added to neutralize solution containing the diazotized reagent, which was then further diluted to a final volume of 0.6 ml by addition of isotonic Ringer's solution. The apical surface was routinely iodinated as described in reference [8] using 2.5 mCi of diazotized ~25I-sulfanilic acid for 15 rain at 2~ The reaction was terminated by removal of the reaction mixture and rinsing of the apical surface with cold isotonic Ringer's solution followed by homogenization buffer. The bladder epithelial cells were then removed by scraping and treated in the homogenation protocol as detailed above.…”