Structural and cytochemical differentiation of membrane elements of the apical membrane of amphibian urinary bladder epithelial cells. A label fracture study

Abstract

It is now generally accepted that ADH-induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze-fractured preparations and their chemical nature is still unknown. In this study, we used the label-fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156-1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze-fractured frog urinary bladder epithelial cells. With label-fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label-fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co-distribution of WGA-gold complexes with the segregated particles on the E face. This indicates that WGA-binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze-fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA-binding sites appeared to be associated to these aggregates.

Wheat germ agglutinin (WGA) reduces ADH-induced water flow and induces cell surface changes in epithelial cells of frog urinary bladder

“…Differences could also result from functional characteristics such as electrical coupling between neighbouring cells or possible differences in the physiological state of the cells. Heterogeneity in lectinbinding properties of the granular cells is possibly related to differences in particle density on Ef described in the frog bladder by Chevalier et alo [6] and to cell-to-ceU differences observecl in WGA-induced endocytosis and simultaneous microvilli collapsus described in this article. Binding of the lectins to receptors has been shown to modify receptor repartitition.…”

“…From a random distribution, they redistribute on the cell apical surface by lateral movement [23]. In the case of WGA in frog urinary bladder, this leads to a patching-like phenomenon [6] in which the carbohydrate chains of the coat comigrate with the large intramembrane particles present on the external membrane leaflet, suggesting relationships between intrinsic glycoproteins and the membrane particles [42]. This could be due to the fact that WGA is a divalent lectin which probably cross-finks and displaces the glycoproteins in the membrane.…”

76 and 14 kDa polypeptides, two major components released from amphibian urinary bladder epithelium. Localization and potential role

“…The gold label appears superimposed with the conventional image of the E face (EF). We previously demonstrated that in label fracture/gold particles are clearly located under the fracture face [10,37]. × 58000.…”

Freeze‐fracture cytochemistry: Review of methods

“…In addition to cell suspensions, label-fracture can be applied to monolayer cell cultures (Boonstra et al, 1985(Boonstra et al, ,1987Carpentier et al, 1985) and intact epithelia (Chevalier et al, 1986;Brown and Orci, 19861, producing E-surface labeling/E-face replica views of the "dorsal" plasma membrane and luminal plasma membrane, respectively. Isolated organelles (Van Berkel et al, 1986) and membranes also provide suitable systems for study; in the former, P-surface labelling is viewed with the membrane's replicated P face2 while the latter can provide both P-surface label/P-face views and E-surface label/E-face views.…”