Determinants of apical membrane permeabilities of barrier epithelia

Abstract

Renal collecting duct and thick ascending limb, as well as stomach, exhibit strikingly low permeabilities to water and solutes. However, the apical membrane characteristics responsible for these unique permeabilities remain unknown. While the lipid composition of artificial membranes governs membrane permeability, exoplasmic and cytoplasmic leaflets of biological apical membranes exhibit striking asymmetries in lipid composition. This asymmetry, as well as the presence of membrane proteins, may be critical to barrier function. To determine the role of bulk lipid composition in apical membrane barrier function, we compared permeabilities to water (Pf), protons, ammonia, and several small nonelectrolytes of gastric apical membrane vesicles [native gastric vesicles (NGV)] and liposomes prepared from lipids quantitatively extracted from these vesicles [gastric lipid large unilamellar vesicles (LUV)]. Permeabilities were measured on a stopped-flow fluorimeter by monitoring self- or pH-sensitive quenching of entrapped carboxyfluorescein. NGV exhibited low Pf (2.8 +/- 0.3 x 10(-4) cm/s) while gastric lipid LUV Pf averaged 1.2 +/- 0.1 x 10(-3) cm/s, a fourfold increase compared with the value in NGV. Gastric lipid LUV also demonstrated higher permeabilities to protons, ammonia, propylene glycol, butyramide, ethanolamine, and acetamide compared with values in NGV. In contrast, gastric lipid LUV exhibited the same or lower permeabilities to urea, glycerol, and ammonia compared with values in NGV. We conclude that lipid composition alone can reconstitute membrane permeabilities to some, but not all, molecules. These results indicate that bilayer asymmetry may be required for the unique permeability of "water-tight" apical membranes and reveal different barrier mechanisms for water and protons, as opposed to ammonia, urea, and glycerol.

Reconstituting the Barrier Properties of a Water-tight Epithelial Membrane by Design of Leaflet-specific Liposomes

“…Proton Permeability-Apparent proton permeabilities were measured using pH-dependent quenching of fluorescence as described previously (3,10,19,20,22). Stopped-flow experiments were performed in which the liposomes were pretreated with 1 M valinomycin and then rapidly mixed with an identical buffer acidified to pH 6.50.…”

“…J Hϩ is the flux of protons, ⌬C is the initial difference in concentration of protons between the inside and the outside of the vesicle, ⌬pH is the change in pH when time equals , the time constant of the single exponential curve describing the initial change in fluorescence as a function of time, and BCV is the buffer capacity of an individual vesicle (3,10,20). NH 3 Permeability-NH 3 permeability was determined using stopped-flow fluorimetry by monitoring the pH-sensitive increase in fluorescence when vesicles equilibrated to pH 6.8 were rapidly mixed with the same buffer containing 20 mM NH 4 Cl as described (3,10,20).…”

“…NH 3 Permeability-NH 3 permeability was determined using stopped-flow fluorimetry by monitoring the pH-sensitive increase in fluorescence when vesicles equilibrated to pH 6.8 were rapidly mixed with the same buffer containing 20 mM NH 4 Cl as described (3,10,20). NH 3 in solution passes through the membrane and becomes protonated to NH 4 ϩ in the vesicle interior.…”

“…An earlier study from our laboratory demonstrated the functional consequences of losing bilayer asymmetry, when gastric apical vesicles, which possessed exceptionally low permeabilities to water, protons, and non-electrolytes, were made symmetrical by quantitative lipid extraction and reconstitution into liposomes. The newly formed liposomes, although made from identical apical membrane lipids, exhibited much higher permeabilities than the native membrane (10). Bilayer asymmetry is maintained by tight junctions between cells that not only isolate the basolateral and apical membranes, but also the exofacial leaflet from the cytoplasmic leaflet of the apical membrane (14,15).…”

Role of Sulfatide in Influenza A Virus Replication

“…MAL complexes with sulfatide might contribute to stabilization and apical sorting of lipid rafts. 48,49) Such complexes and their associated apical sorting might be involved in progeny virus formation at the apical surface of tracheal epithelial cells. Further study on sulfatide function in virus replication may help to reveal mechanisms underlying these biological activities and these diseases, leading to the development of drugs against sulfatide-associated diseases.…”