Nystatin as a probe for investigating the electrical properties of a tight epithelium.

Abstract

90024.n B S T R A C T We show how the antibiotic nystatin may be used in conjunction with microelectrodes to resolve transepithelial conductance G t into its components: Ga, apical membrane conductance; Gbl, basolateral membrane conductance; and Gj, junctional conductance. Mucosal addition of nystatin to rabbit urinary bladder in Na+-containing solutions caused Gt to increase severalfold to ca. 460/~mho/p.F, and caused the transepithelial voltage V t to approach +50 mV regardless of its initial value. From measurements of G t and the voltage-divider ratio as a function of time after addition or removal of nystatin, values for Ga, Gbl, and Gj of untreated bladder could be obtained. Nystatin proved to have no direct effect on Gbl or Gj but to increase Ga by about two orders of magnitude, so that the basolateral membrane then provided almost all of the electrical resistance in the transcellular pathway. The nystatin channel in the apical membrane was more permeable to cations than to anions. The dose-response curve for nystatin had a slope of 4.6. Use of nystatin permitted assessment of whether microelectrode impalement introduced a significant shunt conductance into the untreated apical membrane, with the conclusion that such a shunt was negligible in the present experiments. Nystatin caused a hyperpolarization of the basolateral membrane potential in Na+-containing solutions. This may indicate that the Na + pump in this membrane is electrogenic.

Distinct Apical and Basolateral Membrane Requirements for Stretch-induced Membrane Traffic at the Apical Surface of Bladder Umbrella Cells

“…In the first phase the TEV hyperpolarized, the conductance and I sc increased, and the C T increased to ϳ100% over starting values ( Figure 4A). Because ion transport across the apical membrane of the umbrella cell is the primary contributor to transepithelial conductance (Lewis et al, 1977), and because C T increased, the changes we observed in phase 1 likely reflected increased ion transport and membrane traffic at the apical plasma membrane domain of the umbrella cells. During the second phase of outward bowing the C T , I sc , and TEV rapidly reversed course ( Figure 4A).…”

β-Adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

“…Indeed, recent work has shown that ENaC can be rapidly controlled via changes in internal [Na ϩ ] and [Cl Ϫ ] (8,22), and it is very unlikely that such ionic control of G Na ϩ (8,22) would be detected in studies of basolaterally permeabilized cells. In this experimental situation, transient changes in internal ionic composition may occur as apical conductances are activated, but the relatively large volume of the external solution implies that such changes would be rapidly compensated for by ions entering/leaving the cytoplasm via the permeabilized membrane (21). The present estimates of G Na ϩ were thus made under relatively constant ionic conditions.…”

“…These compounds form pores in cholesterol-containing membranes that are permeable to Na ϩ , K ϩ , and Cl Ϫ but not to divalent cations or highermolecular-weight substances and thus allow experimental control over the cytoplasmic [Na ϩ ], [K ϩ ], and [Cl Ϫ ], whereas intracellular Ca 2ϩ is regulated by the normal, physiological mechanisms. Because the pores formed by amphotericin B have a higher Cl Ϫ conductance than do those formed by nystatin (13,21), the former compound was used to make measurements of GClϪ.…”

“…Basolateral [Cl Ϫ ] was thus raised to 49 mM by isosmotically replacing gluconate without affectQuantification of Na ϩ pump capacity. The method used to determine the capacity of the basolateral Na ϩ pump is described elsewhere (21,36), and so only brief details are presented here. Epithelial monolayers were mounted in Ussing chambers, bathed with the standard physiological solution (i.e., high Na ϩ ), and treated with apical amiloride (10 M) to block the Na ϩ channels in this membrane, which was then permeabilized using nystatin (75 M).…”

Intracellular sodium ion activity and sodium transport in rabbit urinary bladder.

“…The tissue was dissected, mounted according to the procedures of , and placed between modified Ussing chambers (15 ml./chamber) which were designed to eliminate edge damage and to reduce hydrostatic gradients during micro-electrode impalements (Lewis, Eaton, Clausen & Diamond, 1977). The exposed area of tissue was 1 cm2.…”