Electrical properties of the rabbit urinary bladder assessed using gramicidin D

Abstract

Recently, antibiotics have enjoyed widespread usage as tools in studies of epithelial transport. In the present study we assess the usefulness of the pore-forming antibiotic gramicidin D as a means for probing the electrical properties of the tight epithelium rabbit urinary bladder. Addition of 50 micron gramicidin to the mucosal bath (either a NaCl or KCl Ringer's solution) led to a large irreversible increase in the transepithelial conductance (GT) with 800 sec. GT increased by approximately 1200% and 500% in KCl and NaCl Ringer's solutions, respectively. Microelectrode measurements of the resistance ratio (the ratio of apical membrane resistance to basolateral membrane resistance) showed that apical membrane resistance is decreased by the drug. Measurements of the basolateral membrane resistance (Rbl) and tight junctional resistance (Rj) using a new and independent method (based on the perturbation of basolateral membrane electrogenic Na+ pump) demonstrated that Rbl and Rj were unaffected, suggesting that the effects of gramicidin are restricted to the apical membrane for periods of at least 2 hours after drug addition. The selectivity of the gramicidin-induced permeability in the apical membrane was calculated from measurements of the apical membrane potential after ion substitutions using a modified version of the constant field equation. The selectivity sequence for cations was Cs+ greater than K+ greater than Na+ greater than Li+ greater than choline. Unlike the commonly used polyene antibiotics nystatin and amphotericin B, gramicidin did not induce a significant Cl- permeability. In addition, the dose-response curve had a slope of 1. A method is described for calculating membrane resistances directly from transepithelial measurements under some conditions of gramicidin use, without requiring the use of microelectrode measurements.