Functional distinction between two transport mechanisms in rabbit gall‐bladder epithelium by use of ouabain, ethacrynic acid and metabolic inhibitors.

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Abstract
Net fluid transport rate, transepithelial potential difference (p.d.) and resistance, and unidirectional Na+-fluxes were measured in rabbit gall-bladder preparations exposed on both sides to bicarbonate-Ringer solution in vitro. Both ouabain and ethacrynic acid (ETCA) caused dose-dependent decreases of net fluid transport rate; ouabain inhibited fluid transport predominantly from the serosal side, but the inhibitory effect of ETCA was elicited mainly from the mucosal (luminal) side. Applied bilaterally, the ID50 [median inhibitory dose] for ouabain was 2.5 .times. 10-6 M, and for ETCA 2.3 .times. 10-4 M. After maximal inhibition at each concentration level of the 2 inhibitors, fluid transport could not be reversed. 2,4-Dinitrophenol (2,4-DNP) (2 .times. 10-4 M) or substitution of O2 by N2 caused an 80% reversible decrease of net fluid transport. The spontaneous p.d. across the rabbit gall bladder was about 2.7 mV, mucosal side positive. 2,4-DNP, N2 and serosal application of ouabain depressed the p.d. after an initial hyperpolarization. This decrease was reversible during recovery from 2,4-DNP and N2, but irreversible after removal of ouabain at concentrations .gtoreq. 10-4 M. Mucosal application of ETCA (10-3 M) caused no decrease in p.d., which actually increased slightly. Calculated passive serosal-to-mucosal Na+-fluxes changed in the same direction as did changes in conductance. Apparently ETCA does not interfere primarily with the Na-K-ATPase or cellular oxidative metabolism. The data support the proposal that the pump responsible for isomotic transepithelial fluid transfer is located in the luminal end of the cells. This pump is ETCA-sensitive. The ATPase-dependent Na-K pump, which can be inhibited by ouabain, is localized in the serosa-facing cell membrane. The inhibition of net fluid transport by ouabain is indirect and mediated by changes in intracellular ion concentrations. The concept that the transepithelial fluid transport mechanism is electroneutral is supported, and the mucosa positive transepithelial p.d. may be due to differences in electromotive forces arising from ion (mainly K+) diffusion across the mucosal and serosal cell membranes.