Ion flux changes induced by voltage clamping or by amphotericin B in the isolated urinary bladder of the trout.

Abstract

SUMMARY1. When bathed with Ringer solutions on both sides in Ussing chambers, isolated urinary bladders of trout carry out an electrically silent transport of Na+ and Clions from mucosa to serosa. In the present experiments, an electrical gradient was set up across the epithelium mounted under these conditions either by using a voltage-clamp technique or by submitting the mucosal side to the polyene antibiotic Amphotericin B 2. With stable potential differences between -40 and + 40 mV applied to the tissue, both unidirectional fluxes (Jms and Jsm) for Na+ and for Cl-varied as a function of the electrical gradient. This indicates that a sizeable fraction of these ionic exchanges is due to passive diffusion across a low-resistance pathway.3. Amphotericin B (40 ,sg/ml.) applied to the mucosal side induced an immediate and large (up to 50 mV, serosa positive) potential difference and a sustained diminution in tissue resistance. These effects were strictly Na+-dependent and were reduced in the presence of ouabain. Therefore they are due to increased passive entry of Na+ along its electrochemical gradient across the cellular apical membrane. 4. The effect of Amphotericin B was enhanced when Cl-was replaced by an impermeant anion such as gluconate. Therefore the shunting effect of Cl-on the induced potential observed in the control conditions appears to be due to an increase in Cl-permeability across an anion pathway. Flux data indicate that this pathway is transcellular.

Ion‐selective micro‐electrode studies of the electrochemical potentials in trout urinary bladder.

“…In trout urinary bladder, coupled NaCl co-transport is associated with an undetectable transepithelial potential difference (Vt) and short-circuit current (I,,). However, in the presence of the antibiotic ionophore amphotericin B, in the mucosal Ringer, the trout bladder produces a transient serosa positiveVt and ISc (Fossat & Lahlou, 1982). These effects of the antibiotic were found to be strictly Na dependent and were reduced in the presence of ouabain.…”

“…Amphotericin B was used as amphotericin B oxycholate (Fungizone, Squibb) at a final concentration of 40 jug ml-' in the mucosal Ringer. Oxycholate is present in the commercial preparation as an emulsifier and is reported to have no effect on the electrical properties of Necturus gall-bladder (Graf & Giebisch, 1979) nor trout urinary bladder (Fossat & Lahlou, 1982). Ouabain was obtained from Sigma.…”

“…However, we calculate that little change in Ri occurs and furthermore transepithelial Cl-fluxes are not modified by amphotericin B (Fossat & Lahlou, 1982 (Fossat & Lahlou, 1982).…”

“…The technique is similar to that used in the amphotericin B treated rabbit gall-bladder (Cremaschi, Henin, Meyer & Bacciola, 1977;Henin & Cremaschi, 1978), toad urinary bladder (Reuss, Gatzy & Finn, 1978) and in the nystatin treated rabbit urinary bladder (Lewis, Eaton, Clausen & Diamond, 1977;Wills, Lewis & Eaton, 1979 Amphotericin B produces a serosa positive Vt in Necturus gall-bladder (Reuss, 1978), rabbit gall-bladder (Cremaschi etal. 1977) and trout urinary bladder (Fossat & Lahlou, 1982) and several different hypotheses have been advanced to explain the origin of this Vt. In trout bladder the Vt induced by the ionophore may have resulted from (i) a fall in Vmc as a result of a decrease in Ra, (ii) a fall in Ea and/or a rise in Eb such that the net result is an increase in (Ea -Eb), (iii) a serosa positive change in E;, (iv) electrogenic extrusion of Na across the basolateral membrane.…”

“…We used two independent methods to determine the electrical resistance of the apical and basolateral cell membranes and of the paracellular shunt pathway; (a) (Graf & Giebisch, 1979) nor trout urinary bladder (Fossat & Lahlou, 1982 Single-and double-barrel ion-sensitive micro-electrodes were prepared and calibrated as previously described (Harvey & Kernan, 1984a, b). Double-barrelled micro-electrodes were calibrated with a constant background interference of 100 mM-KCl for Na+ electrodes, 25 mM-NaHCO3 for Cl-electrodes and 10 mM-NaCl for K+ electrodes.…”