Effect of mercurial compounds on net water transport and intramembrane particle aggregates in ADH-treated frog urinary bladder

Abstract

It has been suggested that during the oxytocin-induced hydrosmotic response, water crosses the luminal membrane of urinary bladder epithelium cells through membrane-spanning proteins. Although specific inhibitors of osmotic water transport have not been found, certain sulfhydryl reagents such as mercurial compounds may help to identify the proteins involved in this permeation process. We tested the effects of p-chloromercuribenzene sulfonate (PCMBS) and of fluorescein-mercuric acetate (FMA) on the net water flux, the microtubule and microfilament structures of the frog urinary bladder, and the distribution of intramembrane particle aggregates in the luminal membrane. We observed that: (i) 5 mM PCMBS at pH 5 and 0.5 mM FMA at pH 8 added to the mucosal bath at the maximum of the response to oxytocin partially inhibited the net water flux. Inhibition then increased progressively when the preparation was repeatedly or continuously stimulated, until it reached a maximal inhibition at 120 min. This inhibition was not reversed even when cystein was added in the mucosal bath. PCMBS and FMA effects were also observed when cyclic AMP (3',5' cyclic adenosine monophosphate) was used to increase water permeability, (ii) PCMBS mucosal pretreatment did not modify the basal water flux but potentiated the inhibitory effect of PCMBS or FMA on the hydrosmotic response to oxytocin. (iii) Microtubule and microfilament network, visualized in target cells by immunofluorescence, was not affected by PCMBS. (iv) The maximal PCMBS or FMA inhibition was not associated with a reduction of aggregate surface area in the apical membrane. The persistence of the intramembrane particle aggregates associated with the oxytocin-induced hydrosmotic response during the net water flux inhibition by PCMBS, suggests that the PCMBS effect occurs possibly at the level of sulfhydryl groups of the water channel itself.