Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium

Summary In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with³H-uridine (30 min pulse–140 min chase), with or without aldosterone (3.5×10^–8 m, 7×10^–8 m) in the presence or absence of SC-9420 (7×10^–6 m, 2.5×10^–5 m) at molar ratios of 2001 to 2801. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5–20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of³H-uridine (30 min pulse–150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change inscc correlated linearly with the fractional change in³H-uridine of 12S cytoplasmic RNA (r=0.95,p<0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of³H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na⁺ transport.