Kinetics of RNA synthesis in toad bladder epithelium: action of aldosterone during the latent period

Abstract

A B S T R A C T Processing of RNA in the toad bladder was analyzed by polyacrylamide-gel electrophoresis to determine whether aldosterone causes any changes in the 1 hr before it potentiates transport of sodium ion. No change was found in the quantity or in the specific activity of bulk RNA labeled with uridine-5-3H. In vivo and in vitro with either uridine-5-'H or with methionine-(methyl)-'H as precursors, processing of RNA was extremely slow. Heterodisperse RNA was obvious after 30 min of continuous labeling, but labeling of the 40S precursor of ribosomal RNA was not apparent for 60 min. Labeling of mature 28S and 18S RNA first became apparent after 8 hr. -S RNA was the principal fastmigrating species labeled at 30 min, and 4S RNA was not heavily labeled until 1 hr. Aldosterone (5 x 10-mole/liter) produced no changes. If care were not taken to inhibit metabolism of native bacteria colonizing the bladder, bacterial RNA of high specific activity predominated. We conclude that RNA metabolism in the toad bladder is extraordinarily slow, that a major acceleration of de novo synthesis in response to physiologic doses of aldosterone was not demonstrable, and that some reports to the contrary may have been influenced by artifacts from bacterial RNA metabolism. Earlier evidence for obligatory alterations in RNA metabolism during the latent period is not strong.

Kinetics of RNA Labeling in Toad Bladder Epithelium: Effects of Aldosterone and Related Steroids

“…(2) During the first 30 min, the effect of aldosterone on the labeling of the 9S-12S fraction is much greater than the effect, if any, on the 18S and 28S rRNA peaks (Figs. 3 and 4), If all classes of RNA share a common precursor pool, which is likely, these results imply enhanced synthesis of mRNA and are in contrast to those of.Rousseau and Crabbe (13), Vancura et al (14), and Sharp and Komack (23). These earlier studies, however, differed from ours in a number of respects: (1) We used constant temperature conditions (25 0.20), which provided more reproducible and faster kinetics of RNA synthesis; which is in accord with the finding of pronounced temperature dependence in [3H ]uridine uptake (12).…”

“…(5) Well-defined labeling of all of the classes of cytoplasmic RNA was achieved with a 30-min [3H]uridine pulse and a chase of 90-150 min. Some of the earlier studies (13,14,23) used continuous labeling which may have obscured the increment in labeling of the cytoplasmic 9S-12S class imparted by the steroid. We recently found that pulse labeling 150 mim after administration of the hormone resulted in lesser incorporation of [3H]uridine into 9S-12S RNA than a similar pulse during the first 30 mim of exposure to the steroid (unpublished observations).…”

“…Precise definition of regulation of transcription by aldosterone, however, has not yet been achieved. Recent attempts to demonstrate direct effects on the synthesis of discrete classes of RNA were unsuccessful (13,14). …”

Effects of Aldosterone on RNA and Protein Synthesis in the Toad Bladder

“…On the other hand, sedimentation analysis of RNA extracted from the polysomes with dodecylsulfate revealed a trend for the radioactive profile to depart from the control when aldosterone 0.75 0. 50 was present in the incubation medium (Fig.6). The significance of the latter results is difficult to assess because the deviations in the ratio value could not always be exactly superimposed from one set of experiments to another and also because the greatest deviations are observed in fractions of the gradients where radioactivity is very low.…”

“…Indeed, studies on toad bladder and rat kidney have shown that the effect of aldosterone on sodium transport is prevented by inhibitors of RNA and protein synthesis [40,41,, that aldosterone is specifically bound in the cell nucleus [Q-13,431 and that it increases the specific radioactivity of RNA upon exposure of the tissue to labelled precursors [16][17][18]47,48]. However, there are several inconsistencies when results from different laboratories are compared [49,50]. Moreover, all these effects could be accounted for by other interpretations than derepression of specific genes [51].…”