Alkaline Ribonuclease Activity Increase in Rat

Acid azo dyes, most of them naphtholdisulfonic acid derivatives, were given intraperitoneally to rats and their effect on "alkaline" ribonuclease activity was studied in total homogenates of kidney cortex and liver. Acid treatment was used to release bound enzyme activity. Several of the dyes, including trypan blue, increased RNase activity in both organs 3 days after administration of single doses, while others, like Evans blue, were inactive. Activity was apparently bound to the sulfonic substitution in the 3, 6 positions in the naphthalene rings, substitutions in the benzidine rings being not critical. All of the active and most of the inactive compounds were taken up by tubule cells of kidney cortex and by reticular and parenchymal cells of liver. While the effect on both liver and kidney was obtained 1 day after trypan blue administration, RNase remained increased for only about 3 days in the first organ, and for at least a month in the second. However, repeated trypan blue doses increased liver enzyme activity for at least 9 days. Serum RNase activity was decreased after trypan blue administration. Ethionine administration together with trypan blue markedly blocked the effect of the dye on liver RNase activity; simultaneously given methionine partially reversed the action of the antimetabolite. This suggests that de novo synthesis of RNase is induced in liver by trypan blue. The action of ethionine on the kidney RNase response to trypan blue was less marked although significant; in view of the possible kidney uptake of the plasma enzyme, interpretation of this finding must be postponed. Results are discussed with reference to the mechanism of the structural specificity of the compounds used, cytological localization of the dyes and their mechanism of action on liver and kidney RNase. Disclosure of the participation of ribonucleic acids in the mechanism of protein synthesis led to a renewal of interest in enzymes involved in RNA synthesis and degradation (1). Moreover, the findings of Brachet (2) and others of in vivo effects of pancreatic ribonuclease (RNase) on a variety of single-celled and multicellular organisms, pointed to the need of further knowledge on the biochemistry and physiology of interand intracellular hydrolytic enzymes of this group (cf. 3). Previous work from this laboratory has shown that in mammals plasma alkaline RNase is inactivated by the kidneys (4) and suggested that the kidney enzyme could be taken up from the plasma (5). During the course of a study on the effect of administering several proteins on RNase activity of the renal cortex (6), azo dyes known to be bound to proteins were also assayed. It was found that trypan blue and some related dyes increase 105 on A uust 0, 2017 jcb.rress.org D ow nladed fom RNase activity of kidney cortex and liver. The t ime course of this effect, s t ructural requirements of the dye molecules, and experiments bearing on the mechan ism of the phenomenon will be presently described. M A T E R I A L S A N D M E T H O D S Albino rats of the Wistar strain, both males and females, wcighing 100 to 200 gin. were used. The dyes were dissolved in saline and thc solutions were administered intraperitoncally in single or repeated injections of 5 ml. per 100 gin. body weight. The rats received the dyes in doses of from 1.7 to 30 mg per 100 gm. body weight (of. Table I). Control animals received only saline. Trypan bluc was purified by ethanol extraction (7), and in some cxpc~'imcnts the ethanol-soluble "rcd fraction" was recovcrcd and assayed. In a few expcrimcnts animals were simultaneously treatcd with DL-cthionine (0.22 mmolcs per 100 gm. body weight) with or without the administration of nL-methioninc (0.25 or 1.25 mmoles per 100 gin. body weight). Under cther anesthesia kidney cortex and liver samples (weighing 20 and 120 rag., respectively) were obtained, one before and the others at different time periods after experimental treatments were started. Tissues were stored at -10°C. until assayed. Homogenatcs were prepared in 0.05 M citric acid, neutralized with 0.25 M K:HPO4, and assayed at pH 7.3 as previously described (8). Assays were performed in triplicate on 0.17 rag. kidney cortex or 3.0 mg. liver. In some experiments RNase activity of blood serum was determined on 5 #l samples. It is known that acid treatment (0.25 N H2SO4) unmasks liver alkaline RNasc activity (9). Prcliminary experiments have shown that citric acid can be convcnicntly uscd for this purpose, giving similar activities as the H2SO4 treatment. Maximal activities were also obtained by making homogenates with 0.5 X 10 -8 M n-ethyl maleimide (cf. 10). Results of RNase assays are expressed either as arbitrary optical densities or in percentages of the activity of initial biopsies. For histological study of the dye uptake, formolcalcium-fixed pieces of kidney and liver were frozen sectioned, the sections lightly counterstained with hematoxylin and mounted in balsam. Although, for descriptive purposes, means and standard errors are given, in the statistical analysis of the results non-parametric tests (11) were mainly used. The significance level was taken at 0.05.