Incorporation of [14c]adrenaline Into the Nuclei of Rat Liver Cells

Abstract

A direct relationship between a hormone and a template like DNA or nuclear RNA in rat liver nuclei has, so far, not been established. However, it has already been reported (Decallonne, Briquet & Wiaux, 1967) that adrenaline induced some cytoplasmic modifications in rat liver cells with respect to the turnover of both mRNA 16S and rRNA 29S and 18S.We tried to establish differences between the normal incorporation rate of orotic acid into DNA and nuclear RNA and the rate of incorporation into liver tissue treated with [14C]adrenaline in order to find the hormonal site of action.Rat liver slices, weighing about 500 mg., were incubated with [14C]orotic acid hydrate (sp.act. 3\m=.\2 mc/mm). Each portion of liver was suspended in 10 ml. Ringer solution (5. 10\ m=-\ 3 m-tris-HCl buffer of pH 7\m=.\4 at 38\ m=. \ 2\ s=deg\ , kept under O2:CO2 (95 % :5 %) and 0\m=.\1 \g=m\c[14C]orotic acid/100 mg. liver was added at intervals of 10 min. for 10-90 min. The nuclei were obtained from the cells by the method of Roodyn (1962).Each 500 mg. portion of liver was first washed with 5.10-3 M-tris-HCl buffer at pH 7-4 and then homogenized in 6 ml. of a mixture of saccharose : CaCl2 (0-25 M : 0-0018 m) cooled to 4°. The homogenate was then suspended in 6 ml. of a saccharose : CaCl2 solution (0-34 m : 0-0018 m) and centrifugated for 10 min. at 700 g. The resulting pellet was re-centrifugated twice or three times under the same conditions. By this procedure an efficiency of about 50-70 % with respect to the nuclei and with very little contamination was obtained. The nuclei were then broken down with 9 ml. of a 5 % aqueous solution of sodium dodecyl sulphate (SDS)/100 ml. Microscopic examination showed that this treatment for 3 min. at 37°affected practically all nuclei. This was also controlled by u.v. spectrophotometry at 265 rap. The amount of DNA extracted was used as a basis for the incorporation calculations. The result of the DNA deter¬ minations (300 ±15 pg. DNA/100mg. liver) agrees with that of Paoletti (1963). The incorporation of [14C]orotic acid was expressed as counts/min./100/¿g. DNA. An increase from 50 + 10 counts/min. at 10 min. to 5850 ± 10 counts/min. at 90 min. was found with an average background of about 10 counts/min. When adrenaline (0-5 10~3 mg./100 mg.) was added to the incubation medium, together with orotic acid, there was an inhibition of about 95 % in orotic acid incorporation into the nuclei. This pronounced inhibition took place as soon as the hormone was added to the incubate. Since adrenaline inhibits the incorporation of this common precursor of DNA and RNA into the nucleus, it seems likely that adrenaline itself does not enter the cell nuclei but remains in the cytoplasmic fraction where the inhibition takes place.