RNA metabolism in subcellular fractions from rat cerebral cortex and from the visual cortex of rats with retinal degeneration

Abstract

The metabolism of RNA in subcellular fractions from rat cerebral cortex was studied. The following fractions were prepared initially: nuclei (purified through hypertonic sucrose), myelin, mitochondria, synaptosomes, microsomes, and supernatant. The proportion of RNA in each fraction was approximately 10, 3.4, 9.5, 10.1, 33.5, and 24.9%, respectively. This contrasted with the distribution of protein which was 4, 4.4, 20.1, 13.8, 19.5, and 26.5%, respectively. DNA was primarily nuclear but 1–2% was in the mitochondrial fraction. The synaptosomes were further fractionated by osmotic shock and gradient centrifugation and it was found that over 40% of the RNA was associated with the synaptosomal mitochondria and the bulk of the remainder associated with the membrane fraction (characterized by their high Na +-K +-ATPase, glutamine synthetase, and p-nitrophenyl phosphatase activity). Labeling studies using [6- 14C] orotic acid revealed that the nuclear fraction was the most rapidly labeled, reaching a maximum RNA specific activity within 2 hr and decreasing thereafter. The cytoplasmic fractions all reached a maximum specific activity within 48 hr and had the same specific activity as the nuclear fraction α after 72 hr. The supernatant fraction was the most rapidly labeled of the cytoplasmic fractions, the synaptosome fraction the least. However there was no delay in the labeling of the synaptosome fraction. Initially, labeled synaptosomal RNA was labeled in the synaptosomal supernatant but after 48 hr the bulk of the label was associated with the RNA in the synaptosomal membranes. When the nuclear fraction was further separated into its neuronal and glial components it was found that the incorporation of [ 14C] orotic acid was approximately three times greater in the neuronal fraction after 2–4 hr but difference was apparent before 0.5 hr or after 24 hr. The metabolism of RNA in the subcellular fractions of the visual cortex of blind and sighted rats was found to differ significantly, the former having a slower rate of transfer of RNA from nucleus to cytoplasm. The difference in the incorporation rates of [ 14C] orotic acid into neuronal and glial nuclei was also less in the blind visual cortex. The RNA metabolism in the subcellular fractions of the frontal cortex of blind and sighted rats did not differ.