The subcellular localization of tritium after incubation of homogenates of rat brain with 3h-norepinephrine

Abstract

Density gradient centrifugation of rat brain homogenates, after their incubation at 37° with 3H-norepinephrine (NE) revealed an uneven distribution of tritium among the five discrete fractions obtained. The bulk (76 per cent) of the tritium was found in the upper two fractions ( A and B) : the fraction lying between 0.29 and 0.88 M sucrose ( B) contained 46 per cent of the total tritium in the gradient. Pretreatment of the animal with reserpine, or incubation with 3H-NE at 4°, decreased the percentage of tritium in fraction B to approximately 36 per cent. The percentage distribution of tritium was uninfluenced by a fifty-fold increase in the quantity of 3H-NE added to the incubation medium. In contrast, the absolute amount of tritium in B bore a linear relationship to the concentration of added amine. The simultaneous addition of both nonisotopic NE and 3H-NE to the incubation medium decreased the tritium content of fraction B; the decrease was roughly proportional to the amount of nonisotopic NE present. Since over 80 per cent of the tritium in fractions A and B could be accounted for by unchanged norepinephrine, it is suggested that in this study the distribution of tritium in brain homogenates may accurately reflect that of endogenous catecholamine in intact neural structures.