Abstract

The possibility of differentiating between chromaffin vesicles with different catecholamine contents was tested by studying the distribution of rabbit adrenal dopamine β-hydroxylase (EC 1.14.21) and catecholamines, and the buoyant densities of the catecholamine storage vesicles after isopycnic centrifugation of crude storage vesicle fractions in sucrose density gradients. Catecholamine storage vesicles were prepared from adrenal glands of untreated rabbits, rabbits which had received chlorisondamine chloride (10/kg intraperitoneally) to block ganglionic transmission, and rabbits which had received both chlorisondamine chloride and reserpine (1 mg kg). Adrenal glands were examined 1 day after treatmenmt with chlorisondamine and 1 and 8 days after combined treatment with chlorisondamine and reserpine. Intact storage vesicles obtained from glands of untreated animals had a specific gravity of 1.27, while the membranes obtained from vesicles lysed in distilled water had a specific gravity of 1.12. Chlorisondamine had no effect on the total dopamine β-hydroxylase and catecholamine content of the adrenal glands or on the buoyant density, even though there was a slight reduction of the activities in segments A and B of the gradients. Twenty-four hours after treatment with chlorisondamine plus reserpine there was a marked decrease in the catecholamine content, no change in the dopamine β-hydroxylase activity, and a significant decrease in the buoyant density of the storage vesicles; the ratio of dopamine β-hydroxylase to catecholamines in the purified storage vesicles was 2-3 times greater than those of untreated and chlorisondamine-treated animals. Eight days after treatment with reserpine and chlorisondamine the dopamine β-hydroxylase activities were twice those of untreated animals, but the catecholamine content was only 60-70% of the control levels. At eight days the buoyant density of the storage vesicles was still significantly less than that of vesicles obtained from untreated rabbits, but not different from that of chlorisondamine-treated animals. These results suggest that recovery of the catecholamine content following reserpine administration requires synthesis of new storage vesicles, but this may not be the srate-limiting step in the recovery. Isopycnic centrifugation through sucrose density gradient provides a method for differentiating normally filled, partially filled, and empty storage vesicles.