Inactivation by 6-hydroxydopamine of the cerebral factor(s) responsible for the inhibition of the autoxidation of norepinephrine in vitro

Abstract

The effect of extracts from rat cerebral cortex was examined on the stability of norepinephrine-HCl (NE) in 16 mM Na-phosphate buffer, pH 7.4, at 37°C. The autoxidation products of NE were detected spectrophotometrically at 480 nm. Dialysed samples from a synaptosomal preparation and from the 100,000 g supernatant of a crude homogenate were tested. Aliquots from these preparations, in the range of 0.005–5.0 or 0.01–10.0 μg protein/ml, respectively, produced up to 80–85% inhibition of the autoxidation of 100 μM NE for a period of at least 3 h. Similar results were obtained with albumin and ovalbumin at 10- and 10 3-times higher concentrations, respectively. After the preparations were exposed to 0.1–1.0 mg 6-hydroxydopamine-HCl/mg protein for 5 min at 25°C followed by rapid dialysis, the maximal inhibitory effect was reduced to between 95% to less than 5% of control values. The percent inactivation by a given quantity of 6-hydroxydopamine (6-OHDA) was inversely related to the potency of the untreated sample. Additional observations are presented which suggest that the destruction of the antioxidant activity is caused by breakdown products of 6-OHDA reacting with nucleophilic sites of the preparation. Similar inactivating substances are expected to be formed from other autoxidizing catecholamines, although at a slower rate.