Differential effects of ascorbate and EDTA on high-affinity binding of 3H-apomorphine and 3H-ADTN to calf caudate membranes.

Recent reports describe effects of ascorbate on binding of dopaminergic agonists to membrane preparations of brain tissue. We now report that with calf caudate membranes, in the absence of EDTA, ascorbate (up to 10 mM) lowered the binding of [3H] (-)apomorphine (3H-APO) by up to 34%, and the proportion "specific," from 69% to 36%, and lowered binding of [3H] (+/-)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (3H-ADTN) by up to 38%, with little effect on the proportion of its specific binding. EDTA (5 mM), alone, also reduced binding of both ligands by 40-50% but had no effect on the proportion specific. With ascorbate plus EDTA, although total binding was lowered, the proportion of specific binding of 3H-APO rose to a maximum of 82% at 2.5 mM ascorbate, whereas that of 3H-ADTN was only slightly increased. Moreover, TLC revealed that the antioxidants were required during incubation to prevent temperature- and time-dependent degradation of APO much more than ADTN. Thus, while each additive, alone, lowered binding of 3H-APO and of 3H-ADTN, ascorbate plus EDTA increased the proportion of specific binding, especially of 3H-APO, and protected both from degradation.