Ascorbic acid and membrane ageing: critical determinants of the in-vitro binding of [3]ADTN to rat striatal tissue

Abstract

Abstract Tritiated(±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([3H]ADTN), binding (0˙125-4˙0 nM) to rat striatal membranes was investigated using Tris-HCl buffer containing Na2EDTA, nialamide and varying concentrations of ascorbic acid. In the absence of ascorbic acid [3H]ADTN exhibited a high affinity (KD 1˙26 nm) saturable binding (Bmax 138 fmol mg−1 protein) (Scatchard analysis). This was not modified by 10−6 or 10−5m ascorbic acid used immediately or 60 min after preparation. However, 10−4m ascorbic acid added (within) 15 min after its preparation reduced the number of bindings sites and added 60 min after it also reduced affinity. Ascorbic acid 5.7 mm reduced affinity whether used within 15 min or 60 min of its preparation. When ascorbic acid 10−4m was used within 15 min of preparation, and membranes were used immediately, the binding of 2 nm [3H]ADTN was specifically displaceable by nm or sub-nm concentrations of dopamine agonists and antagonists. However, when membranes were used 1–2 h after their preparation there was an increasing loss of the high affinity binding sites displaceable by sub-nm concentrations of (±)-ADTN. Thus, [3H]ADTN can be shown to exhibit high affinity stereoselective binding to rat striatal membranes when these are freshly prepared and the assay is performed using Tris-HCl buffer containing nialamide, Na2EDTA and 10−4m ascorbic acid prepared within 15 min of use. The characteristics of this binding can be markedly modified if the concentration of ascorbic acid is increased, if its preparation time is extended, or if the membranes are allowed to ‘age’.