Molecular Pharmacology, Vol 19, 372-378, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Binding of [³H]Amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene to Rat Striatal Membranes

Effects of Purine Nucleotides and Ultraviolet Irradiation

¹ Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262

The binding of the rigid dopamine analogue [³H]amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene ([³H]ADTN) to rat striatal membranes was characterized in the presence and absence of purine nucleotides. At 37°, when assays were carried out in the presence of ATP or GTP, the binding of [³H]ADTN increased for approximately 5 min and then progressively declined. However, when assays were carried out at 20°, [³H]ADTN binding reached equilibrium and was stable in both the absence and presence of purine nucleotides. Scatchard analysis of binding isotherms showed that the affinity of the binding sites for [³H]ADTN decreased by approximately 3-fold in the presence of either 0.3 mM ATP or GTP. An unexpected finding was that the density of binding sites increased by approximately 5-fold. In contrast, the nonhydrolyzable purine nucleotide analogues guanylylimidodiphosphate and adenylylimidodiphosphate did not affect the binding of [³H]ADTN. The pharmacological specificity of [³H]ADTN binding, determined either in the absence or presence of ATP or GTP, was characteristic of binding to dopamine receptors in that dopamine, epinine, and (±)-ADTN were more potent than (-)-norepinephrine or (-)-epinephrine in inhibiting [³H]ADTN binding, and binding was inhibited by a variety of neuroleptics including (+)-butaclamol and ()-flupenthixol. However, there were effects of purine nucleotides on the affinities of the receptor for the partial agonist apomorphine and for a variety of antagonists. In the presence of nucleotides, antagonists were 2 to 10 times less potent in inhibiting [³H]ADTN binding, whereas apomorphine was 300-fold less potent. Exposure of striatal membranes to UV irradiation (_max = 310 nm) for 30 min reduced [³H]ADTN binding observed in the absence of nucleotide by 60% and eliminated the increase in binding observed in the presence of nucleotide. Neither [³H]spiroperidol binding nor dopamine- or ADTN-stimulated adenylate cyclase activity was affected by this treatment. The results suggest that, in the presence of ATP or GTP, [³H]ADTN binds to a second class of binding sites in the striatum. These additional sites (B_max 20 pmoles/mg of protein) are not associated with either [³H]spiroperidol binding or dopamine-stimulated adenylate cyclase activity.

Note:
ACKNOWLEDGMENTS We thank Ms. Candace Collamer for excellent secretarial assistance and Drs. Barry Wolfe, Ken Minneman, Rita Huff, Rick Rabin, and Greg Weiland for their helpful discussions. Dr. James Maller generously donated the Walsh inhibitor.