Characterization of opioid-binding sites in zebrafish brain

Abstract

The pharmacological profile of opioid-binding sites in zebrafish brain homogenates has been studied using radiolabeled binding techniques. The nonselective antagonist [(3)H]diprenorphine binds with high affinity (K(D) = 0.27 +/- 0.08 nM and a B(max) = 212 +/- 14.3 fmol/mg protein), displaying two different binding sites with affinities of K(D1) = 0.08 +/- 0.02 nM and K(D2) = 17.8 +/- 9.18 nM. The nonselective agonist [(3)H]bremazocine also binds with high affinity to zebrafish brain membranes but only displays one single binding site with a K(D) = 1.1 +/- 0.09 nM and a B(max) = 705 +/- 19.3 fmol/mg protein. Competition binding assays using [(3)H]diprenorphine and several unlabeled ligands were performed. The synthetic selective agonists for mammalian opioid receptors DPDPE ([DPen(2),D-Pen(5)]-enkephalin), DAMGO ([D-Ala(2),NMe-Phe(4),Gly(5)-ol]-enkephalin), and U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] failed to effectively displace [(3)H]diprenorphine binding, whereas nonselective ligands and the endogenous opioid peptides such as dynorphin A showed good affinities in the nanomolar range, although several of the endogenous peptides only displaced approximately 50% of the specifically bound [(3)H]diprenorphine. Our results provide evidence that, although the selective synthetic compounds for mammalian receptors do not fully recognize the opioid-binding sites in zebrafish brain, the activity of the endogenous zebrafish opioid system might not significantly differ from that displayed by the mammalian opioid system. Hence, the study of zebrafish opioid activity may contribute to an understanding of endogenous opioid systems in higher vertebrates.