Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Abstract: Opioid receptors have been characterized in Drosophila neural tissue. [³H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (K_D= 8.8 ± 1.7 nM; B_max= 2.3 ± 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [³H]dihydromorphine ([³H]DHM), a μ‐selective ligand: K_D= 150 ± 34 nM; B_max= 3.0 ± 0.6 pmol/mg of protein. Trypsin or heat treatment (100°C for 15 min) of the Drosophila extract reduced specific [³H]DHM binding by >80%. The rank order of potency of drugs at this site was levorphanol > DHM > normorphine > naloxone « dextrorphan; the μ‐specific peptide [d‐Ala²,Gly‐ol⁵]‐enkephalin and δ‐, κ‐, and μgm‐ligands were inactive at this site. The other site was labeled by (‐)‐[³H]ethylketocyclazocine {(‐)‐[³H]EKC}, a κ‐opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (K_D= 212 ± 25 nM; B_max= 1.9 ± 0.2 pmol/mg of protein). (‐)‐[³H]EKC binding could be displaced by κ‐opioids but not by μ‐, δ‐, or μgm‐opioids or by the κ‐peptide dynorphin(1–17). Specific binding constituted ∼70% of total binding at 1 nM and ∼50% at 800 nM for all three radioligands ([³H]etorphine, [³H]EKC, and [³H]DHM). Specific binding of the δ‐ligands [³H][d‐Ala²,d‐Leu⁵]‐enkephalin and [³H][d‐Pen²,d‐Pen⁵]‐enkephalin was undetectable in this preparation. These findings demonstrate the presence of morphine (μ‐like) and EKC (κ‐like), but not δ like, binding sites in Drosophila and indicate that vertebrate opioid peptides are inactive in this preparation. To determine whether opioid sites in Drosophila nervous tissue mediate a functional response, morphine sulfate was administered to Drosophila larvae throughout development. Morphine produced a dose‐dependent delay in pupation of larvae and in eclosion of adults and significantly reduced the ability of larvae to survive to the adult stage. Together, these results indicate that morphine elicits a physiological response in Drosophila larvae and suggest a possible function for opioid receptors in the Drosophila nervous system.