Characterisation of dopamine receptors in insect (Apis mellifera) brain

In vitro binding experiments using the vertebrate D₁ dopamine receptor ligand ³H]SCH23390 and the vertebrate D₂ dopamine receptor ligand ³H]spiperone were conducted on membrane preparations of honey bee (Apis mellifera) brain. Specific binding of ³H]SCH23390 was saturable and reversible. Analysis of saturation data gave an apparent K_d of 6.3 ± 1.0 nM and B_max of 1.9 ± 0.2 pmol/mg protein for a single class of binding sites. The specificity of high affinity ³H]SCH23390 binding was confirmed in displacement experiments using a range of dopaminergic antagonists and agonists. The rank order of potency for antagonists was: R(+)-SCH23390 > cis-(Z)-flupentixol ≥ chlorpromazine > fluphenazine> S(+)-butaclamol > spiperone. R(±)-SKF38393 and dopamine were the most effective agonists tested. ³H]SCH23390 labels a site in bee brain that is similar, but not identical to the vertebrate D₁ dopamine receptor subtype. ³H]Spiperone also bound with high affinity to bee brain homogenates. Scatchard analysis of ³H]spiperone saturation data revealed a curvilinear plot suggesting binding site heterogeneity. The high affinity site had a apparent K_d of 0.11 ± 0.02 nM and B_max of 9.2 ± 0.5 fmol/mg protein. The calculated values for the low affinity site were a K_d of 19.9 nM and B_max of 862 fmol/mg protein. Kinetic analyses also indicated that ³H]spiperone recognises a heterogeneous population of sites in bee brain. Furthermore, agonist competition studies revealed a phenolaminergic as well as a dopaminergic component to ³H]spiperone binding in bee brain. The rank order of potency of dopaminergic antagonists in competing for ³H]spiperone binding was: spiperone > fluphenazine> S(+)-butaclamol > domperidone> R(+)-SCH23390 > S(−)-sulpiride.