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 Kd of 6.3 +/- 1.0 nM and Bmax 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 > or = 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 Kd of 0.11 +/- 0.02 nM and Bmax of 9.2 +/- 0.5 fmol/mg protein. The calculated values for the low affinity site were a Kd of 19.9 nM and Bmax 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.