Binding of octopamine to membranes from the white-eyed and wild-type housefly heads

In attempting to develop an octopamine (OA) receptor preparation with ready access to large amounts of tissue, we examined the binding of OA to membranes from the heads of white and red houseflies (Musca domestica L.). Binding was dependent on the presence of L-ascorbic acid in the medium. However, equilibrium was reached only over 24–36 h at 4°C and reversal of binding was also slow and incomplete. Scatchard analysis revealed at least two binding sites in the white-eyed housefly. A high-affinity site (K_d = 13.9 nM and B_max = 3.9 pmol/mg protein) was present, but the majority of the binding had low affinity (K_d = 1130 nM and B_max = 165 pmol/mg protein). Scatchard analysis revealed a low affinity in the red-eyed housefly (K_d = 240 nM and B_max = 12 pmol/mg protein). Catecholamines were the best competitors for OA binding followed by phenolamines such as OA and synephrine. 5-Hydroxytryptamine was less effective. Phentolamine and mianserin, which are good antagonists of the ability of OA to stimulate adenylate cyclase in housefly head membranes, and formamidine and imidazolines, which are potent partial agonists of this adenylate cyclase, were poor competitors of OA binding. The slow kinetics, low affinity, large amount, and unconventional pharmacological profile of this binding is not congruent with it being a neuroreceptor. When the brain was dissected free from the head, less than 10% of the total specific binding of OA was found in the brain membrane fraction. This suggests that most of the binding of OA may be to cuticular sites that possibly are associated with the metabolism of catecholamines in cuticular synthesis. Thus, binding studies made with labeled catecholamines and phenolamines on insect tissues containing significant cuticular elements should be interpreted with caution.