Molecular and pharmacological analysis of an octopamine receptor from American cockroach and fruit fly in response to plant essential oils.

Octopamine receptors from American cockroach, Periplaneta americana (Pa oa1), and fruit fly, Drosophila melanogaster (OAMB), were cloned and permanently expressed in HEK-293 cells, and found to activate adenylate cyclase activity and increase [Ca2+]i levels through G-protein coupled receptor signaling pathways. Sequencing information (GenBank accession number AY333178) and functional data of Pa oa1 were recently published. Saturation binding analysis with 3H-yohimbine was performed with Pa oa(1) and OAMB expressed in COS-7 cells. The K(d) values were determined to be 28.4 and 43.0 nM, respectively. B(max) was determined to be 11.8 and 8.04 pmol receptor/mg protein, respectively. Competitive binding data using cell membranes expressing either OAMB or Pa oa1 demonstrated significantly decreased binding activity in binding assays performed in the presence of plant essential oils, eugenol, cinnamic alcohol, and trans-anethole. Eugenol decreased cAMP level in HEK-293 cells expressing Pa oa1, but trans-anethole increased cAMP in HEK-293 cells expressing OAMB. All three chemicals increased [Ca2+]i level in both cell models. Toxicity data against fruit flies and American cockroaches demonstrated species differences in response to treatment with tested plant essential oils. The toxicity of tested chemicals against wild type and octopamine mutant (iav) fly strains suggested that an octopamine receptor mediates the toxicity of cinnamic alcohol, eugenol, trans-antehole, and 2-phenethyl propionate against fruit flies. Collectively, the data suggest a correlation between cellular changes induced by tested plant essential oils and their toxicity against fruit fly and American cockroach.

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana.

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.