Pharmacological characterization of a Bombyx mori α‐adrenergic‐like octopamine receptor stably expressed in a mammalian cell line

Abstract

Series of agonists and antagonists were examined for their actions on a Bombyx morialpha-adrenergic-like octopamine receptor (OAR) stably expressed in HEK-293 cells. The rank order of potency of the agonists was clonidine>naphazoline>tolazoline in Ca(2+) mobilization assays, and that of the antagonists was chlorpromazine>yohimbine. These findings suggest that the B. mori OAR is more closely related to the class-1 OAR in the intact tissue than to the other classes. N'-(4-Chloro-o-tolyl)-N-methylformamidine (DMCDM) and 2-(2,6-diethylphenylimino)imidazolidine (NC-5) elevated the intracellular calcium concentration ([Ca(2+)](i)) with EC(50)s of 92.8 microM and 15.2 nM, respectively. DMCDM and NC-5 led to increases in intracellular cAMP concentration ([cAMP](i)) with EC(50)s of 234 nM and 125 nM, respectively. The difference in DMCDM potencies between the cAMP and Ca(2+) assays might be due to "functional selectivity." The Ca(2+) and cAMP assay results for DMCDM suggest that the elevation of [cAMP](i), but not that of [Ca(2+)](i), might account for the insecticidal effect of formamidine insecticides.

Characterization of a β-adrenergic-like octopamine receptor from the rice stem borer (Chilo suppressalis)

“…The first insect octopamine receptor was isolated from Drosophila (Han et al, 1998), which was assigned to the OctR class. Activation of these receptors primarily leads to an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) and has a minor effect on intracellular cAMP concentration ([cAMP] i ) elevation (Grohmann et al, 2003;Han et al, 1998;Huang et al, 2010). The OctR class is subdivided into three subclasses and they each specifically increase [cAMP] i levels in response to octopamine.…”

A Secreted Placental Alkaline Phosphatase-Based Reporter Assay System for Screening of Compounds Acting at an Octopamine Receptor Stably Expressed in a Mammalian Cell Line

“…6,16) The EC 50 values of OA and DMCDM, calculated as the concentration to reach half the maximal SEAP activity at 10 À5 M and 10 À6 M respectively, were at least one order of magnitude lower than those obtained in our previous cAMP assays ( Table 1), indicating that the SEAP reporter assay is a more sensitive method to examine the potency of agonists. If the reporter SEAP expression is amplified by cAMP cascade reactions, the expression levels should reach maximum earlier than cAMP.…”

“…Thus, the rank order of the two antagonists was yohimbine > chlorpromazine, the same as determined by Ca 2þ mobilization assays. 16) Yohimbine at a high concentration, 10 À5 M, did not completely inhibit the OA-induced increase in luminescence (inhibition, 94.1%). When tested as an agonist at 10 À9 -10 À5 M, yohimbine at 10 À5 M induced a small increase in SEAP activity in HEK-BmOAR1 cells (data not shown).…”

“…5,[12][13][14] The formamidine insecticide chlordimeform (CDM) is N-demethylated in insect bodies, and the metabolite demethylchlordimeform (DMCDM) acts as an OA receptor agonist, showing pesticidal and pestistatic effects. 15) To date, a variety of compounds have been synthesized and assayed to identify more effective and selective OA receptor agonists, 3,16) but previous assay systems include time-consuming and onerous steps: repeated homogenization and centrifugation to prepare membrane fractions from nerve tissues or cell lines and extraction of cAMP produced from OA receptorexpressing cell lines. Hence, the number of assay samples in a given batch experiment has been limited.…”

“…Because SEAP is secreted into the cell culture medium, aliquots of the medium in the wells of 96-well plates are used directly to detect secreted SEAPdependent luminescence. By leaving cells intact, one can screen OA agonists or antagonists more simply and rapidly than we could in our previous efforts, 6,7,16,19,20) and other laboratories could through cAMP assays. [8][9][10][11]21) As an additional advantage, medium supernatants, including secreted SEAP, can be reserved in a freezer until use, because SEAP activity was unchanged by freezing and thawing of the media (data not shown).…”