Abstract

Octapamine receptors are widely distributed in invertebrate species, yet little is known about their biochemical structure or tissue localization, in part because there exist no high affinity or irreversible ligands for these receptors. This paper characterizes 2-(2,6-diethyl-4-azidophenylimino)imidazolidine (NC-5Z), a new, high affinity octopamine receptor probe that binds reversibly and, under photolyzing conditions, irreversibly to membrane-associated octopamine receptors. Under reversible conditions NC-5Z is a full agonist, 50-100 times more potent than octopamine in activating the highly enriched and specific octopamine-sensitive adenylate cyclase of the firefly light organ. NC-5Z shows a similar potency in cockroach muscle and thoracic ganglia and in tobacco hornworm nerve cord. Activation of light organ adenylate cyclase by NC-5Z is nonadditive to that caused by octopamine and can be blocked by antagonists, including mainserin (Ki = 0.9 microM), cyproheptadine (Ki = 5 microM), phentolamine (Ki = 20 microM), and propranolol (Ki = 75 microM). These constants agree well with those for the same antagonists in inhibiting stimulation due to octopamine. In physiological studies, NC-5Z mimics the action of, but is more potent than, octopamine in stimulating light emission in intact firefly tails and in disrupting motor behavior and feeding of tobacco hornworms. Under reversible conditions, [3H]NC-5Z, the tritiated derivative of NC-5Z, binds to light organ membranes with an apparent affinity (0.59-0.7 microM) similar to that (0.35-0.7 microM) for NC-5Z in activating adenylate cyclase. Under photolyzing conditions, NC-5Z irreversibly activates light organ adenylate cyclase, and this can be blocked by an excess of octopamine. Under similar conditions, [3H]NC-5Z binds irreversibly to light organ membranes and to membranes from tobacco hornworm nerve ganglia, fat body, and gut. This binding is reduced by prior incubation with octopamine agonists, including octopamine, demethyl-chlordimeform, and 2-(phenylimino)imidazolidines, but not by norepinephrine, dopamine, serotonin, or histamine. Irreversible binding is also reduced by prior incubation with antagonists, most effectively (55% of total binding) by mianserin. The apparent affinity of [3H]NC-5Z for membrane binding, as reflected by its ability to be displaced by mianserin, is altered by GTP. In autoradiographic studies of whole tissue, [3H]NC-5Z shows irreversible, mianserin-displaceable labeling of intact firefly light organs. Taken together, these data indicate that NC-5Z and [3H]NC-5Z are potent and selective agonists of octopamine receptors in a variety of tissues.(ABSTRACT TRUNCATED AT 400 WORDS)