Characterization of Octopamine-Sensitive
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Proc. NatI. Acad. Sci. USA Vol. 82, pp. 599-603, January 1985 Neurobiology Characterization of octopamine-sensitive adenylate cyclase: Elucidation of a class of potent and selective octopamine-2 receptor agonists with toxic effects in insects (phenyliminoimidazolidines/cyclic AMP/pesticides/firefly/tobacco hornworm) JAMES A. NATHANSON Department of Neurology, Harvard Medical School, and Neuropharmacology Research Laboratory, Massachusetts General Hospital, Boston, MA 02114 Communicated by Vincent G. Dethier, September 13, 1984 ABSTRACT Octopamine-2 receptors, associated with ac- histamine-activated adenylate cyclase. For these reasons, tivation of adenylate cyclase, mediate a number of the impor- the firefly light organ appears to be an excellent tissue with tant hormonal and neurotransmitter functions of octopamine which to characterize octopamine-2 receptors. in invertebrates. By utilizing the highly enriched octopamine- Using this tissue in an attempt to develop better sensitive adenylate cyclase present in the firefly light organ, it octopamine receptor agents, I have found that certain sub- has been possible to pharmacologically characterize octopa- stituted 2-(phenylimino)imidazolidines are extremely potent mine-2 receptors and to define a new class of highly potent and selective agonists of octopamine-activated adenylate and selective octopamine-2 agonists. At low concentrations, cyclase, having greater activity than previously described these substituted phenyliminoimidazolidines stimulate light compounds. I also report that these agents exert potent emission when injected into fireflies. At somewhat higher physiological and toxicological effects in insects, including concentrations, these compounds, when ingested by tobacco stimulation of light emission in the firefly and disruption of hornworms, cause disruption of motor and feeding behavior, motor activity (including feeding) in the tobacco hornworm. leading to insect death. The effects of these compounds are markedly potentiated by phosphodiesterase inhibitors and MATERIALS AND METHODS mimicked by other activators of octopamine-sensitive adenyl- Specimens of Photinus pyralis were collected in summer, ate cyclase, including octopamine itself. Because octopamine-2 frozen on dry ice, and stored in liquid N2. Under these receptors appear to be present primarily in invertebrates, conditions, octopamine-sensitive enzyme activity remains these findings, together with other data, raise the possibility stable for 6 months or longer (13). After thawing at 40C, tail that potent and selective octopamine agonists could be useful sections were opened, and the light organs were removed as insect toxins with low toxicity in vertebrates. from the ventral cuticle, cleaned of any adhering nonlantern tissue, and homogenized (10 mg/ml) in 6 mM Tris maleate, Biochemical, physiological, and anatomical studies have pH 7.4. A washed particulate (P2) fraction was prepared as established that octopamine [1-(p-hydroxyphenyl)-2-amino- described (13) and kept at 0°C until used. In some experi- ethanol] is an important neurotransmitter and neurohormone ments, adenylate cyclase was measured in P2 fractions in invertebrates (reviewed in refs. 1-4). Many of the physi- prepared from the ventral nerve cords (including head and ological functions of octopamine appear to be mediated by a tail ganglia) of adult cockroaches (Periplaneta americana) class of octopamine receptors ["octopamine-2 receptors" and of 50-mm-long tobacco hornworms (Manduca sexta). In (2)] specifically coupled to the activation of adenylate cy- other experiments, P2 fractions were prepared from caudate clase. Since octopamine-activated adenylate cyclase was nucleus (10 mg/ml), liver (50 mg/ml), and heart (50 mg/ml) described over a decade ago (5), there have been a number of of 3-month-old Sprague-Dawley rats. efforts to characterize octopamine-2 receptors by studying Adenylate cyclase activity of all tissues was measured in the production of cyclic AMP in invertebrate nerve and test tubes containing (in 0.3 ml) 80 mM Tris maleate, pH 7.4; muscle tissue (6-8). However, because several other hor- 10 mM theophylline; 8 mM MgCl2; 0.1 mM GTP; 0.5 mM mone receptors (e.g., those for dopamine, serotonin, and EGTA; 2 mM ATP; 0.06 ml of P2 fraction; and various proctolin) are also positively coupled to cyclic AMP produc- compounds to be tested. Prior experiments had shown that, tion in these tissues (3, 4), it has been very difficult (due to under these conditions, adenylate cyclase activity was opti- the confounding effects of these other receptors) to define mized (13).The enzyme reaction (4 min at 30°C) was initiated the pharmacological characteristics of octopamine-2 recep- by addition of ATP and stopped by heating at 90°C for 2 mirn. tors. The reaction mixture was then centrifuged at 1000 x g for 15 Recently, studies from this and other laboratories have min to remove insoluble material. Cyclic AMP in the super- suggested that octopamine is the neurotransmitter involved natant was measured by protein binding assay (14). Under in neural control of light emission in the firefly light organ, a these assay conditions, enzyme activity was linear with tissue which, in certain respects, is analogous to the respect to time and enzyme concentration, and phosphodi- cholinergically innervated electroplax organ of electric esterase activity was nearly completely inhibited. Protein fishes (9-12). The firefly light organ is highly enriched in concentration was determined by the Lowry method. Acti- octopamine-activated but not other amine-activated adenyl- vation constants (Kact) and inhibitory constants (Ki) were ate cyclases. Thus, octopamine can cause a >50-fold stimu- calculated as described (13). lation of adenylate cyclase in this preparation, which, unlike To measure the effects of drugs on firefly light emission, invertebrate nerve cord or brain, contains no significant an isolated tail (terminal three abdominal segments, contain- amount of dopamine-, norepinephrine-, serotonin-, or Abbreviations: DDCDM, didemethylchlordimeform; NC5, NC7, The publication costs of this article were defrayed in part by page charge and NC9, the 2,6-diethyl, 2-methyl-4-chloro, and 2,4-dimethyl de- payment. This article must therefore be hereby marked "advertisement" rivatives, respectively, of 2-(phenylimino)imidazolidine; i-BuMe- in accordance with 18 U.S.C. §1734 solely to indicate this fact. Xan, 3-isobutyl-l-methylxanthine. 599 Downloaded by guest on September 25, 2021 600 Neurobiology: Nathanson Proc. Natl. Acad. Sci. USA 82 (1985) ing the light organ) of a fresh adult Photinus pyralis male was plied by CIBA-Geigy, and cyproheptadine [4-(5H-dibenzo- mounted on a 30-gauge stainless-steel needle and placed at [a,d]cyclohepten-5-ylidene)-1-methylpiperidine], by Merck. the focal point of an optical system connected to a photometer/photomultiplier/chart recorder combination. RESULTS AND DISCUSSION Drug [dissolved in insect saline (15)] was injected (in 3 1ul) To characterize octopamine receptors regulating cyclic AMP into the abdominal cavity dorsal to the lantern and light production, I first evaluated the hormone sensitivity of emission was recorded for 45 min, after which the next adenylate cyclase in P2 fractions of Photinus light organs. (larger) dose of drug was injected. In the case of animals Fig. LA shows that the Kact of octopamine for stimulating injected with drugs other than octopamine, 10 nmol of enzyme activity in the light organ was -20 pM. In the same octopamine (a maximally effective dose) was injected after tissue, dopamine and the f3-adrenergic agonist isoproterenol the last dose. caused only a small degree of enzyme stimulation even at To measure the effects of drugs on motor and feeding high concentrations. The a-adrenergic agonist phenyleph- behavior of tobacco hornworms, drugs (dissolved in water) rine (data not shown) was also of low potency (Kact > 200 were applied as an aerosol to isolated, hydrated tomato ILM). leaves and allowed to dry. A group of six 3-day-old Manduca Table 1 lists the kinetic constants of receptor-mediated sexta larvae (reared on artificial medium) were then placed enzyme activation for several well-known phenylethyla- on each leaf; the amount of leaf remaining 72 hr later was mines and phenylethylamine metabolites. With the excep- measured. The "antifeeding activity" observed was the net tion of synephrine (N-methyloctopamine), which is slightly result of motor-behavioral disruption and not necessarily more potent than octopamine, no other phenylethylamine due to a specific effect on feeding (16) (see below). In other analog (among these and several dozen others that were biochemical experiments, isolated Manduca nerve cords examined) had more activity than octopamine. In all cases, treated with phenyliminoimidazolidines or octopamine those analogs that had partial activity satisfied criteria (data showed increases in cyclic AMP content that were enhanced not shown) that strongly suggested that their effects on in the presence of the phosphodiesterase inhibitor cyclic AMP production were due to their activation, at high 3-isobutyl-1-methylxanthine (i-BuMeXan). Thus, in these concentrations, of the octopamine-specific adenylate cy- behavioral experiments, i-BuMeXan (0.1 mg per leaf) was clase. Thus, at optimally effective concentrations, none of included in both control and experimental leaves. (At the the compounds showed activity that was additive to the dose used, i-BuMeXan itself had little or no effect