Pharmacological Characterization of the Locust Air Sac Octopamine Receptor

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO.

55, 218–225 (1996)

0051

Pharmacological Characterization of the Locust Air Sac Octopamine Receptor H. ZENG, K. R. JENNINGS,

AND

B. G. LOUGHTON1

Biology Department, York University, North York, Ontario M3J 1P3 Canada Received November 16, 1995; accepted August 15, 1996 The pharmacology of the locust air sac octopamine receptor was investigated by measuring changes in the concentrations of cyclic 3*,5*-adenosine monophosphate (cAMP) after isolated abdominal air sacs were treated with octopamine and related agonists and antagonists. Among the agonists tested only AC6, clonidine, and compound 1 caused significant increases in cAMP. AC-6 was a full agonist whereas clonidine increased cAMP to 10% that of the octopamine-stimulated level. Compound 1 increased cAMP levels to only 4% of the octopamine-stimulated level. The data from the present study lend weight to the proposal that AC-6’s insect toxicity is a result of its activity at the octopamine receptor. All the antagonists tested except yohimbine proved capable of inhibiting octopamine-induced increases in air sac cAMP concentration. The rank order of inhibition efficiency was Mianserin, phentolamine, metoclopromide, and chlorpromazine. This rank order suggested that the air sac receptor is a type 3 receptor, like the locust neural receptor. However, the high Ki value of metoclopromide for the air sac differed from that for the neural receptor. A novel effect was observed with chlorpromazine, which enhanced octopamine-induced increases in air sac cAMP at low concentrations but had no effect on its own. At higher concentrations chlorpromazine acted as a competitive inhibitor. The sum total of agonist and antagonist effects on the air sac octopamine receptor suggests that it is similar to but not identical to the type 3 neural octopamine receptor described by T. Roeder (Life Sci. 50, 21–28, 1992). q 1996 Academic Press

INTRODUCTION

Since the pioneering study of octopaminesensitive adenylate cyclase in cockroach nerve cords (2), many octopamine-sensitive adenylate cyclases have been investigated in various tissues in a wide variety of insects (3 – 11), and the enzyme has been considered an indicator for some classes of the octopamine receptor. The relationship of the octopamine receptor to adenylate cyclase activity has been examined (12, 13) and pharmacological studies of the octopamine receptor have been conducted by measuring adenylate cyclase activity in the presence of octopamine agonists and antagonists (4, 14 – 18) although the initial determination of octopamine receptor classification in locust extensor tibiae muscle was based on the oc1 To whom correspondence should be addressed. Fax: (416) 736-5690.

topamine-mediated electrophysiological response (19). This classification was later refined using receptor – ligand binding (1). Octopamine receptors have been shown to be analogous to the a-adrenergic receptor in vertebrates because they are sensitive to aadrenergic agonists and antagonists. However, the lack of specific octopamine agonists has hindered the progress of studies on octopamine receptor pharmacology (20). Therefore, efforts have been made to synthesize new octopamine agonists and their specificity has been tested in several tissues from different species (21–27). These studies not only provide the basis for exploring octopamine receptor pharmacology, but also provide the means to develop new selective pesticides because the octopamine target site is not present in vertebrates (28). A previous study in this laboratory demonstrated that there is an adenylate cyclase present in locust air sac tissue which is highly

218 0048-3575/96 $18.00 Copyright q 1996 by Academic Press All rights of reproduction in any form reserved.

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sensitive to octopamine (29). The present study focused on the pharmacology of the air sac octopamine receptor in an attempt to classify the receptor type and to provide data for the assessment of potential pesticides. MATERIALS AND METHODS

Locusta migratoria of phase gregaria were reared at a day temperature of 367C, which fell at night to 237C (30). Air sacs were obtained from Day 6 to Day 12 male adult locusts which had been isolated at fledging and starved on the day of use. All dissections were undertaken in ice-cold locust saline (140 mM NaCl; 1.36 mM CaCl2r2H2O; 1.97 mM MgCl2 ; 5.88 mM KH2PO4 ; 4.0 mM KH2CO3). The fat body was carefully dissected away by fine forceps (No. 55, A. Dumont & Fils). The dissection of air sacs took about 15 min for each animal. All incubations were carried out at room temperature (207C) for 20 min. Incubations were terminated by transferring the tissues to 1.5-ml polypropylene test tubes containing 200 ml of ice-cold 3% perchloric acid. Tissues were homogenized (20 strokes per sample) on ice with 1-ml conical glass pestle tissue grinders (Canadawide Scientific). After neutralization with chilled 2.5 M potassium bicarbonate (pH 5.5–6), the homogenate was centrifuged at 12,000g for 12 min. The supernatant was analyzed for cAMP, using a cAMP assay kit (Diagnostic Products Corp., LA) and the pellet, dissolved in 200 ml of 0.5 M NaOH, was used for protein determination by using a Bio-Rad assay kit with bovine serum albumin as standard. The chemicals used in the experiments were as follows: 3 - isobutyl-1-methyl-xanthine (IBMX),2 DL-octopamine, mianserin, chlorpromazine (all from Sigma Chemical Co., St. Louis, MO), clonidine, UK-14,304, p-aminoclonidine, phentolamine, metoclopramide, yo2 Abbreviations used: AC-6, 2-(4-chloro-o-toluidino)-2-oxazoline; OA, octopamine; IBMX, 3-isobutyl-l-methyl-xanthine; BU224, 2-(4,5-dihydroimidaz-2-yl)quinoline hydrochloride; BU239, 2-(4,5-dihydroimidaz-2-yl)-quinoxaline hydrochloride; compounds 1 and 2, dihydrooxadiazines.

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himbine (RBI Co.), 2-(4,5-dihydroimidaz-2yl)quinoline hydrochloride (BU224), 2-(4,5dihydroimidaz-2-yl)quinoxaline hydrochloride (BU239) (Tocris Cookson Ltd., UK), 2-(4-chloro-O-toluidino)-2-oxazoline (AC-6) (American Cyanamid Co., Princeton, NJ), and two dihydrooxadiazines (compounds 1 and 2) (gifts from Uniroyal Chemical Research Labs, Guelph, Ontario, Canada). All the chemicals were dissolved in ethanol and diluted in saline immediately prior to use. The final concentration of ethanol in control and experimental salines was 6% or less and the concentration of IBMX used in all experiments was 5 1 1004 M. Least-squares nonlinear regression was used to determine the best fit line to the experimental data. Curves were fitted to cAMP elevations using the logistic equation for activation of a single enzyme. A computer program (Sigma Plot) provided parameter estimates and the standard error of the mean, including an indication of goodness of fit and the EC50 . RESULTS DL-Octopamine, hereafter referred to as octopamine, elevated cAMP concentrations in abdominal air sacs in a dose-dependent manner (Fig. 1). At a concentration of 1 1 1004 M octopamine, the concentration of cAMP in air sacs was 40-fold over the control level. Tests were conducted on the effects of three conventional a-adrenergic agonists, two imidazoline agonists (BU244 and BU239), two dihydrooxadiazines (compounds 1 and 2), and an aminooxazoline (AC-6). The results are presented in Table 1. Among the three conventional a-adrenergic agonists, only clonidine had any effect on cAMP elevation (P õ 0.05) with 10.87% of the potency of octopamine. The other two, p-aminoclonidine and UK-14,304, did not have significant agonist activity on the air sac octopamine receptor. The two imidazoline agonists, BU224 and BU239, did not show any significant effects either. Compound 1 had a very limited effect with only 3.75% of the potency shown by octopamine (P õ 0.05), while compound 2

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sented in Table 3. At low concentrations (1 1 1006 to 1 1 1008 M), chlorpromazine potentiated the effect of octopamine on cAMP production by more than threefold over control (Fig. 3, expressed as negative inhibition). Figure 3 also shows that chlorpromazine at higher concentrations (1 1 1004 M) inhibited cAMP production. Yohimbine was the only a-adrenergic blocker tested that had no effect on octopamine-sensitive adenylate cyclase activity (Table 3). DISCUSSION

FIG. 1. Dose–response curve for the action of octopamine and AC-6 on cAMP increase in locust air sacs. The maximal increase in octopamine was 4076 pmol/mg protein above the basal level (basal level was 100 pmol/ mg protein). Each value is mean { SEM for 10–12 determinations. The two values (OA and AC-6) at 1 1 1004 M are not different (P ú 0.05,ttest).

had no effect on cAMP production. AC-6 was the only agonist examined in the experiments that was a potent adenylate cyclase activator, with a maximal stimulation of 70% (p õ 0.01) that of octopamine. Like octopamine, AC-6 also elevated cAMP levels in intact air sac tissue in a dose-dependent manner with a threshold of 1 1 1007 M and it reached the maximal stimulation at 10 mM with an EC50 of 1.48 mM (Fig. 1). To establish that AC-6 acts on air sac octopamine receptors, rather than other aminergic receptors present on air sacs, octopamine antagonists were used. Table 2 shows that the octopamine antagonists effectively block the AC-6 effect of the cAMP production in the same order of efficacy as for octopamine. Putative antagonist compounds tested were efficient octopamine antagonists inhibiting cAMP production by 88 to 98% at 1 1 1004 M (Table 3 and Fig. 2). The rank order of antagonist potency, measured by their ability to inhibit production of cAMP in air sacs, was mianserin ú phentolamine ú metoclopramide ú chlorpromazine. The IC50’s and the estimated pKi values of the antagonists are pre-

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The effects of the various agonists and antagonists on the air sac octopamine receptor allow us to distinguish the air sac receptor from the receptors characterized in other tissues. An analysis of the ability of potential octopamine agonists to stimulate cAMP production by air sac cells showed that only three of the compounds caused a significant increase in intracellular cAMP. AC-6 proved to be a potent agonist. None of the other potential agonists raised cAMP levels by more than 10% that of octopamine. cAMP in air sac cells is raised 40 times over baseline levels after octopamine treatment (1 1 1005 M). Clonidine causes a quadrupling of baseline levels (P õ 0.05), whereas p-aminoclonidine more than doubled control concentrations (but not significantly different from controls). Uniroyal compounds 1 and 2 also doubled the concentration of cAMP over baseline levels. Only the increases caused by compound 1 were statistically significant (see Table 1). The other compounds tested as agonists increased cAMP but none of the changes were significantly different from controls. The efficacy of the aminooxazoline AC-6 in stimulating the air sac octopamine receptor confirmed the observations of Lange and Orchard (31) on locust fat body. Since we now believe that the octopamine-sensitive adenylate cyclase is present on the air sac cells and not the fat body itself (29), this result is not surprising. AC-6 was a less effective agonist on locust oviduct (25) and cockroach nerve cord (24). Clonidine exhibited only weak agonist activity on the air

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AIR SAC OCTOPAMINE RECEPTOR TABLE 1 cAMP Increase by Octopamine Agonists

a

Expressed as the percentage of cAMP maximal increase by octopamine (maximal increase is 4076.48 pmol cAMP/mg protein at 1 1 1004 M octopamine). b Results are means { SEM for four to six determinations. Compounds were tested at 1 1 1005 M. ** Significantly different from control at P õ 0.01 (t test). * Significantly different from control at P õ 0.05 (t test).

sac adenylate cyclase. Octopamine receptors on different locust tissues show a wide range of sensitivity to clonidine (17, 18, 32). The potency of AC-6 and clonidine for the air sac

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octopamine receptor differs substantially for that of the oviduct and the glandular lobe of the corpus cardiacum. In addition it would appear that the air sac receptor also differs in

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ZENG, JENNINGS, AND LOUGHTON TABLE 2 Antagonist Effect on cAMP Increase by AC-6 Compounds

Maximal inhibition

Mianserin Phentolamine Metoclopramide

105.75 { 0.62a 96.97 { 2.58 96.08 { 2.18

a Expressed as percentage of inhibition at 1 1 1004 M AC-6. Values are means { SEM for three determinations.

some degree from that of nervous tissue in that it is more sensitive to AC-6 and less sensitive to clonidine. The first classification of insect octopamine receptors was based on the physiological response of the locust extensor tibia muscle to a number of agonists and antagonists (19). This analysis allowed the distinction of three receptor subtypes, octopamine 1 and octopamine 2a and 2b receptors. Later, using adenylate cyclase activity to assay for octopamine and octopamine analogue activity, it was observed that most octopamine receptors belonged to the type 2 class (15–17, 19, 21, 33). More recently, Roeder (1) measured octopamine binding to locust brain membranes in the presence of octopamine antagonists and proposed a further receptor subtype, termed octopamine 3. This receptor was of neural origin. The rank order of antagonist affinity for

the type 3 receptor octopamine binding site was mianserin ú phentolamine ú chlorpromazine ú metoclopramide. This pattern of affinity closely resembles the rank order of antagonist inhibition of air sac cAMP production by octopamine except that the estimated pKi value of chlorpromazine is slightly lower than that of metoclopramide in air sac receptors (Table 3). Thus it would appear that the air sac octopamine receptor most closely resembles the type 3 receptor defined by Roeder (1) and suggests that it has the characteristics of a neural octopamine receptor. It is quite clearly different from the type 1 and type 2 receptors defined by Evans (19) and Roeder (1). The finding that chlorpromazine at low concentration potentiated the stimulation of cAMP production by octopamine while inhibiting octopamine activity at high concentrations (Fig. 3) in part confirms the observation of Orchard and Lange (17) who found that chlorpromazine in the presence of octopamine potentiated the production of cAMP in locust oviduct. The oviduct receptor appears to be a type 2b receptor following Roeder’s classification. This difference in receptor type may explain why Orchard and Lange (17) do not mention any inhibiting effect of chlorpromazine on their preparation. It is important to note that chlorpromazine alone did not stimulate cAMP production by air sac cells at any concentration

TABLE 3 Octopamine Antagonist Rank Order

IC50

Estimated pKi b

38.0 nM 1.17 mM 8.29 mM 10.5 mM —

9.18 7.69 6.84 6.73 —

Maximal inhibitiona

Compounds Mianserin Phentolamine Metoclopramide Chlorpromazine Yohimbine

98.98 91.89 88.06 92.54

{ 0.57c* { 1.39* { 5.64* { 2.16* ned

Expressed as percentage of inhibition at 1 1 1004 M octopamine. Estimated by Cheng–Prusoff equation (35). c Results are means { SEM for six determinations. d No inhibition effect. *Significantly different from control at P õ 0.01 (t test). a b

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FIG. 2. Dose–response curve for the action of octopamine antagonists on octopamine-stimulated cAMP increase in air sacs. Each value is the mean { SEM for six determinations. The inhibition was calculated as the relative difference between the octopamine-stimulated level of cAMP and the level determined when octopamine was incubated in the presence of inhibitor.

tested (data not shown). This suggests that chlorpromazine does not recognize and activate the octopamine receptor binding sites at low concentrations. This interpretation is consistent with the ligand binding data of Roeder (1), who showed that chlorpromazine could compete with octopamine for the octopamine binding site only at high concentrations. Thus it would appear that at low concentrations chlorpromazine alters the octopamine receptor system to make it more sensitive to octopamine. We believe that it may act as an allosteric enhancer of the octopamine receptor. While agonist and antagonist studies are valuable in classifying the octopamine receptor, agonist data also provide useful information for pesticide assessment. Since octopamine receptors have not been found in vertebrate tissues, potent octopamine agonists could serve as selective pesticides (34). AC6, one of the 2-aminooxazolines, is a biorationally synthesized, potent octopamine agonist and has been examined for both the octopamine sensitive adenylate cyclase activity and octopamine receptor binding (24, 25, 32). AC-6 also showed direct toxic effects on in-

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sects and acarids with properties similar to those of the formamidines (24). The data from the present study lend weight to the proposal that AC-6 acts as a selective pesticide through the octopamine receptor target site. A second group of octopamine agonists, the dihydrooxadiazines (26), possessed only weak agonist activity on the locust air sac receptor. This pharmacological study of octopamine agonists and antagonists has allowed the characterization of the octopamine receptor in locust air sacs. In addition, the agonist data provided information for pesticide assessment. It has been shown that firefly light organ has a highly enriched octopamine-activated adenylate cyclase with a 50-fold stimulatory effect over the control level (21). It is the highest level of stimulation seen in an octopaminergic system examined to date. The locust air sac system also showed a similarly high adenylate cyclase activity in response to octopamine. Therefore, the locust air sac serves as another good system for octopamine receptor pharmacology studies.

FIG. 3. Dose–response curve for the action of chlorpromazine on octopamine-stimulated cAMP increase in air sacs, showing the inhibition and the potentiation effect of the antagonist at different concentrations. Each value is the mean { SEM for five to six determinations. The inhibition was calculated as the relative difference between the octopamine-stimulated level of cAMP and the level determined when octopamine was incubated in the presence of inhibitor.

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ACKNOWLEDGMENTS This work was supported by Cyanamid Canada Inc. and by an operating grant from the Natural Sciences and Engineering Research Council of Canada to B.G.L.

REFERENCES 1. T. Roeder, A new octopamine receptor class in locust nervous tissue, the octopamine 3 (OA3) receptor, Life Sci. 50, 21–28 (1992). 2. J. A. Nathanson and P. Greengard, Octopamine-sensitive adenylate cyclase: Evidence for a biologic role of octopamine in nervous tissue, Science 180, 308– 310 (1973). 3. A. J. Harmar and A. S. Horn, Octopamine-sensitive adenylate cyclase in cockroach brain: Effects of agonists, antagonists and guanylyl nucleotides, Mol. Pharmacol. 13, 512–520 (1977). 4. P. D. Evans, A modulatory octopaminergic neuron increases cyclic nucleotide levels in locust skeletal muscle, J. Physiol. 348, 307–324 (1984). 5. G. L. Orr, J. W. D. Gole, and R. G. H. Downer, Characterization of an octopamine-sensitive adenylate cyclase in haemocyte membrane fragments of the American cockroach Periplaneta americana L, Insect Biochem. 15, 695–701 (1985). 6. J. A. Nathanson, Octopamine receptors, adenosine 3*,5*-monophosphate, and neural control of firefly flashing, Science 203, 65–68 (1979). 7. J. A. Nathanson and E. J. Hunnicutt, Neural control of light emission in Photuris larvae: Identification of octopamine-sensitive adenylate cyclase, J. Exp. Zool. 208, 255–262 (1979). 8. A. B. Lange and I. Orchard, Identified octopaminergic neurons modulate contractions of locust visceral muscle via adenosine 3*,5*-monophosphate (cAMP), Brain Res. 363, 340–349 (1986). 9. R. A. A. Worm, Involvement of cyclic nucleotides in locust flight muscle metabolism, Comp. Biochem. Physiol. 67C, 23 (1980). 10. C. Suter, Does cyclic AMP mediate the action of octopamine on insect neurons? Comp. Biochem. Physiol. 84C, 189–193 (1986). 11. I. Orchard, B. G. Loughton, J. W. D. Gole, and R. G. H. Downer, Synaptic transmission elevates adenosine 3*,5*-monophosphate (cyclic AMP) in locust neurosecretory cells, Brain Res. 258, 152–155 (1983). 12. Y. Dudai and S. Zvi, High-affinity [3H] octopamine binding sites in Drosophila melanogaster: Interaction with ligands and relationship to octopamine receptors, Comp. Biochem. Physiol. 77C, 145–151 (1984). 13. N. Minhas, J. W. D. Gole, G. L. Orr, and R. G. H. Downer, Pharmacology of [3H] mianserin binding in the nerve cord of the American cockroach, Periplaneta americana, Arch. Insect Biochem. Physiol. 6, 191–201 (1987).

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PEST 2226

/

al03$$$$63

14. J. W. D. Gole, G. L. Orr, and R. G. H. Downer, Octopamine-mediated elevation of cyclic AMP in haemocytes of the American cockroach, Periplaneta americana L., Can. J. Zool. 65, 1509–1514 (1987). 15. I. Orchard, J. W. D. Gole, and R. G. H. Downer, Pharmacology of aminergic receptors mediating an elevation in cyclic AMP and release of hormone from locust neurosecretory cells, Brain Res. 288, 349–353 (1983). 16. T. Pannabecker and I. Orchard, Pharmacological properties of octopamine-2 receptors in locust neuroendocrine tissue. J. Insect Physiol. 32, 909–915 (1986). 17. I. Orchard and A. B. Lange, Pharmacological profile of octopamine receptors on the lateral oviducts of the locust, Locusta migratoria, J. Insect Physiol. 32, 741– 745 (1986). 18. Z. Wang, R. G. H. Downer, J. W. D. Gole, and G. L. Orr, Characterization and pharmacological studies of an octopamine-sensitive adenylate cyclase from nerve cord of Locusta migratoria, Arch. Int. Physiol. Biochim. Biophys. 99, 189–193 (1991). 19. P. D. Evans, Multiple receptor types for octopamine in the locust, J. Physiol. 15, 317–474 (1981). 20. P. D. Evans, Octopamine, in ‘‘Comparative Insect Physiology, Biochemistry, and Pharmacology’’ (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 11, pp. 499–527, Pergamon Press, Oxford, 1985. 21. J. A. Nathanson, Characterization of octopamine-sensitive adenylate cyclase: Elucidation of a class of potent and selective octopamine-2 receptor agonists with toxic effects in insects. Proc. Natl. Acad. Sci. USA 82, 599–603 (1985). 22. J. A. Nathanson, Identification of octopaminergic agonists with selectivity for octopamine receptor subtypes. J. Pharm. Exp. Ther. 265, 509–515 (1993). 23. J. A. Nathanson,and G. Kaugars, A probe for octopamine receptors, synthesis of 2-[(4-azido-2,6-diethyphenyl)imino] imidazolidine and its tritiated derivative, a potent reversible-activator of octopaminesensitive adenylate cyclase, J. Med. Chem. 32, 1795– 1799 (1989). 24. K. R. Jennings, D. G. Kuhn, C. F. Kukel, S. H. Trotto, and W. K. Whitney, A biorationally synthesized octopaminergic insecticide: 2-(4-chloro-o-toluidino)-2-oxazoline, Pest. Biochem. Physiol. 30, 190– 197 (1988). 25. A. B. Lange and P. K. C. Tsang, Biochemical and physiological effects of octopamine and selected octopamine agonists on the oviducts of Locusta migratoria, J. Insect Physiol. 39, 393–400 (1993). 26. S. M. M. Ismail, M. A. Dekeyser, and R. G. H. Downer, Effect of dihydrooxadiazines on the octopamine-sensitive adenylate cyclase complex of the two-spotted spider mite, Tetranychus urticae, Koch, Pest. Biochem. Physiol. 47, 1–7 (1993). 27. A. Hirashima, H. Tarui, H. E. Taniguchi, and M. Eto,

12-10-96 10:48:36

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AP: PEST

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AIR SAC OCTOPAMINE RECEPTOR

28. 29.

30.

31.

Structure-activity studies of some putative octopaminergic agonists in ventral nerve cord of Periplaneta americana, Pest. Biochem. Physiol. 50, 83–91 (1994). T. Roeder, Biogenic amines and their receptors in insects, Comp. Biochem. Physiol. 107, 1–12 (1994). H. Zeng, K. R. Jennings, and B. G. Loughton, Tissue specific transduction systems for octopamine in the locust (Locusta migratoria), J. Insect Physiol., in press. S. S. Tobe, and B. G. Loughton, An investigation of haemolymph protein economy during the fifth instar of Locusta migratoria migratorioides, J. Insect Physiol. 15, 1659–1672 (1969). A. B. Lange, and I. Orchard, The action of phenyliminoimidazolidines and 2-aminooxazolines on octopamine receptors on locust fat body, Pest. Biochem. Physiol. 37, 24–29 (1990).

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/

al03$$$$63

32. T. Roeder, Pharmacology of the octopamine receptor from locust central nervous tissue (OAR3), Br. J. Pharmacol. 114, 210–216 (1995). 33. M. Lafon-Cazal, and J. Bockaert, Pharmacological characterization of octopamine-sensitive adenylate cyclase in the flight muscle of Locusta migratoria L, Eur. J. Pharmacol. 119, 53–59 (1985). 34. K. R. Jennings, D. G. Kuhn, S. H. Trotto, and W. K. Whitney, Monoamines as targets for insecticide discovery, in ‘‘Trace Amines: Their Comparative Neurobiology and Clinical Significance’’ (A. R. Boulton, A. V. Juorio, and R. G. H. Downer, Eds.), pp. 53– 56, Humana Press, New Jersey, 1987. 35. Y.-C. Cheng and W. H. Prusoff, Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099–3108 (1973).

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