Pharmacology of octopamine- dopamine- and 5-hydroxytryptamine-stimulated cyclic amp accumulation in the corpus cardiacum of the american cockroach Periplaneta americana L.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 5:119-128 (1987) Pharmacology of Octopamine-, Dopamine-, and 5-Hydroxytryptamine-Stimulated Cyclic AMP Accumulation in the Corpus Cardiacum of the American Cockroach, Periplaneta americana L. JohnW.D. Gole, Gregory L. Om, and Roger G.H. Downer Department of Biology, University of Waterloo, Waterloo, Ontario, Canada A variety of potential receptor agonists were incubated with isolated corpora cardiaca from the American cockroach Periplaneta americana t o determine their effects o n cyclic AMP production in this gland. Octopamine, dopamine, and 5-hydroxytryptamine elevated cyclic AMP levels in a dose-dependent manner with estimated K, values of 15.8, 1.7, and 1.1 pM, respectively, and their stimulation was found t o be additive. Several vertebrate receptor antagonists were tested against the three amines and a preliminary pharmacological profile developed. Key words: biogenic amine, amine receptor, adenylate cyclase INTRODUCTION Octopamine mediates several important physiological processes in insects [l] including the regulation of (neuro)hormonal release from the CC.* The release of AKH from the glandular lobes of the CC in locusts is regulated by axons of NCCII [2,3] through the mediation of an octopamine-sensitive adenylate cyclase located on the postsynaptic membrane of the hormonecontaining cells [4,5]. In the American cockroach, the release of hypertrehalosemic hormone(s) from CC is influenced by adrenergic components of NCCII  and electrical stimulation of the nerve results in elevated levels of cyclic AMP and release of hypertrehalosemic factor(s) from the CC [7J. Cyclic *Abbreviations: CC = corpus cardiacum; AKH = adipokinetic hormone; NCCll = nervi corporis cardiacurn 11; GTR = glucose-trehaloseringers; IBMX = 3-isobutyl-I-methylxanthine; DCDM = N’-demethylchlordimeform. Received August 5,1986; accepted December 8,1986. Address reprint requests to Dr. R.G.H. Downer, Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3C1. 0 1987 Alan R. Liss, Inc. 120 Cole, Orr, and Downer AMP levels in the CC are also increased by octopamine, dopamine, and 5three putative neurotransmitters that have been hydroxytryptamine identified in this tissue [8,9]. However, octopamine is the only monoamine Thus the release of this that elicits release of a hypertrehalosemic factor (these) hormone(s) appears to be under the control of octopamine, whereas dopamine and 5-hydroxytryptamine may regulate the release of other (neuro)hormones. The present study was undertaken to confirm the existence of three separate monoaminergic receptors in the corpus cardiacum of Peeriplaneta ameericana and to provide a pharmacological characterization of octopamine-, dopamine-, and 5-hydroxytryptamine-stimulatedcyclic AMP accumulation. [n, [n. MATERIALS AND METHODS Experimental Animals Adult male cockroaches were taken from a colony of Peeriplaneta ameericuna L. maintained under standard conditions in this laboratory [lo]. Insects were taken between 1and 5 months after the final molt and isolated 24 h prior to dissection. Incubation of Corpora Cardiaca The CC was dissected free of adhering tissue and placed in 1.5 ml microcentrifuge tubes containing 100 pl physiological saline containing glucose and trehalose [11,12] for 30 min at room temperature. The incubation medium was then removed and replaced with 100 pl fresh GTR containing the test compound and 0.5 mM IBMX. Following a further 10 min incubation (aminestimulated increases in cyclic AMP were linear with respect to time for 10 min), the reaction was terminated by the addition of 0.5 m10.4 N perchloric acid. Following perchloric acid treatment, the solution was brought to pH 6.2 with 2.5 M potassium bicarbonate and centrifuged at 2,OOOg for 5 min. A 100 pl aliquot of the supernatant was used to determine the concentration of cyclic AMP. In experiments involving blocking agents, the 30 min preincubation was followed by a 5 min incubation in fresh GTR containing the antagonist. This solution was removed and replaced with GTR containing both the agonist and antagonist, in addition to IBMX, and this was allowed to incubate for 10 min before the reaction was terminated with perchloric acid. Calcium-free incubation conditions were maintained by using GTR with no added CaC12 for the 30 min preincubation and by including 2.0 mM ethyleneglycol-bis-(Paminoethyl ether) in the CaC12-free GTR containing the test compound. Measurement of Cyclic AMP Production The measurement of cyclic AMP was performed by radioimmunoassay using a kit obtained from New England Nuclear Research Products (Boston, MA). This system utilizes the method of Steiner et al.  as modified by Cailla et al. . Amine-Stimulated CAMP Accumulation in Corpus Cardiacum 121 Chemicals N’-Demethylchlordimeform was generously provided by Dr. I. Orchard, University of Toronto. The following compounds were also received as gifts from the indicated suppliers: flupenthixol (Lundbeck and Co., Copenhagen, Denmark), cyproheptadine (Merck, Sharp and Dohm, Kirkland, Quebec, Canada), promethazine (Poulenc Ltd., Montreal, Quebec, Canada), phentolamine (Ciba-Geigy, Dorvall, Quebec, Canada). Mianserin and butaclamol were purchased from Research Biochemicals Inc. (Wayland, MA), forskolin from Calbiochem (San Diego, CA), and all other reagents from Sigma Chemical Co. (St. Louis, MO). Determination of and Ki Values The IC50value was defined as the concentration of antagonist required to cause a 50% inhibition of agonist-mediated production of cyclic AMP. The Ki, or inhibitor constant, refers to the dissociation constant of the receptorinhibitor complex, assuming that competitive inhibition is occurring. The Ki was calculated from the equation Ki = IC50/(l + SIK,), where S is the concentration of agonist used and K, is the concentration of agonist causing half-maximal cyclic AMP production, and IC50was determined as previously described . RESULTS Effect of Potential Agonists on Cyclic AMP Production Data summarized in Table 1indicate that octopamine, dopamine, and 5hydroxytryptamine cause statistically significant increases in cyclic AMP levels (4.9-fold, 3.7-fold, and 2.7-fold, respectively). The absence of calcium from the incubation medium does not affect the octopamine-stimulated increase in cyclic AMP levels. These data, although not conclusive, suggest that octopamine is acting through a receptor that is coupled to adenylate cyclase rather than effecting the release of an endogenous substance that in turn causes increased cyclic AMP levels. Ergonovine maleate, which is a dopamine agonist/5-hydroxytryptamineantagonist causes a 2.6-fold increase in cyclic AMP levels, whereas ergotamine tartrate, a dopamine agonist and antagonist of 5-hydroxytryptamine and a-adrenergic receptors is without effect. The formamidine DCDM elevates cyclic AMP levels (1.9-fold) and the diterpene forskolin causes the largest increase observed (22-fold over controls). Octopamine, dopamine, and 5-hydroxytryptamine increase cyclic AMP levels in a dose-dependent manner (Fig. l), with octopamine eliciting a maximal 13-fold increase in cyclic AMP production, whereas the maximal production caused by dopamine and 5-hydroxytryptamine is about 9-fold greater than that of the control. The estimated K, values (50% of maximal response) for octopamine, dopamine, and 5-hydroxytryptamine are 15.8, 1.7, and 1.1pM, respectively. Ergonovine maleate also causes a dose-dependent increase in cyclic AMP with an estimated K, of 23.0 pM and a maximal production that is 3.3-fold greater than that of the control. Additivity studies 122 Gole, Orr, and Downer TABLE 1. Effect of Various Potential Receptor Agonists on CAMPProduction in CC of Peridaneta americana Treatmenta Octopamine Octopamine (-Ca2+) Dopamine 5-Hydroxytryptamine Synephrine Ergonovine maleate DCDM Forskolin (0.1 mM) Phenylephrine Isoproterenol Apomorphine Tyramine Norepinephrine Clonidine 0-Phenylethylamine Phenylpropanolamine Norphenylephrine Ergotamine tartrate Hydroxymandelic acid Ephedrine CAMPincrease (pmollminlCC) 0.171 0.165 0.120 0.077 0.118 0.070 0.040 0.926 -0.008 -0.014 -0.008 -0.003 0.005 0.012 -0.005 -0.011 0.013 0.005 -0.012 -0.004 f 0.019 (14)b,* f 0.029 (5)* f 0.019 (5)* f 0.021 (5)* f 0.021 (5)* f 0.078 (5)* f 0.007 (5)* f 0.055 (5)* f 0.004 (10) f 0.003 (10) *f 0.002 (5) 0.006 (5) 0.008 (5) f 0.008 (5) f 0.008 (5) f 0.002 (5) & 0.011 (4) f 0.008 (4) f 0.003 (4) 0.004 (4) aTreatment concentrations were 10 pM unless otherwise stated. bValues indicate mean f SEM for the number of determinations in parenthesis. *Values significantly different from control values; P = 0.01, Dunnett’s test [control value = 0.044 f 0.003 (14) pmollminlCC]. using octopamine, dopamine, and 5-hydroxytryptamine at maximally effective concentrations of each agonist demonstrate that the cyclic AMP production is completely additive (Table 2), suggesting that these compounds are working through separate receptor populations. Effects of Antagonists on the Octopamine-, Dopamine-, and 5Hydroxytryptamine-Mediated Increases in Cyclic AMP To distinguish further between the various amine responses, a variety of vertebrate receptor antagonists were tested for their effectiveness in inhibiting the increase in cyclic AMP levels. The most effective inhibitor of the octopamine-mediated response is the a-adrenergic antagonist and 5-hydroxytryptamine inhibitor mianserin. Gramine, an inhibitor of 5-hydroxytryptamine, phentolamine, a general a-adrenergic antagonist, and cyproheptadine, a 5-hydroxytryptaminelhistamineantagonist, are approximately 10fold less potent. Promethazine, a phenothiazine derivative with antihistamine properties, the antipsychotic drugs flupenthixol and +butaclamol, which block dopamine receptors, and the P-adrenergic blocking agent propranolol are poor antagonists of the octopamine-mediated response (Table 3). The dopamine-response is blocked most effectively by cis-flupenthixol, with gramine, +butaclamol, and mianserin being less effective (1.3-, 1.9-, and 6.6-fold, respectively; Table 4). The 5-hydroxytryptamine-mediatedincrease in cyclic AMP levels is most effectively blocked by cyproheptadine. Amine-Stimulated cAMP Accumulation in Corpus Cardiacum 123 0.5 - 04. \ c . E ;Y" 03. 0 -E w v) 9 U 02. V L a I Q, 01. - 0.0 1 ~ - 7 10-6 10-5 10-4 10-3 10-2 AMINE CONCENTRATION (MI Fig. 1. Effect of octoparnine (V),dopamine (O), and 5-hydroxytryptarnine (m) on cAMP production in incubated cockroach CC. Each point represents the mean f SEM for five to 15 determinations. Cyclic AMP production in control incubations was 0.031 f 0.001 pmol/min/ CC (mean of three experiments). TABLE 2. Additivity of Octopamine-, Dopamine-, and 5-Hydroxytryptamine-Mediated Increases in cAMP in CC of Periplaneta americana Treatmenta Octopamine Dopamine 5-Hydroxytryptamine Octopamine + dopamine Dopamine + 5-hydroxytryptamine Octopamine + dopamine + 5-hydroxytryptamine cAMP increaseb (pmoliminlCC) 0.351 f 0.040 (15)' 0.314 f 0.028 (15) 0.311 & 0.035 (10) 0.674 f 0.092 (5) 0.634 f 0.049 (5) 1.046 & 0.174 (5) aAll treatments were 0.1 mM. bControl value was 0.031 k 0.005 (9). CValuesindicate mean f SEM for the number of determinations in parenthesis. Mianserin is 39-fold less effective, whereas the other compounds tested are relatively poor antagonists (Table 5). DISCUSSION The present study provides the first pharmacological characterization of amine-stimulated cyclic AMP accumulation in the CC. Incubation of potential receptor agonists with this tissue (Table 1)indicates pharmacological similarities with other insect systems [16-221. The cardiotonic diterpene forskolin enhances adenylate cyclase activity in a variety of intact and broken tissue 124 Cole, Orr, and Downer TABLE 3. Effect of Receptor Antagonists on the Octopamine-Mediated Increases in cAMP in CC of Periplaneta americana Antagonist Mianserin Gramine Phentolamine Cyproheptadine cis-Flupenthixol Promethazine Propranolol -Butaclamol trans-Flupenthixol +Butaclamol IC50 (PM)' 0.45 2.82 5.01 6.92 17.80 105.00 120.00 Ki (PM) 0.37 2.32 4.13 5.70 14.70 86.50 98.90 % Inhibition with 0.1 mM antagonist 100.0 100.0 99.8 78.8 60.2 49.1 45.0 32.0 24.2 15.0 Toncentration of octopamine used was 5.0 pM. TABLE 4. Effect of Receptor Antagonists on Dopamine-Mediated Increases i n cAMP i n CC of Peridaneta americana Antagonist cis-Flupenthixol Gramine +Butaclamol Mianserin Promethazine Phentolamine Propranolol Cyproheptadine -Butaclamol trans-Flupenthixol IC50 bMIa Ki (PM) 1.60 2.14 3.16 10.60 20.00 42.20 236.00 310.00 - 0.89 1.19 1.76 5.91 11.20 23.50 132.00 173.00 - - - Yo Inhibition with 0.1 mM antagonist 100.0 86.6 82.6 92.5 89.8 58.1 37.6 1.0 24.2 6.2 aConcentration of dopamine was 5.0 pM. TABLE 5. Effect of Receptor Antagonists on 5-Hydroxytryptamine-Mediated Increases in cAMP in CC of Periplaneta americana Antagonist Cyproheptadine Mianserin cis-Flupenthixol +Butaclamol Promethazine Phentolamine Gramine trans-Flupenthixol Propranolol -Butaclamol IC50 (PM)' Ki (PM) 0.10 0.40 15.80 37.20 58.90 70.80 321.00 616.00 3.79 8.93 14.10 17.00 77.10 148.00 - - - - ~ aConcentration of 5-hydroxytryptamine was 5.0 pM. - % Inhibition with 0.1 mM antagonist 96.2 91.2 80.1 72.7 53.8 38.5 31.6 20.1 10.0 3.4 Amine-Stimulated CAMPAccumulation in Corpus Cardiacum 125 preparations  by activation of the catalytic subunit of adenylate cyclase. Forskolin-enhancement of cyclic AMP production by the cockroach CC suggests similarities between insect and other eukaryotic adenylate cyclases. The increase in cyclic AMP resulting from DCDM treatment confirms the proposal that formamidine pesticides may exert some of their effects through interaction with octopamine-sensitivereceptors coupled to adenylate cyclase [24-27]. Formamidines also interact to a lesser degree with dopamine- and 5hydroxytryptamine-sensitive adenylate cyclase , so some of the effects of these pesticides may result from interaction with receptors other than those that are specific for octopamine. Synephrine (n-methyloctopamine), which has not been detected in insects, probably stimulates cyclic AMP production in the cockroach CC by interacting with octopamine receptors [21,22]. The other phenylethylamine derivatives tested are typically poor agonists of insect adenylate cyclase (K, values greater than 10-fold those of octopamine), since substitution at either the /3-hydroxyl or p-hydroxyl group on the octopamine molecule substantially reduces potency [21,22]. In general, the data presented in Table 3 confirm earlier studies on insect octopamine receptors in that the a-adrenergic antagonist phentolamine and the 5-hydroxytryptaminelhistamine inhibitor cyproheptadine are effective antagonists of the octopamine-stimulated cyclic AMP increase, whereas /3adrenergic blockers, such as propranolol, are ineffective [1,20-22,28,29]. In addition, gramine, a 5-hydroxytryptamine antagonist, which is a potent inhibitor of octopamine-sensitive receptors in locust CC 1291, was also effective in cockroach CC (Table 3). For cockroach CC (Table 3), nerve cord , and hemocytes , the most potent inhibitor of the octopamine response is mianserin. Nerve cord octopamine-sensitive adenylate cyclase is only 1-3fold less sensitive to blockage by phentolamine, gramine, and cyproheptadine than mianserin [lo], whereas these compounds are 11-100-fold less effective than mianserin in CC (Table 3) and hemocytes . Therefore, based on the equal sensitivity to mianserin, phentolamine, gramine, and cyproheptadine, the octopamine-sensitive receptors of the nerve cord appear to be pharmacologically distinct from those present in CC and hemocytes. The octopamine receptors associated with CC and hemocytes can be further differentiated on the basis of their response to gramine, cyproheptadine, and promethazine. Gramine and cyproheptadine are better antagonists in the CC than in hemocytes relative to mianserin, whereas the reverse is true of promethazine. In all three cockroach tissues, the dopamine blockers flupenthixol and butaclamol are relatively weak antagonists of the octopamine response. A classification of multiple octopamine-receptor subtypes has been proposed by Evans  in the locust extensor-tibiae muscle. In this classification, the antagonists chlorpromazine and metochlopramide are used to distinguish between octopamine-1 and octopamine-2 receptors. However, in cockroach hemocytes, metochlopramide and chlorpromazine are equally poor antagonists ; therefore, although the agonist profile approximates that of locust octopamine-2 receptors, the antagonist profile is not supportive. The classification proposed by Evans  is based on physiological responses 126 Cole, Orr, and Downer (myogenic rhythm and twitch tension) rather than effects on adenylate cyclase, as in the study of cockroach hemocytes , and pharmacological characterization of the octopamine-sensitive adenylate cyclase of locust tibiae muscle indicates poor correlation with the agonist profile developed on the basis of twitch tension . Therefore, pharmacological classification of the octopamine receptor(s) may vary according to the particular response being studied. More specific antagonists for octopamine may be required before a complete classification is possible. The dopamine-stimulated increase in cyclic AMP in cockroach CC (Table 4) is sensitive to inhibition by vertebrate dopamine antagonists. Thus the most effective compound is cis-flupenthixol with +butaclamol being only 2fold less effective, whereas the inactive isomers of these compounds are poor antagonists. Phentolamine and cyproheptadine are relatively poor antagonists of the dopamine response in cockroach CC (Table 4), which is in agreement with previous studies in locust salivary glands  and cockroach brain  and nerve cord . The ergot-alkaloid ergonovine maleate causes a dose-dependent increase in cyclic AMP (Fig. 2), and additivity studies indicate that it is additive with the octopamine response but nonadditive with dopamine (unpublished observations). This suggests a possible interaction with dopamine receptors. In vertebrates, ergonovine acts as a dopaminergic agonist at both D1 and D2 receptors, whereas another ergot-alkaloid, ergotamine, is more effective as a D2 agonist . The lack of response to ergotamine tartrate and the stimulatory effects of ergonovine maleate on cockroach CC cyclic AMP levels (Table 1)suggest not only similarities between vertebrate and cockroach dopamine 1 - . . - 0.10. V 0 .s E 0.08. e Ln -: 006. W In 5K V z 0 0.04. 002. 0.OoJ 1o-’ 10-6 10-5 10-4 10-3 ERGONOVINE-MALEATE CONCENTRATION (M) Fig. 2. Effect of ergonovine maleate on cyclic AMP production in cockroach CC. Points SEM for five determinations. Cyclic AMP production in control represent the mean incubations was 0.029 f 0.004 prnol/rnin/CC. Amine-Stimulated CAMPAccumulation in Corpus Cardiacum 127 receptors but also indicate that CC dopamine receptors may be pharmacologically similar to vertebrate D1 receptors. The 5-hydroxytryptamine-stimulatedincrease in cyclic AMP in cockroach CC is the most sensitive to inhibition by cyproheptadine (Table 5). In nerve cord, cis-flupenthixol is approximately 30-fold more effective than cyproheptadine, followed by gramine, which is 2-fold more effective than cyproheptadine . By contrast, gramine is a poor antagonist of CC 5-hydroxytryptamine response (Table 5). Therefore, based on the relative potency to cyproheptadine cis-flupenthixol and gramine, the 5-hydroxytryptamine-sensitive receptors of cockroach CC and nerve cord appear to be pharmacologically distinct. This may be analogous to the situation that prevails in vertebrate neural tissue in which distinct adenylate cyclase-coupled 5-hydroxytryptamine receptor subtypes have been proposed . Comparison with other studies of 5-hydroxytryptamine pharmacology in insects is difficult because of the limited number of compounds used. The 5-hydroxytryptamine-sensitive adenylate cyclase of cockroach nerve cord [16,34] and CC (Fig. 1)and that present in the ventral nerve cord of Manduca sexta  have a K, of approximately 1pM, similar to that of many vertebrate 5-hydroxytryptamine receptors . Furthermore, compounds such as cyproheptadine and mianserin, which are known to block vertebrate 5-hydroxytryptamine receptors , are also antagonists of cockroach 5-hydroxytryptamine-sensitive adenylate cyclase (Table 5) 116,341. A more detailed pharmacologic study, including ligand-binding studies of insect 5-hydroxytryptamine receptors, is necessary before meaningful comparisons can be made. LITERATURE CITED 1. Orchard I: Octopamine in insects: neurotransmitter, neurohormone and neuromodulator. Can J Zoo1 60, 659 (1982). 2. Rademakers LHPM: Identification of a secretomotor centre in the brain of Locusfa migrutoriu, controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum. 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