LIBRARY Michigan State University PLACE IN RETURN BOX to remove We checkout from your record. TO AVOID FINES return on or before due due. DATE DUE DATE DUE DATE DUE [ JL— l TT—n I! MSU le An Afflrmetive Action/Equal Opportmlty Institution PHARMACOLOGY AND SIGNAL-TRANSDUCTION MECHANISMS OF OCTOPAMINE RECEPTORS IN INSECTS BY Nailah Orr A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology and Neuroscience Program 1990 ABSTRACT PHARMACOLOGY AND SIGNAL-TRANSDUCTION MECHANISMS OF OCTOPAMINE RECEPTORS IN INSECTS BY Nailah Orr Octopamine (0A) is found in high concentrations in insects and has been physiologically compared to the vertebrate fight-or-flight hormones. The existence of 0A receptors has been demonstrated by a number of techniques and most 0A receptors are coupled to an OA-sensitive adenylate cyclase (OSAC). Studies of this enzyme provide a reliable method for characterizing the pharmacology and biochemistry of the 0A receptor. The objectives of the present study were: (1) to identify a high-affinity ligand for the 0A receptor, (2) to identify an insect cell-line model system for studying the 0A receptor, and (3) to investigate the presence of phosphoinositide hydrolysis- coupled 0A receptors. The imidazoline, 2,3-rylylaminomethyl-2’-imidazoline, XAMI, was shown to have the highest reported affinity for insect OSAC, with a Ka 187, 158, 165, and 81 times more effective than 0A in cockroach nerve cord, brain and hemocytes and tobacco hornworm nerve cord, respectively. XAMI-mediated cAMP production is dependent upon GTP and enhanced by forskolin, suggesting that XAMI activates adenylate cyclase (AC) via interaction with a receptor-G- protein complex. Additivity studies suggest that XAMI interacts specifically with the 0A receptor. These effects of XAMI are reversible, exhibit agonist-induced desensitization and are pharmacologically similar to other OA-mediated effects. Thus, because of its specificity and high affinity, XAMI may be a good general probe for the 0A receptor. Sf9 cells from Spodoptera frugiperda were shown to contain a G-coupled OSAC. The pharmacology of this receptor is similar to that of other Lepidopteran 0A receptors but differs from the cockroach receptor, suggesting the existence of species-specific OA-receptor subtypes. Therefore, Sf9 cells can be used as a readily available, homogeneous model system for studying Lepidopteran 0A receptors. Using a radioreceptor assay, the ability of 0A agonists to elevate inositol 1,4,5-trisphosphate (1P3) levels in cockroach brain slices was demonstrated. This effect is blocked by phentolamine and is not an indirect consequence of elevated intracellular cyclic AMP levels. This is the first report of OA-mediated inositol phosphate hydrolysis. Copyright by NAILAH ORR 1991 This dissertation is dedicated to my parents, Maqsood A. and Ruqaiyya Minhas, who instilled in me an appreciation for hard work and provided me with unending love and support. I also dedicate this work to my husband, Dr. Gregory L. Orr, my mentor and best friend, who has helped me in every step of the way and without whom I could not have accomplished as much. ACKNOWLEDGMENTS I wish to thank Dr. R.M. Hollingworth for his unfailing academic and moral support throughout the progress of my work. I would also like to thank the members of my committee, Drs. M.L. Contreras, G.I. Hatton, K.L. Klomparens and R.A. Pax for their helpful suggestions and discussions during the course of this study; It greatly appreciate the time and care they have taken on my behalf. I thank Mr. Guoping Shi for synthesizing NCS and Dr. R.M. Hollingworth for synthesizing XAMI and DCDM. I wish to extend a special thanks to the many people I have had the opportunity to work with, especially members of the Center for Electron Optics, Neuroscience Program and Departments of Zoology and Pharmacology and Toxicology. A special thanks is due to Capt. J.M. Wierenga for his invaluable friendship and helpful discussions during the course of this study. I would also like to extend a very special thanks to Drs. J.E. Steele and R.G.H. Downer for introducing me to the fascinating field of insect pharmacology and biochemistry and teaching me the virtues of science. A special thanks to my husband and family for their unending support. Words do not do justice to their contributions. vi TABLE OF CONTENTS Page LIST OF TABLES.........................................xi LIST OF FIGURES........................................xii GENERAL INTRODUCTION...................................1 CHAPTER 1. Characterization of a potent agonist of the insect octopamine-receptor-coupled adenylate cyclase.................................23 INTRODUCTIONOOOOOOOOOOOO00.000.00.000.0....0.00.0.24 MATERIALS ANDMETHODS...0.0.0.0.0.000000000000000025 Insects......................................25 Tissue preparation...........................26 Preparatory period......................26 Dissection of cockroach nerve cords.....27 Dissection of hornworm nerve cords......27 Dissection of cockroach brain...........28 Hemocyte collection.....................28 Cockroach and hornworm nerve cord homogenate preparation..... ........ 29 Cockroach brain and nerve cord membrane preparation....................29 Analytical procedures........................29 Chemicals...............................29 Protein assay...........................30 Cyclic AMP radioimmunoassay.............31 Pharmacological procedures...................38 Determination of V ax and Ka value......38 Determination of IE values............38 Determination of K- values..............38 Additivity studies......................38 Reversibility studies...................39 Statistical procedures............... ..... ...40 RESULTSOCOOOOOO0......OOOOOOOOOOOOOOOO...0.0.0.0.040 Identification of a novel stimulator of cockroach neural adenylate cyclase........41 Characterization of the XAMI-mediated cyclic AMP production in cockroach nerve cord...................................43 Antagonist profile for the XAMI- mediated production of cyclic AMP in cockroach nerve cord.........................45 vii Tissue and species-specific effects Of XAMI O O O O O O O O O O O O O O O O O O O O O O O O O O O I O I O O O O O O O O 49 DISCUSSION. 0 O O O O O O O O O O O O O O O C O O O O O O O O O O O O O O O O O O O O O I 50 Identification of a potent agonist of insect adenylate cyclase.....................50 Pharmacological characterization of the XAMI-mediated elevation in cyclic AMP 1evels............................59 Comparative effectiveness of XAMI in several insect systems......... ........... 60 CHAPTER 2. Identification and pharmacological characterization of an insect cell line as a model system for studying octopamine receptors.........................................63 INTRODUCTIONOOOOCOOOOO00......0.0.0.0000000000000064 MATERIALS ANDMETHODSOOOOOOCOOOOOOOOO0..0.0.0.0...66 Cockroach nerve cord preparation.............66 Tissue culture...............................67 Sf9 cell preparation.........................68 Intact cells............................68 Sf9 membranes...........................7O Analytical Procedures........................70 Chemicals...............................70 Protein assay...........................71 Cyclic AMP radioimmunoassay.............71 Desensitization experiments..................72 Pharmacological procedures...................73 Statistical procedures.......................73 RESULTSOOOOOOOOOOOOOOO00.0.0.0...O...O. ........... 73 Identification of an insect cell line expressing octopamine-sensitive adenylate cyclase............................73 Characterization of the adenylate cyclase-coupled octopamine receptor in intact Sf9 cells..........................75 Characterization of the adenylate cyclase-coupled octopamine receptor in Sf9 membranes.............................75 Pharmacology of the Sf9 adenylate cyclase......................................85 Pharmacology of the cockroach adenylate cyclase............................92 Agonist-induced desensitization in intact Sf9 cells..........................96 viii CHAPTER 3. DISCUSSION........................................99 Identification and characterization of an octopamine-sensitive adenylate cyclase in Sf9 cells............... ........ ..99 Agonist-induced desensitization of the Sf9 adenylate cyclase.................100 Pharmacology of the octopamine- sensitive adenylate cyclase- coupled receptor.............. ............... 102 Identification of a novel signal- transduction mechanism for insect-octopamine receptors: elevation of inositol 1,4,5- trisphosphate levels................... ........... 113 INTRODUCTION......... ............ . .............. ..114 MATERIALS AND METHODS......... ..... . ............ ..117 Insects... ....... .... ..................... ...117 Cell culture.......................0.........117 Preparatory period ....... ........... ..... ....117 Dissection of cockroach brain................118 Sf9 intact cell collection...... ....... ......118 Crude brain-slice preparation................119 Trichloroacetic acid extraction of inositol 1,4,5-trisphosphate.. .......... ..120 Removal of trichloroacetic acid from extracts......... ................ ..121 Analytical procedures............ .......... ..122 Chemicals......... ........ . ............. 122 Protein assaYeeeeeeeeeeeeeeeeeeeeeeeeeee122 Inositol 1,4,5-trisphosphate radioreceptor assay......... ............ 122 Statistical procedures..... .......... .. ...... 126 RESULTS........................... ............... .126 Characterization of the IP radioreceptor assay for use with insect tissues.................... ......... ..126 Pharmacology of IP3 production.. ...... .......130 DISCUSSION........................................132 Characterization of an inositol 1,4,5-trisphosphate radioreceptor assay in insects................. ............ 132 ix Pharmacology of IP production in cockroach brain slices.......................136 CONCLUSIONS.................................. ......... .143 APPENDIX ..............................................l47 Compounds used in the determination of the pharmacological properties of octopamine- sensitive adenylate cyclase in Sf9 cells and cockroach nerve cord membranes................148 REFERENCES.............................................151 Table 10. 11. LIST OF TABLES Page Proposed classification of octopamine receptors from locust extensor-tibiae muscle (Evans, 1981)...................................018 Additivity studies on cyclic AMP production in cockroach nerve cord..........................46 Effects of various potential antagonists on XAMI-mediated elevation of cyclic AMP in nerve-cord membrane preparations of Eeriplaneta americana............................51 Comparative potencies of agonists of octopamine-sensitive adenylate cyclase...........56 Effects of various agents on cyclic AMP production in whole-cell preparations Of Sf9 cells............................... ...... 74 Effects of various octopaminergic and adrenergic agonists on the production of cyclic AMP in whole-cell preparations of Sf9 cells.............................. ....... 89 The effects of various antagonists on octopamine-mediated cyclic AMP production in intaCt Sf9 ceIISeeeeeeeeeeeeeeeeeeeeeeeeeeeeee91 Effects of various octopaminergic and adrenergic agonists on cyclic AMP production in cockroach ventral nerve-cord membrane preparations.................93 Effects of various antagonists on octopamine—mediated cyclic AMP production in cockroach ventral nerve-cord membrane preparations..................... ....... 95 Effects of 24 hr pretreatment of Sf9 cells with octopaminergic compounds on octopamine-mediated cyclic AMP production ....... 98 Effect of various compounds on 1P3 production in cockroach-brain slices........ ..... 131 xi LIST OF FIGURES Figure Page 1. Chemical structures of Octopamine and Norepinephrine..............................00.0.2 2. Cyclic AMP standard curve (0.01 to 25.0 pmol) 3. obtained with a non-acetylated, iodinated radioimmunoassay kit. Each point represents the mean of two determinations with SEM of less than 5%.....................................34 Cyclic AMP production as a function of the concentration of cockroach ventral nerve cord membranes for basal (5 = 0.986) and 1 mM octopamine-mediated (r = 0.988) stimulation. Values are the mean of 3 experiments with a SEM of less than 10%. Values for 100% protein were in the range of 48 to 53 ug.................36 Time course of octopamine (1 mM) and sodium fluoride (10 mM) stimulated cyclic AMP production in cockroach ventral nerve cord membranes. Each point represents the mean of 3 experiments with SEM of less than 7%..........37 Chemical structures of octopamine agonists used in the present study.................. ..... 41 Effects of 0A, NCS, DCDM and XAMI on cyclic AMP production in membrane preparations of cockroach nerve cords. All four agonists were tested on the same preparation (experiment) to standardize the response. Values are the mean 3 SEM of 5 separate experiments, performed in duplicate. Control values for cyclic AMP production = 198.4 1 17.6 pmol/min/mg......................42 Effects of 0.05 mM GTP and 0.01 pM forskolin on XAMI—mediated cyclic AMP production in cockroach nerve cord membranes. Values are the mean i SEM of 3 separate experiments, performed in duplicate. Control cyclic AMP production in the presence of GTP = 83.5 i 5.3 and GTP + forskolin = 86.0 i 7.0, and in the ab ence of GTP = 63.5 i 4.2 pmol/min/mg. Denotes points that are significantly different from the equivalent GTP-treated value (p<0.05)......................44 xii 10. 11. 12. 13. 14. Reversibility of XAMI-mediated cyclic AMP production in membranes of cockroach nerve cord. Membranes were either (1) untreated and assayed for cyclic AMP production in the presence of 1‘pM XAMI, or were pre-exposed to (2) buffer or (3) IuM XAMI and washed three times. Values indicate the mean i SEM of two separate experiments, assayed in duplicate.......................................47 Effect of various antagonists on XAMI- mediated elevation of cyclic AMP in nerve cord membranes of Periplaneta amezicaga. XAMI was included at 0.1‘pM. Values indicate the mean i SEM of 3 to 4 separate experiments, assayed in duplicate...............48 Effect of a fixed concentration of various antagonists on XAMI-mediated cyclic AMP production in cockroach nerve cord membranes. Values are the mean i SEM of 3 separate experiments, assayed in duplicate. Control cyclic AMP production was 170.3 3 17.5 pmol/min/mg................................52 Agonist-induced cyclic AMP production in cockroach brain membranes. Values indicate the mean 1 SEM of 4 experiments, assayed in duplicate. Control cyclic AMP production was 255.3 1 4.2 pmol/min/mg. All agonists were tested in the same preparation........ ..... 53 Agonist-induced cyclic AMP production in cockroach hemocytes. Points indicate the mean i SEM of 3 separate experiments, assayed in duplicate. Control cyclic AMP production = 12.1 i 1.4 pmol/10 min/mg. All agonists were tested in the same preparation.............54 Agonist-induced cyclic AMP production in tobacco hornworm nerve cord homogenates. Values are the mean 1 SEM for 3 separate experiments assayed in duplicate. Control cyclic AMP production was 419.9 3 88.8 pmol/10 min/mg. Both agonists were tested in the same preparation.........................55 Representative growth curve for Sf9 cells grown in Ex-cell 400 medium. Cell viability ranged from 85 to 95%...........................69 xiii 15. 16. 17. 18. 19. 20. Effect of Sf9 intact-cell protein concentration on basal and octopamine- mediated (0.1 mM) cyclic AMP production. Values are the mean of two experiments assayed in duplicate, with SEM of less than 14%. The values for 100% protein ranged from 0.38 to 0.41 mg. The correlation coefficients for basal and octopamine-mediated effects were 0.995 and 0.998, respectively......76 Effect of temperature on cyclic AMP production in membrane preparations of Sf9 cells. (A) Histogram of control and octopamine- mediated (0.1 mM) cyclic AMP production. (B) Data from Figure 16A is expressed as an increase in cyclic AMP production. Control cyclic AMP production at 10, 23, 27 and 35°C was 3.32 i 0.46, 4.36 i 0.52, 19.97 i 1.3 and 32.23 i 0.98 pmol/min/mg, respectively.......................77 Time-dependent production of octopamine- mediated (0.1 mM) cyclic AMP production in intact Sf9 cells. Values are the mean of 3 to 6 separate experiments. The correlation coefficient for the range of points between 1 and 5 min = 0.979............78 Octopamine-mediated (1 mM) cyclic AMP production in Sf9 membranes as a function of protein concentration. Values are the means of 2 expsriments, with SEM of less than 20% (r = 0.982). The values for 100% protein were in the range of 9.1 to 14.9 ug.................... ..... 80 Time-dependent production of octopamine- mediated (1mM) cyclic AMP in membrane preparations of Sf9 cells. Values are the mean i SEM of 2 separate experiments ........ 82 Dose-dependent octopamine-mediated cyclic AMP production in Sf9 membranes in the presence (0.05 mM) and absence of GTP and in the presence of GTP + 0.5 ‘pM forskolin. Values are the mean i SEM of 3 separate experiments. Control values, in the absence of GTP = 14.1 i 0.4 pmol/min/mg and in the presence of GTP and GTP + forskolin = 17.8 i 0.7 and 44.2 1; 2.8 pmol/min/mg, respectively....................................83 xiv 21. 22. 23. 24. 25. 26. Cyclic AMP production as a function of the GTP concentration in control and octopamine-mediated (0.1 mM) Sf9 membranes. Values are the mean 1 SEM of 6 experiments. Basal cyclic AMP production (in the absence of GTP) for control and octopamine-treated samples was 13.8 i 0.1 and 16.7 i 0.9 pmol/min/mg, respectively....................................84 Dose-dependent effects of forskolin, sodium fluoride (NaF) and 5'-guanylylimidodiphosphate (GppNHp) on basal production of cyclic AMP in Sf9 membranes. Values are the mean 1 SEM of 3 separate experiments. Control cyclic AMP production = 15.6 i 1.3 pmol/min/mg..........................86 Effects of octopamine agonists on cyclic AMP production in intact Sf9 cells. Values are the mean i SEM of 3 to 6 separate experiments. Control cyclic AMP production was 3.0 i 0.2 pmol/min/mg.................................87 Agonist-induced desensitization of octopamine- mediated cyclic AMP production in intact Sf9 cells. Values are the mean 1 SEM of 3 to 6 separate experiments. Denotes points which are significantly different from the equivalent control (p<0.05)....... ..... 97 Representative D-myo-inositol 1,4,5- trisphosphate standard curves (0.12 to 12.0 pmol) obtained with a tritiated radioreceptor assay. Each curve represents a separate experiment with new and old standard indicating the use of the standard (without refreezing) within 4 and by 6 days of solubilization, respectively............125 Representative protein linearity curves for the basal production of inositol 1,4,5- trisphosphate in cockroach brain slices. (A) Protein linearity expressed in terms of brain equivalents per sample (r2 = 0.993). (B) Data from Figure 26A e ressed as protein concentration per sample (r = 0.999)...........127 27. Inositol 1,4,5-trisphosphate production and spike (2 pmol 1P3) recovery as a function of protein concentration. IP recovery for insect saline, cockroach brains slices and intact Sf9 cells ranged from 95-100%, 87-90% and 85-90%, respectively. Data are from a represenzative experiment with 100% protein 8 4 x 10 cells/sample and 6 brains/sample, respectively for cockroach brain slices and Sf9 intact cells. Correlation coefficients for the six curves, buffer, buffer + spike, brain slices, brain slices + spike, Sf9 cells, Sf9 cells + spike are 1.00, 0.99, 0.97, 0.99 and 0.99, respectively...... ......... 129 xvi 1 GENERAL INTRODUCTION The biogenic amine octopamine (0A) exhibits a widespread distribution in the insect nervous system and is found throughout the animal kingdom. This phenolic analog of norepinephrine (NE) was first discovered in 1951 by Erspamer and Boretti in the posterior salivary glands of the octopus, QQLQPJE vulgaris, hence receiving its name. Interestingly, however, 0A had been synthesized some fOrty years prior to this by Dale in 1910 (Evans, 1985). Octopamine is a monohydroxy analog of NE, lacking the meta- hydroxyl group (Figure 1). In the vertebrate nervous system, which contains substantially more NE than 0A, it has been very difficult to distinguish effects mediated by 0A through its own receptors from those mediated through noradrenergic interactions (Evans, 1985). In many invertebrates, however, the virtual absence of NE allows one to study the specific actions of 0A in isolation. Prior to 1969, 0A was referred to as a "false transmitter”, suggesting that it could be taken up, stored and released from catecholaminergic terminals (Robertson, 1981). The availability of a sensitive and specific radiochemical-enzymatic assay, along with HPLC-EC procedures has further helped to assign a putative transmitter role to 0A (Molinoff g; 31., 1969; Downer g; a1., 1985). In invertebrates, a single function cannot be ascribed to 0A. It is well known that 0A subserves a variety of OH H OH H N OH N \\H \\H OH ' 0H OCTOPAMINE NOREPINEPHRINE Figure 1. Chemical structures of Octopamine and Norepinephrine. 3 roles which include its actions as a neuromodulator, neurohormone, and neurotransmitter (Orchard, 1982). Octopamine's numerous physiological functions and its release into the circulatory system during excitation (Goosey and Candy, 1982; Bailey gt al., 1984) indicate that in insects, 0A may be the functional equivalent of the vertebrate fight-or-flight hormones, epinephrine and NE. Localization Octopamine is found in high concentrations in many invertebrate nervous systems (David, 1984) as well as non- nervous and neurohemal tissues such as the corpora cardiaca of locust and cockroach (David and Lafon-Cazal, 1979; Goosey and Candy, 1982). It has been suggested that D(-)-OA is the naturally occurring isomer in insect hemolymph (Goosey and Candy, 1980) and nervous tissue (Staratt and Bodnaryk, 1981). Octopamine is also found in very high concentrations in the light organs of fireflies (Robertson and Carlson, 1976). The optic lobes of insects also tend to contain large amounts of 0A (Evans, 1980). Although 0A measurements have only been performed in a limited number of insect species, the distribution patterns appear to be fairly similar. Attempts at developing a specific OA-antibody have had limited success (Konings gt g1., 1988) and therefore OA- containing cells are primarily identified by their lack of fluorescence with the Falck-Hillarp histochemical procedure and positive staining with neutral red dye (Evans, 1985). 4 Octopamine has been found in brain and sympathetically innervated tissues of every vertebrate examined (Molinoff and Axelrod, 1972); however, it is present in very small quantities [e.g. 1601 ng/g tissue in cockroach brain vs. 4.7 ng/g in rat brain (David and Coulon, 1985)]. The ratio of 0A and NE has been studied during fetal development in rats and its functional significance has been a center of heavy debate (Saavedra gt; g)“, 1974; David, 1984). It is suggested that 0A is a metabolic by-product found in noradrenergic neurons. In fact, any treatment which decreases NE levels, affects 0A in a similar fashion (Molinoff and Axelrod, 1972). Recently, electrolytic lesioning in the locus coeruleus of rats has been unable to resolve whether or not 0A is a "false transmitter" in vertebrates (Hicks gt gl., 1987). Hence, at present, there is no direct evidence for any specific octopaminergic neuronal system in vertebrates. Neurochemistry The biosynthetic pathways of 0A have been studied primarily by the use of radioactive precursors and specific blockers. Studies using [3H]tyrosine suggest a biosynthetic scheme consistent with the conversion of tyrosine to tyramine, which is then converted to 0A. In the arthropods and crustaceans, two main enzymes are proposed to be involved in the synthesis of 0A (David and Coulon, 1985): tyrosine decarboxylase and tyramine B-hydroxylase which 5 catalyze the conversion of tyrosine to tyramine and tyramine to 0A, respectively. In vertebrates, the synthesis of 0A requires an intact sympathetic system and it has been suggested that it may follow a scheme similar to that seen in invertebrates (David, 1984). Inhibition of dopamine B- hydroxylase by FLA-63 (Brandau and Axelrod, 1972) decreases 0A levels in the vertebrate brain, and the 0A precursor, tyramine can be detected in mammalian and other vertebrate brains (Boulton, 1973). Studies also suggest a high rate of turnover of 0A in vertebrates; this is evident from the fact that the levels of the 0A metabolite, para-hydroxymandelic acid in the urine are as high as the levels of 5- hydroxyindole acetic acid and homovanillic acid, the metabolites of 5-hydroxytryptamine (5-HT) and NE and epinephrine, respectively (Kakimoto and Armstrong, 1981). In order for CA to function as an effective chemical messenger in insects, it must be inactivated both in the circulating hemolymph and locally within the tissues. This can be accomplished in two ways; either by a high-affinity uptake mechanism or by enzymatic inactivation, and both these mechanisms have been demonstrated in invertebrates (Robertson, 1981; Wierenga and Hollingworth, 1990). The major means of inactivation of 0A in the insect is via the enzyme N-acetyltransferase (NAT) which is found in high levels, whereas monoamine oxidase (MAO) which is primarily responsible for the inactivation of NE in vertebrates is 6 found in very small quantities, and its role is as yet unresolved (Evans, 1984). In insects, studies on the mode of 0A inactivation are complicated by the presence of high concentrations of metabolites of biogenic amines used in cuticular tanning. Also, since insect nervous systems are surrounded by a sheath of specialized glial cells, it is not clear if the metabolism of CA by isolated intact nerve cords, is due to a peripheral non—neural enzymatic barrier, or to an inactivation mechanism within the nervous system itself (Evans, 1985). Insect neural tissues also contain a high-affinity uptake mechanism for CA (Evans, 1978) which is specific for B-hydroxylated compounds and has a greater affinity for phenolic amines than catecholamines. In cockroach nerve cord, a high-affinity sodium-dependent and sodium- insensitive components and a low-affinity sodium-dependent uptake mechanism for CA have been demonstrated. Evans (1980) has suggested that the high-affinity mechanisms may be involved in the uptake of amines released by synaptic activity and the low-affinity mechanism for the uptake of amines in non-nervous tissue such as glial cells. Although 0A uptake has not been studied for the meta- and ortho- isomers, the D(-)-0A isomer exhibits greater affinity for this site (Evans, 1978). In 1972 and 1973, Robertson and Steele reported that 7 tyramine, a potent inhibitor of OA-uptake, stimulated glycogen phosphorylase activity in intact insect nerve cord. They suggested that tyramine, by inhibiting 0A uptake, increased the concentrations of endogenous CA at receptor sites in the tissue, leading to activation of glycogen phosphorylase. In vertebrates, the uptake of 0A is similar to that of NE and takes place at the presynaptic membrane. This uptake is temperature sensitive and has a requirement for sodium; the uptake results in the accumulation of 0A in catecholaminergic neurons (Baldessarini and Vogt, 1971). Octopamine in Vertebrates Although 0A is found in many vertebrate tissues, including heart, spleen, liver, brain and adrenals (Ibrahim gt 11., 1985), it appears that mammals are the only vertebrates studied in this field and no OSAC or OA-mediated protein phosphorylation has been described (Huang and Daly, 1972; Nathanson, 1977). However, Nathanson (1976) did find 0A to be effective in stimulating a non-NE-sensitive AC in rat pineal gland. As mentioned earlier, Hicks gt g_. (1987) recently studied this theory by measuring the effects of electrolytic lesions of the locus coeruleus on endogenous 0A and NE levels in rat brains by testing at post-lesion intervals of 4 to 18 days. This experimental paradigm was used because neurochemical lesions of central catecholaminergic systems with 6-hydroxydopamine or 8 reserpine deplete 0A and related trace amines as well as NE and dopamine. Also, inhibition of MAC or pretreatment with p-chlor-ophenylalanine, antidepressants, or with common precursors, elevate levels of CA as well as NE, thus making it difficult to test the "false transmitter" hypothesis. These researchers found that in the anterior cortex, posterior cortex and striatum the rates of depletion of NE mirrored those of 0A. In the cerebellum, hypothalamus, hippocampus and pons/medulla, they found that the levels of 0A diminished later than those of NE, with an initial increase within the first week post-lesion. In the midbrain, however, they found that the pattern was reversed, i.e., DA levels initially decreased, parallel with NE, but eventually recovered to significantly higher values. Therefore, there is no unequivocal support for the "false transmitter" hypothesis, however, neither is there clear evidence to support earlier contentions that 0A is likely to play a role in central neurons as a synaptic transmitter, independent of the noradrenergic system. In dogs and cats, CA has been shown to have sympathomimetic effects on blood pressure and heart rate, with D(-)-0A being the more effective isomer (Korol gt gl., 1968), and 0A administration has been shown to result in positive chronotropic and inotropic responses (Chiba, 1976 in David and Coulon, 1985); however, the cardiovascular effects of 0A are less potent than those of NE. Injection 9 of p-OA into the lateral ventricles of rats, depending on the dose, can cause a transient increase or decrease in blood pressure (Delbarre gt g_., 1980 in David and Coulon, 1985) . In spontaneously hypertensive rats (SHR Kyoto), the levels of OA are elevated (David, 1978 in David and Coulon, 1985) and the levels increase with age and appear to be correlated to blood pressure. It has been proposed by Fischer and Baldessarini (1971) that the neurological and cardiovascular complications of hepatic coma might be related to high OA levels since there appears to be a correlation between 0A levels in serum, urine or brain, and the degree of encephalopathy or hepatic coma. David and Coulon (1985) do suggest, however, that the role of 0A in renal dysfunction remains unclear since during dialysis OA levels increase. They feel that this increase may be due to the stress induced by dialysis. In patients with Reye’s syndrome, Lloyd gt :1. (1977) found a post- mortem decrease of NE and a 700% increase of 0A. A deficiency in OA has been observed in depressive patients and CA has been shown to act as a lipolytic factor in rats (David and Coulon, 1985). Other effects of 0A in vertebrates include some antidepressant activities (David and Coulon, 1985) and a production of hypothermia which appears to work through cholinergic mechanisms. It has been suggested that 0A is an antagonist of induced hyperthermia and via these actions 10 acts as an antagonist of the central serotoninergic system (Jagiello-Wojtowicz gt g;., 1982 in David and Coulon, 1985). Recently it has been suggested that intravenous injection of the formamidine insecticides (which act via the OA receptor in invertebrates) into rats produces dose- dependent mydriasis and bradycardia (R.M. Hollingworth, personal communication). These effects were blocked by the xz-adrenergic antagonist idazoxan but not by the «1- adrenergic antagonist prozosin, suggesting that these effects were mediated by dz-adrenoceptors. A similar conclusion was presented by Costa gt _l. (1987, 1988) who found that in mouse brain, formamidines inhibit the binding of [3H]clonidine to 4’2-adrenoceptors and [3H]WB4101 to 0(1- adrenoceptors, with respective IC50 values of 18.2 and 87 uM. These insecticides exhibited weak activities at other receptor sites (e.g. B-adrenergic, GABAA, benzodiazepine and muscarinic). Again, these data support the belief that the formamidines (and CA by inference) may interact directly in yittg with “Fadrenergic receptors, suggesting that in yiyg, OA may mediate its actions via these receptors. Raffa gt gt. (1989) have shown that administration of p-OA by intracerebroventricular or intrathecal routes, but not orally, produced antinociception in acetylcholine- induced abdominal constriction. This effect was abolished by reserpine or pretreatment with the 4¥adrenergic antagonist phentolamine. Naloxone did not block these 11 effects. Similarly, the potent 0A agonist, XAMI, has been shown to be very effective in this test as well as in a 48°C hot-plate test and as with GA, this effect is blocked by phentolamine (R.B. Raffa, personal communication). It is possible that OA is producing analgesia via non-naloxone sensitive mechanisms which may involve imidazoline- preferring receptors and/or ahadrenoceptors involved in analgesia. Regardless of the exact mechanism of action, one cannot escape the conclusion that OA does have certain significant activities in vertebrates and the development of high-affinity OA-specific ligands in invertebrates will help answer some of these questions in vertebrates. Physiological Role of Octopamine in Insects Evidence suggests that OA functions as a neuromodulator, neurotransmitter and neurohormone in insects (Orchard, 1982) . However, it should be noted that these three terms designate different regions in a continuous spectrum of modes of intercellular chemical communication. Octopamine as a Neuromodulator in Insects Octopamine is a well established modulator of neuromuscular transmission and contraction of insect skeletal muscle (Evans, 1982). The only detailed study of an identified octopaminergic neuron has been on the DUMETi (dorsal unpaired median cell) neuron innervating the extensor tibiae muscle of the hind leg of locusts (Hoyle gt 12 11., 1974). Administration of OA mimics the actions of DUMETi in a dose-dependent fashion. Electrical activity of DUMETi also has a modulatory effect upon the action of the slow excitatory motorneuron (SETi) (O'Shea and Evans, 1979) and this action is mimicked in a dose-dependent fashion by the application of OA. DUMETi does not form synapses with the muscle fibers, but simply releases 0A within the vicinity of the muscle and the SETi neuromuscular junctions via "blind" neurosecretory terminals (Orchard, 1981). Presumably, 0A diffuses from these terminals to the site of action where it modulates the amount of transmitter released from SETi by an action on presynaptic receptors, thus acting as a neuromodulator (Evans, 1985). Another example is seen in the lateral oviduct of the locust, Locustg mistatgtig, which is innervated by octopaminergic neurons that modulate muscle activity (Orchard and Lange, 1986). Octopamine as a Neurotransmitter in Insects In the firefly light organ (Nathanson, 1979) and in the glandular lobe of the corpus cardiacum of the locust (Orchard and Loughton, 1981) and cockroach (Downer gt gl., 1984) , there is strong evidence that 0A is performing a neurotransmitter role. In addition, Nathanson and Greengard (1973) have reported the existence of a specific OA- sensitive adenylate cyclase (OSAC) in insect neural tissue, leading to a variety of reports which implicate OA as a central neurotransmitter. 13 In insects, a large number of neurosecretory cells have their cell bodies within the central nervous system (CNS). These cells are typically unipolar and can release their products directly within the parent ganglion, or may liberate neurohormones within the CNS via distal processes which extend throughout the ventral nerve cord. Alternatively, their axons may run outside the CNS, along peripheral nerves and liberate neurohormones directly into the hemolymph (Osborne, 1986). This release is believed to occur in a diffuse way from individual axons as they course along the nerves or, in a coordinated manner from specialized neuroendocrine (neurohemal) organs such as the corpora cardiaca, corpora allata and perisympathetic organs. Some of these neurosecretory cells contain OA and have their cell bodies in the pars intercerebralis of the brain from where axons travel to the corpus cardiacum and presumably release OA from this location and/or control the release of neurohormones (Orchard, 1982). The glandular cells of the locust corpus cardiacum release hyperlipemic hormones such as adipokinetic hormone (AKH). Orchard and Loughton (1981) found that AKH is released upon electrical stimulation of the nervus corpus cardiacum II (NCCII) and this effect can be blocked by the octopaminergic antagonist, phentolamine. Hence, on the basis of the presence of OA, along with the fact that NCCII axons take up tritiated amines without showing fluorescence, 14 these researchers suggested that 0A could be acting as a transmitter regulating the release of hyperlipemic hormone. Recently, OSAC has been demonstrated in short-term cultures of corpora allata from locusts (Lafon-Cazal and Baehr, 1988), and CA has been shown to be the transmitter responsible for the release of juvenile hormone III (Lafon- Cazal and Baehr, 1988). It has been speculated that OA may in fact be involved in the release of other hormones from insect neurohemal organs (Downer gt 91., 1984). Octopamine as a Neurohormone in Insects As mentioned previously, CA has been shown to be present in locust and cockroach hemolymph (David and Lafon- Cazal, 1979; Goosey and Candy, 1980; Bailey gt 1.1., 1983). These observations along with increases in OA titre associated with physiological events clearly show GA to be a neurohormone released during stress and flight (Orchard gt 31., 1981). An excitation-induced hypertrehalosemic (EXIT) response was demonstrated by Downer (1979) and.OA was proposed as the probable mediator of this response. 0A was shown to rapidly increase upon excitation (Bailey 3 11., 1983) and enhance the production of trehalose from glycogen in isolated fat body via a cyclic AMP-mediated process (Cole and Downer, 1979). It has also been shown that increased hemolymph levels of OA can activate muscle trehalase (Jahagirdar gt g” 1984). Other neurohormonal functions may include the ability of 0A to 15 increase the activity of fat body glycogen phosphorylase, to stimulate glycogenolysis in the cockroach nerve cord (Robertson and Steele, 1973) and increase the rate of glucose oxidation in locust flight muscle (Candy, (1978). Octopamine has behavioral effects in insects and has been implicated in the control of activity patterns (Fuzeau- Braesch gt 11., 1979; David and Verron, 1982), feeding behavior (Hollingworth and Johnstone, 1982; Mercer and Enzel, 1982; Davenport and Evans, 1984) and olfactory responses (Sombati and Hoyle, 1984). Overall, much like the actions of NE and epinephrine (sympathetic activation) in vertebrates, the majority of the effects of OA seem to bring about a change from a passive to a more active state in the insect. Similarly, the formamidine pesticides, a class of agricultural chemicals that exhibit potent acaricidal (mite and tick insecticides) activities act through 0A receptors in target insects (Beeman, 1982) causing behavioral changes sufficient to kill the insect or lower its reproductive capacity by interfering with mating behavior, hatching of eggs, feeding and/or host-insect interactions (Hollingworth and Lnnd, 1983). Generally, these compounds act as partial rather than full agonists of OA-mediated events, however in some tissues they may act as full agonists (Hollingworth and Johnstone, 1982). The insecticidal actions of formamidines clearly demonstrate the importance of 0A in coordinating 16 behaviors in the insect and the potential for exploring this receptor as a target for future insecticides. Octopamine-Receptor Pharmacology and Mode of Action In invertebrates, the primary mode of action of OA involves the receptor-mediated activation of adenylate cyclase (AC) (Uzzan and Dudai, 1982). However, this does not rule out the possibility of other second messenger systems being coupled to 0A receptors. Inhibition of OA- sensitive AC has been very difficult to study but at this time it cannot be ruled out as another possible second messenger system (G.L. Orr, personal communication; unpublished data). Recently, Arakawa gt g1. (1990) reported that they had cloned an OA-l-type receptor isolated from finggamggilg ‘which attenuated..AC activity. However, this study has been strongly criticized and work by Saudou gt :1. (1990) claims this to be a tyramine and not an 0A receptor. I have demonstrated recently that OA and its most potent agonist XAMI (Orr gt g;., in press) are both able to enhance inositol 1,4,5-trisphosphate (1P3) production in cockroach brain (N. Orr and G.L. Orr, unpublished observation). There is also mounting evidence to suggest that OA may result in increased intracellular calcium in insect preparations (Evans, 1981; Jahagirdar gt g1., 1987; J.E. Steele, personal communication). The possibility of a variety of second messenger systems should not come as a surprise, considering the widespread distribution and undeniable importance of 17 this biogenic amine in insects. Generally, 0A receptors exhibit greater affinity for monophenolic amines with a single hydroxyl group on the aromatic ring (Evans, 1980), whereas catecholamine receptors have a higher affinity for amines with two hydroxyls on the benzene ring. Evans (1981) proposed the only pharmacological classification of 0A receptors based on studies of the DUMETi neuron (known to contain and release OA) of locust (Table 1). Type 1 (GA-1) receptors are involved in the slowing of myogenic rhythm in the extensor tibiae muscle of this preparation, whereas Type 2A (OA-2A) receptors increase the slow excitatory motorneuron (SETi)- mediated twitch amplitude and Type 28 (OA-ZB) increase the relaxation rate of SETi-induced tension. Type 2A and 28 receptors are believed to mediate their responses via an OA- sensitive AC, whereas OA-1 receptors are believed to be coupled to calcium-gating. The application of this classification to other systems has been hampered by a lack of consistency in the use of pharmacological agents and the problem of comparing the pharmacology of physiological and biochemical events. The pharmacological properties of the 0A receptor have been studied in a variety of preparations (Evans, 1980) principally by monitoring the effects of potential agonists and antagonists on OA-mediated electrophysiological responses (Orchard and Lange, 1986), or OA-mediated 18 Table 1. Proposed classification of octopamine receptors from locust extensor-tibiae muscle (Evans, 1981). Receptor Classification Based On: Type Physiology Bharmasologx l Slowing of myogenic Antagonists: rhythm. chlorpromazine,yohimbine >> metoclopramide Agonists: clonidine > naphazoline 2A Increase in SETi Antagonists: amplitude. i) metoclopramide >> chlorpromazine, yohimbine ii) cyproheptadine, mians- erin, metoclopramide better in 2A than 28. Agonists: i) naphazoline > clonidine ii) naphazoline >> tolazol- ine 28 Increase in SETi Antagonists: relaxation. i) metoclopramide >> chlorpromazine, yohimbine ii) chlorpromazine better in 28 than 2A Agonists: i) naphazoline > clonidine ii) tolazoline >> clonidine 19 activation of AC (Sullivan and Barker, 1975; Gole gt g1 ., 1979; Orr g g;., 1985) . A general characteristic of all 0A receptors is that they are stereospecific for the D(-) isomer of OA (Roberts and Walker, 1981). In the majority of preparations studied, OA and its N-methylated analog, synephrine (which is found in vertebrates but not in invertebrates) are potent agonists of the OA receptor; these include the firefly light organ (Nathanson, 1979), the locust extensor tibiae (Evans, 1981) and cockroach brain homogenates (Harmer and Horn, 1977) . The relative potency of OA and synephrine, however, varies with the preparation (Evans, 1985). Another potent class of OA-agonists is the phenyliminoimidazolines (PII's) (Hollingworth and Lund, 1983; Nathanson, 1985a; 1985b). These compounds are clonidine analogs and have been used to study structure- activity relationships in firefly, cockroach and tobacco hornworm (Nathanson, 1985b; Orr e_t _;., 1989; Orr gt gt” in press); they have been shown to act as full or partial agonists, and to be up to 20 fold more potent than OA (Nathanson, 1985a) . The most potent PII, XAMI, has been shown to have a Ka at least 200 times better than that of OA itself (Orr gt g1., 1989; Orr gt g)", in press). Receptor binding studies of the 0A receptor in insects have been limited by the requirement for large amounts of tissue acquired through tedious dissection. However, there have been some studies using [3H]OA to label 0A receptors 20 (Dudai and 2vi, 1984; Hashemzadeh gt gl., 1985; Konings gt g1., 1989). As would be expected when using a natural ligand, these have been difficult to perform because of the high levels of non-specific binding, problems with metabolism and inactivation of CA as well the fact that the [3H]OA consisted of the racemic mix (i.e. D- and L-isomer). In order to circumvent some of these problems, binding studies have been performed in cockroach nerve cord and brain using the most potent antagonist of OA-sensitive AC [3H]mianserin (Minhas gt a_l., 1987; Orr e_t_ gt” in press). Although this has provided a relatively simple assay for screening the octopaminergic potential of various compounds, the very high non-specific binding (up to 50%) is problematical. Subsequent to these studies, Nathanson (1989) has used the photoaffinity analog of another PII [3H]NCSZ to study OA-receptors in firefly light organ but this ligand also exhibits high levels of non-specific binding. In general, OA receptors show some similarity to vertebrate «Ladrenergic receptors in that compounds such as phentolamine are potent antagonists of this site (Evans, 1980). 0A receptors are not affected by B-adrenergic compounds such as propranolol, although it is the 8- adrenergic receptors in vertebrates that mediate their actions via activation of AC rather than d-adrenergic receptors. Also, other compounds such as cyproheptadine, 21 gramine and mianserin which are 5-HT and histamine antagonists in vertebrates, are very potent OA antagonists (Gole gt gl., 1983; Downer gt g1., 1985; Orr gt g1., 1985), suggesting that based on the antagonist data, 0A receptors are unlike any vertebrate biogenic amine receptor. Recently, the az-adrenergic receptor antagonist, idazoxan, has been shown to be an effective blocker of the AC coupled OA receptor (Orr gt g;., 1989) whereas the aQ-antagonists, yohimbine and rauwolscine are ineffective (Orr gt gl., 1990). It is interesting to note that like other vertebrate receptors, 0A receptors are found both pre- and postsynaptically (David and Coulon, 1985) and appear to exhibit not only interspecies but also intertissue differences (Orr gt .11., in press) making classification based on a single-species study very difficult. It is interesting to speculate whether the 0A receptor in invertebrates is an ancestral d—adrenergic/serotoninergic receptor and/or whether it is in some way related to the imidazoline preferring sites that have been recently described in vertebrates (Bricca gt g;., 1989). The Current Study It is evident from the above account that several major gaps exist in our understanding of the pharmacology of insect octopamine receptors. The current investigation has used the American cockroach, Egtiplangtg gmerigan L., and the Sf9 (Spodoptera ftugiperga) cell line as model systems 22 for studying a number of these topics with particular emphasis on the following: i) The identification and characterization of a novel, high-affinity agonist for OSAC in cockroach nerve cord, hemocytes, brain and tobacco hornworm nerve cord. ii) The identification and characterization of an insect cell line to serve as a model system for studying the OA-receptor. Pharmacological characterization of this Sf9 OA-receptor and comparison with the cockroach nerve cord OA- receptor. iii) Investigation of the existence of an inositol- phosphate-coupled OA-receptor in cockroach brain slices and intact Sf9 cells. CHAPTER 1 Characterization of a Potent Agonist of the Insect Octopamine-Receptor-Coupled Adenylate Cyclase 23 24 INTRODUCTION Octopamine is a major biogenic amine found in high concentrations in invertebrates (Molinoff and Axelrod, 1972; Axelrod and Saavedra, 1977). In insects, this monohydroxyphenolic analog of noradrenaline has been shown to function as a neurotransmitter (Evans, 1978; Nathanson, 1979; Downer gt L., 1984), neuromodulator (Orchard and Lange, 1985; Klaasen and Kammer, 1985) and neurohormone (Robertson and Steele, 1972; Gole and Downer, 1979; Evans, 1984). Many of the physiological functions of 0A are mediated by the activation of an octopamine-sensitive AC (Nathanson, 1979; Orr e_t gt” 1985). Data compiled on the pharmacology of the 0A receptor indicate that it does not conform to the receptor categories that have thus far been recognized in vertebrates but may have certain similarities with az-adrenergic receptors (Harmer and Horn, 1977; Gole gt 11., 1983; Minhas gt g;., 1987). Radioligand binding studies of this receptor have been restricted to the use of [3H]OA (Dudai, 1982; Hashemzadeh gt £11., 1985; Konings gt 1L, 1989), the high-affinity antagonist [3H]mianserin (Minhas gt g)“, 1987; Orr gt 1L, in press) and most recently, [3H]NCSZ (Nathanson, 1989). All three of these ligands exhibit high levels of non-specific binding. The identification of other high-affinity probes is essential for the pharmacological characterization of this site(s). Furthermore, certain compounds with high affinity for the 25 OA-receptor (e.g. formamidines and imidazolines) have pesticidal activity (Hollingworth and Murdock, 1980; Nathanson and Hunnicutt, 1981; Hollingworth and Johnstone, 1983 ; Nathanson, 1985) and the development of additional high-affinity ligands could aid in the discovery of novel pesticides. In the present study, the imidazoline 2,3- xylylaminomethyl-2’-imidazoline (XAMI) which has previously been shown to have strong octopamine-like actions .111. 11192 was evaluated for its effects on OSAC in neural and non- neural tissues in the American cockroach, Petiplanetg gmgtiggng L. and the tobacco hornworm, Magducg sexta. This pharmacological characterization indicates that in the cockroach, XAMI has the highest reported affinity of any OA- receptor agonist and suggests that XAMI and related high- affinity analogues may serve as good ligands for studying the 0A receptor. In addition, this compound could provide the basis for development of compounds having pesticidal activity due to their action at the 0A receptor. MATERIALS AND METHODS Insects Adult males between 1 and 3 months after the final molt were taken from a stock colony of American cockroaches, W gmeticana L., maintained at the Pesticide Research Center, Michigan State University. The insects 26 were fed, gg lititgm, a diet of Purina Dog Chow° and water. The insects were kept in plastic garbage bins with adequate ventilation and with sheets of corrugated cardboard to provide running space and concealment. The temperature in the insectary was maintained at 28°C with a relative humidity of 50% and a 12L/12D photoperiod. Tobacco hornworms (nanduga sexta) were purchased from Carolina Biological Supply Co. (Gladstone, OR) either as ISt instar larvae or eggs and reared on an artificial medium (Bell and Joachim, 1976) under standard conditions in the above mentioned insectary. Larvae were used at either the 4th late 3rd or early instar. Tissue Preparation Preparatory Period Prior to dissection, cockroaches were held without food, in individual petri dishes, for 4-24 hours. Adequate water was placed in each petri dish. This isolation period ensured that the insect was in a post-absorptive and non- or minimally excited state (Downer, 1979). In the case of the tobacco hornworm, the insects were removed from the main colony and placed in a glass beaker no longer than 1 hour prior to dissection. The beaker was then placed in the refrigerator for 10 to 20 min, depending on the number of hornworms per beaker. This anesthetized the insects for dissection and they remained in this state as 27 long as the beaker was placed on ice and a 4°C temperature was maintained. Dissection of Cockroach Nerve Cords Following the removal of legs and wings, the insect was pinned, dorsal side up, on a wax dissecting dish containing no more than 2 to 3 ml of ice-cold insect saline (0.17 M NaCl, 6.0 mM KCl, 2.0 mM NaHCO3, 17.0 mM glucose, 6.0 mM NaH2P04.H20, 2.0 mM CaC12.2H20, 4.0 mM MgC12.6H20), pH 7.0. The cuticle was cut longitudinally and pinned back to expose the internal organs. The gut was carefully removed or pushed aside; care was taken not to let any of the contents leak into the hemocoel. After removal of fat body, the entire nerve cord (including the thoracic and abdominal ganglia) was removed. Care was taken to avoid contamination of nerve cord tissues with tracheae during dissection. The nerve cords were rinsed and stored in fresh insect saline and placed in 3 ml test tubes, which were kept on ice. Generally, the nerve cords were maintained in this manner for no longer than 30 min prior to homogenization. Dissection of Hornworm Nerve Cords An incision was first made in the caudal region of the hornworm larvae allowing drainage of hemolymph. The insect was then pinned, dorsal side up, on a wax dish and was dissected, rinsed and stored in a manner similar to that 28 described for the cockroach nerve cord. Dissection of Cockroach Brain The head was removed and pinned, ventral side up, in a wax dish containing ice-cold insect saline. A longitudinal cut was made in the head capsule from the rostral to the caudal end of the head. Two perpendicular cuts, one to the right and one to the left of the medial cut, were made and the cuticle was pinned back to expose the two cerebral lobes. Any adhering tissues were removed and the optic lobes were severed. The brains were gently removed, being careful not to include the underlying corpus cardiacum. The brains were rinsed and stored as described above. Hemocyte Collection Hemocytes were obtained in a manner similar to that described by Orr and Hollingworth (1990). Insects were slowly injected with 2.0 ml Hoyle’s insect saline containing 10.0 mM ethylenediamine tetraacetic acid (EDTA) and 5.0 mM d-l,4-dithio-L-threitol (DTT) while the diluted hemolymph was drained into an ice-cooled centrifuge tube through the wound formed by excision of two metathoracic legs. The sample was centrifuged at 900 g for 10 min at 4°C and the supernatant discarded. The resultant hemocyte pellet was gently resuspended in Hoyle's insect saline. Microscopic examination of this suspension showed only 29 intact hemocytes to be present. Cockroach and Hornworm Nerve Cord Homogenate Preparation Nerve cords were removed from the insect saline and transferred to ice-cold Tris—EGTA [ethyleneglycol-bis- (B-aminoethyl ether) N,N’-tetraacetic acid] buffer (6.0 mM Tris, 2 mM EGTA), pH 7.2 and homogenized using a glass- teflon homogenizer (10 to 15 strokes at 400 rpm) and the resulting homogenate was appropriately diluted and used for determination of cyclic AMP (cAMP) content. Cockroach Brain and Nerve Cord Membrane Preparation Brains and nerve cords were removed from insect saline and transferred to ice-cold Tris-EDTA/EGTA/DTT buffer (10.0 mM Tris, 1.0 mM EDTA, 2.0 mM EGTA, 1.0 mM DTT) pH 7.2 and homogenized using a glass-teflon homogenizer (15 strokes at 400 rpm). The resulting homogenate was centrifuged .at 27,000 g for 15 min at 4°C and the pellet was resuspended in a 10.0 mM Tris buffer containing 1 mM DTT (pH 7.2). The tissue suspension was centrifuged at 27,000 g for 15 min at 4°C and resuspended in an appropriate volume of Tris-DTT buffer for determination of cAMP. Analytical Procedures Chemicals XAMI was synthesized according to the method of Kristinsson and Traber (1981) and N-demethylchlordimeform 30 (DCDM) was synthesized by the method of Hollingworth (1976). NC5 was synthesized via the N-aryl-S-methylthiuronium intermediate (Timmermans gt gt” 1978) and recrystallized from aqueous ethanol. All three compounds were tested for purity’ using' thin-layer' chromatography; melting-point determinations, nuclear magnetic resonance and mass spectrometry. The last two procedures were performed at the NMR facility of the Department of Biochemistry and the Mass Spectrometry facility of the Department of Chemistry, Michigan State University, MI. The following compounds were kindly provided by the respective companies: idazoxan (Reckitt and Colman, Kingston-upon-Hull, England), phentolamine (CJBA-GEIGY Corp., Summit, NJ), and spiperone (Janssen, Belgium). Mianserin and (+)-butaclamol were purchased from Research Biochemicals Inc., Natick, MA. All other drugs and chemicals were purchased from Sigma Chemical Co., St. Louis, MO. Protein Assay Protein concentration was estimated using the method of Lowry gt gt. (1951) with bovine serum albumin as the protein standard. Aliquots of the various reconstituted membrane and homogenate preparations used for cAMP determinations were assayed for protein using this procedure. In the case of whole haemocytes, a homogenized sample was assayed. Optical density was determined at 750 31 nm using a Mbdel 340 spectrophotometer (Sequoia-Turner Corp., CA) and was found to increase in a linear fashion in the range of protein required for sample determination (r2=0.95-0.99). Cyclic AMP Radioimmunoassay Cyclic AMP measurements were performed using a commercially available radioimmunoassay (RIA) kit (non- acetylated) obtained from New England Nuclear Research Products, MA. This assay utilized the method of Steiner gt g1. (1972) with the modifications of Brooker gt Q}. (1979) and is based on the principle of competition between radioactive and non-radioactive antigen for a fixed number of antibody binding sites. The presence of non-radioactive antigen in the sample results in a decrease in the amount of radioactive antigen bound by the antibody. Therefore, the amount of radioactivity present in the antibody pellet is inversely proportional to the antigen content of the sample. The succinyl-tyrosine- (12 5I) -methyl ester of cAMP is used as the radioactive antigen and separation of bound antigen from free antigen is achieved by the use of a prereacted primary and secondary antibody complex. The primary antibody is generated in rabbits immunized with a succinyl-cyclic AMP-albumin complex and the secondary antibody is generated in sheep against rabbit globulin. Samples, appropriate blanks and standards were incubated in 12 X 75 mm glass test tubes and AC activity was 32 measured following incubation of 100 pl of tissue in a medium containing 75.0 mM Tris, 0.1 mM 3-isobutyl-1-methyl- xanthine (IBMX), 10.0 mM magnesium acetate, 0.05 mM GTP and 0.5 mM ATP in a final volume of 200 ul. The reaction was started by the addition of ATP and the incubation proceeded at 30°C in a shaking water bath. The reaction was terminated by boiling the samples for 3 min in a water bath. Following appropriate dilution (i.e., such that the majority of sample would be represented in the linear portion of the cAMP standard curve), 100 pl aliquots of samples were removed and placed in 12 x 75 mm glass test tubes. To these were then added either 100 pl of 125I-succinyl-cAMP (approximately 12,000 cpm) or 100 pl of 125I-succinyl-—cAMP and 100 ul of cAMP antiserum to a final volume of 300‘pl. The samples were mixed by shaking vigorously by hand (1 to 2 min), covered and stored at 4°C for 16 to 18 hr. The reaction was terminated based on modifications developed by Dr. J.W.D. Gole (personal communication) by the addition of 0.5 ml sodium acetate buffer (0.05 M, pH 6.2) containing 5% polyethyleneglycol (PEG; 8000 MW); 2.0 ml of normal rabbit serum was included per 40 ml of the sodium acetate/PEG buffer. The samples were immediately centrifuged at 3900 g in. a swinging-bucket rotor (4°C) for 20 min. The supernatant was aspirated and the radioactivity of the pellet determined using a gamma counter (Packard Multi-prias I, IL). 33 In earlier experiments, duplicate cAMP standard curves were generated, however, since these curves were reproducible (with an error of less than 5% of the mean), only single replicates of the curve were generated for all future experiments. A new standard curve (0.01 to 25.0 pmol) was generated for each experiment. Cyclic AMP standards were stored at 4°C and discarded after 4 to 7 days. A typical example of the average net counts per minute for each cAMP standard as a percent of the average net counts per minute of the zero cAMP blank (i.e., %B/B°), as calculated by the RIA program of the gamma counter, is shown in Figure 2. The compatibility of this assay for use with a variety of insect tissues (including neural tissues, haemocytes and insect cell lines) has been previously demonstrated by Gole (1984), showing greater than 98% recovery of cAMP. In those tissues, the samples exhibited both linearity and parallelism, indicating the lack of non-specific interfering compounds (Brooker gt g;., 1979). These studies also confirmed the high degree of specificity claimed for the cAMP-RIA by studying the degree of cross reaction by compounds structurally related to cAMP. The values obtained were similar to those reported by New England Nuclear Research Products, with cyclic GMP showing the greatest degree of cross-reactivity (<0.01% at the 50% 8/8 Based o)- 100- 80‘ 60- %B/Bo 40- 20- (101 OJ LO i101) V{00£f CYCLIC AMP (pmol) Figure. 2. Cyclic AMP standard curve (0.01 to 25.0 pmol) obtained with a non-acetylated, iodinated radioimmunoassay kit. Each point represents the mean of two determinations with SEM of less than 5%. 35 on these data, this assay is clearly suited for use with insect tissues. Production of cAMP by the tissue under investigation should be linear with respect to protein (Brooker gt g1., 1979). As shown in Figure 3, cAMP production in cockroach ventral nerve cord membranes was linear with respect to the sample protein concentration (4.8 to 53.0 pg). All subsequent experiments were performed using sample protein levels within the ranges indicated. Finally, if the optimal physical and biochemical environment is maintained and sufficient substrate is provided, cAMP production in a given tissue should be linear with respect to time for the length of the experiment (Brooker gt LL, 1979). Time course experiments were conducted for cockroach nerve cord membranes and as shown in Figure 4 were linear from 1 to 7 min for OA-stimulated and sodium fluoride-stimulated. production of cAMP. .All subsequent cockroach nerve cord and brain experiments were conducted with a reaction period of 5 min, or within the linear portions of the range. Cockroach hemocyte and hornworm nerve cord samples were incubated for 10 min. This incubation period was selected for the hemocytes based on the method of Orr and Hollingworth (1990). For the hornworm nerve cord a time course was not determined and 10 min was chosen as an appropriate incubation period. 600.0 . O CONTROL ff. 2 . O CTO PAM IN E . 9 500.0 « B e,» D A if g E 400 .0 - ,.- . 0. \ x". . 0. '6 300.0 « .. 2 E f" o V d ,3"... 3 200.0 >- .«O U 100.0 « , f. C) I“ C) -AEk-*{3"”‘€}T——_flfln{}~—- O .0 ’.......-:--tv, ---—w r Y x I 0 20 40 60 80 100 ' x PROTEIN CONCENTRA‘HON Figure 3. Cyclic AMP production as a function of the concentration of cockroach ventral nerve cord membranes for basal (r2=0.986) and 1 mM octopamine-mediated (r =0.988) stimulation. Values are the mean of 3 experiments with SEM of less than 10%. Values for 100% protein were in the range of 48 to 53 pg. 6i) OOCTOPAMINE .NOF 5.0« OJ 3 “A 4.0" 005 2.6 CL:> 31)- :0 <§ 0 :V 2.0- 0 )- 0 1 1.0- 000- r r I l T f r I 0 2 4 5 8 10 12 14 16 TIME (min) Figure 4. Time course of octopamine (1 mM) and sodium fluoride (10 mM) stimulated cyclic AMP production in cockroach ventral nerve cord membranes. Each point represents the mean of 3 experiments with SEM of less than 7%. 38 Pharmacological Procedures Determination of Vma and Ka Values X The relative maximum efficacy (Vm ) of an agonist ax was derived from dose-response curves and the activation constant (K3) was calculated as the concentration of agonist which was required to elicit half maximal response or (1/2 vmax’ ° Determination of IC50 Values The IC50 was defined as that concentration of an antagonist required to cause a 50% inhibition of agonist- mediated production of cAMP (in this study, XAMI-mediated production of cAMP). IC50 values were calculated from plots of percent inhibition of cAMP production caused by a fixed dose of the agonist (XAMI at 0.1 pH) with increasing doses of antagonists. Determination of Ki Values The Ki' or inhibition constant, refers to the equilibrium dissociation constant of the receptor-inhibitor complex, assuming competitive inhibition is occurring. The Ki (Cheng and Prusoff, 1973): Ki value can be calculated using the following equation = Icso/(1 + S/Ka), where S is the concentration of agonist used, Ka is the concentration causing half maximal cAMP production, and IC50 is calculated as previously mentioned. Additivity Studies 39 In order to determine if XAMI and other compounds that stimulate cAMP production interact with the same or different receptor types coupled to AC, additivity studies were performed. Compounds were applied separately at maximally stimulating concentrations (as determined by dose- response curves). The tissue was then challenged with various combinations of these compounds and cAMP production compared with the response elicited when these compounds were applied individually. If for example two compounds are interacting with separate receptor populations and AC is not limiting, then the cAMP produced by the combination of these two agents would be equal to the additive production caused by the drugs alone. If the combined effect is non-additive, then competition for the same receptor is implied. Reversibility Studies Cockroach nerve cord membranes were used to investigate the reversible nature of the XAMI-mediated production of cAMP. The tissue was divided into three groups: 1) untreated 2) control- or buffer—pretreated, and 3) XAMI- pretreated (1‘pM). Groups 2 and 3 were pretreated for 5 min at 30°C in a shaking water bath. The buffer- pretreated and XAMI-pretreated groups were diluted to four times the original volume with Tris-DTT (pH 7.2) and centrifuged at 27000 g (4°C) for 15 min. This washing step was repeated a total of three times. Finally, for all three groups, basal and 1 pM XAMI-mediated cAMP production was 40 determined. Statistical Procedures All descriptive statistics were calculated using a Texas Instrument TI-60 calculator. Group means were compared using a two-tailed t-test (Zar, 1974). Standard curves for the cAMP RIA and sample cAMP concentrations were calculated from the relative amounts of radioactivity present with the aid of a computer program associated with the gamma counter (Packard Multi-prias I, IL) . RESULTS Identification of a Novel Stimulator of Cockroach Neural Adenylate Cyclase As seen in Figure 5, three families of compounds have been recognized as having high affinity for the 0A receptor in insects: the phenylethylamines (e.g. 0A), the formamidines (e.g. DCDM) and the imidazolines (e.g. XAMI and NC5). The abilities of OA, NC5, DCDM and XAMI to increase cAMP production were tested in membrane preparations of cockroach ventral nerve cord (Figure 6). At a maximally stimulating concentration of 1 mM, OA, NC5, DCDM and XAMI respectively increased cAMP production by 5.4, 4.9, 3.0 and 4.3 fold over control. Control values for these experiments were 198.4 i 17.6 pmol/min/mg, however for this entire study, control values in cockroach nerve cord samples were 41 o H H j' N . l N\\\//ll\‘ N\\ N H \ H OH CH3 C143 OCTOPAMINE 2 , 3 -XYLYAMINOMETHYL-2 ’ -IMIDAZOLINE (OA) (XAMI) ? cuzcus H I N N §/ \c H 3 NY" H-N] C! 0113 ’ C H 2 C H 3 ‘ N-DEMETHYLCHIDRDIMEFORM 2- (2 , 6-DIETHYLPHENYLIMINO) IMIDAZOLIDINE (DCDM) (NC5) Figure 5 . Chemical structures of octopamine agonists used in the present study. 42 1 200 O XAMI - . NCS A 0A A DCDM CO 0 O 400 ‘ CYCLIC AMP INCREASE (pmoles/min/mg protein) o. n... M-.. e W. 2--.. 10-1010-9 10-3 10-7 10-5 10-5 10-4 10-3 CONCENTRATION (M) Figure 6. Effects of OA, NC5, DCDM and XAMI on cyclic AMP production in membrane preparations of cockroach nerve cords. All four agonists were tested on the same preparation (experiment) to standardize the response. Values are the mean i SEM of 5 separate experiments, performed in duplicate. Control values for cyclic AMP production 8 198.4 1 17.6 pmol/min/mg. 43 in the range of 78.2 to 216.0 pmol/min/mg. The respective Ra's for OA, NC5, DCDM and XAMI are 5.6, 0.7, 1.1 and 0.03 ‘pM. It is evident from the Vmax values, respectively 100, 90, 56 and 80% of control, that both XAMI and DCDM are partial agonists of this receptor. Although the degree of agonism in this tissue by NC5 varied from 87 to 100% of OA, it can be considered a full agonist. Characterization of the XAMI-Mediated Cyclic AMP Production in Cockroach Nerve Cord The GTP dependence of XAMI-mediated cAMP production was investigated (Figure 7), along with the effect of the diterpene forskolin, on this response. In the absence of GTP, there is negligible stimulation of cAMP production. The addition of 0.05 mM GTP significantly enhances XAMI- mediated production, as does forskolin at a threshold concentration of 0.01 M. In these two respective cases, maximal production represents a 4.5 and 5.3 fold (Vmax) increase over control. There was a significant difference (p<0.05) in this parameter between the two treatments. For this data, the Ka for XAMI in the presence of GTP is 0.032 ‘pM and in the presence of both GTP and forskolin, is 0.035 pH. Additivity studies were performed to determine whether XAMI was acting solely at the 0A receptor or whether it interacts with other amine receptors found coupled to AC in the cockroach nerve cord. As described in the previous 500 ' O +GTP 1 e -GTP 1. * A +GTP + Forsk A A 400 ~ . O 300 - ‘ ' 200 « 100‘ CYCLIC AMP INCREASE (pmoles/min/mg protein) O O A . . 04M- -1----.I, I--:,—- 10-9 10-8 10-7 10-5 10-5 10-4 XAMI CONCENTRATION (M) Figure 7. Effects of 0.05 mM GTP and 0.01 uM forskolin on XAMI-mediated cyclic AMP production in cockroach nerve cord membranes. Values are the mean 1 SEM of 3 separate experiments, performed in duplicate. Control cyclic AMP production in the presence of GTP 8 8 .5 i 5.3 and GTP + forskolin = 86.0 i 4.2 pmol/min/mg. Denotes points that are significantly different from the equivalent GTP-treated value (p<0.05). 45 section (Materials and Methods), each compound was used at the concentration required to elicit maximal production of cAMP (Table 2). Because of a variable response to S-HT in membrane preparations, nerve cord homogenates were used to check for interactions at this receptor. The data indicate that XAMI is nonadditive with octopaminergic agonists (i.e. OA and DCDM) but is additive with 5-HT and DA. The reversible nature of the XAMI-mediated production of cAMP was examined (Figure 8). As described previously (Materials and Methods), cockroach nerve cord membranes were assayed for cAMP production in the presence of 1 pM XAMI or were pre-exposed to buffer or 1 pM XAMI, and washed three times. In each of these three treatments, basal and XAMI- mediated cAMP production were determined and compared. Under all three conditions, no differences were observed in either the basal or the XAMI-stimulated cAMP production. For this effect to be reversible, however, a minimum of three washes were required. Antagonist Profile for the XAMI-Mediated Production of Cyclic AMP in Cockroach Nerve Cord The pharmacological nature of the XAMI-mediated enhancement of cAMP production was investigated by comparing the ability of various antagonists to inhibit this stimulation. Inhibition curves were generated for these antagonists using a fixed dose (0.1,pM) of XAMI (Figure 9). A minimum of eight concentrations were used to generate mucoefiuoexo ououoeom mIm no 2mm H some Ono ucomouoou mooam> I o aaououo mE\CHE\HOEQ H.o H o.aa I Houucou I n caououa me\cae\aoea m.o H N.ooH I Houucoo I m v.mm o.ma H m.moH exIm + Hz ASE a. Hzvm Zoom + «o b.@@@ b.mv H «.mmv zoom + H24x m.>mm «.mm H v.amw Hzmx + «o H.mss e.- H m.m~m so + so H.mNHH m.>v H m.HNm Hz¢x + «0 II m.m~ H «.mvm Axe Ho.ov zooo II «.mfl H m.H~m Axe Ho.o. stx II ~.vH H o.mba ASE a. «o II om.ma H m.mom Axe av 40 meseunsoz venues Ame\cfie\aoeev O>wufioom madam mum muoommm scauoocoum COADONMQONm ma ammouocH mz¢o Hmofioouoone mzco c“ camouocH useEumoue commas .Ouoo o>uoc nomouxooo cw cofiuooooum mZd OHHO>0 co moaooum >ua>aufioo< .N manna 47 [:1 Bosol XAMI 800 . O) O O CYCLIC AMP PRODUCTION (pmoles/min/mg protein) N O O _IW 7///////////////////////.~ V///////////////////////// -. 7/////////////////////// Untreated Buffer XAMI Pretreoted Pretreoted Figure 8. Reversibility of XAMI-mediated cyclic AMP production in membranes of cockroach nerve cord. Membranes were either (1) untreated and assayed for cyclic AMP production in the presence of 1 pM XAMI, or were pre-exposed to (2) buffer or (3) 1 pM XAMI and washed three times. Values indicate the mean :3; SEM of 3 to 4 separate experiments, assayed in duplicate. 120 0 MSN. T e PHENT. 1004 80- _ O 60- % XAMI STIMULATION 20- o ' "'"'I ' """'l ' "'""T Tfi'vrv'w j "'""r T f " ‘7 - 10-1010-910-810-7 10-510"5 10-4 10-3 ANTAGONIST CONCENTRATION (M) Figure 9. Effect of various antagonists on XAMI-mediated elevation of cyclic AMP in nerve cord membranes of mm W. XAMI was included at 0.1 pM. Values indicate the mean 1 SEM of 3 to 4 separate experiments, ' assayed in duplicate. 49 these curves, from which Ki values were derived (Table 3). These data indicate that mianserin was the best antagonist (Ki = 0.007 pM) followed by phentolamine, cyproheptadine and the potent dz-adrenergic antagonist idazoxan, which had Ki values in the 0.3 to 0.5 pM range. The XAMI-mediated response is not affected by dopaminergic antagonists such as (+)-butaclamol and spiperone (Ki's > 100 pM) or the 8- adrenergic antagonist propranolol (Ki > 100‘pM). Figure 10 indicates ‘that at a fixed concentration, cyproheptadine (1 pM), phentolamine (0.5 pM) and mianserin (0.01 pM), produced a rightward, approximately parallel shift in the XAMI dose-response curve (i.e. increasing the K for XAMI from 0.03 pM to 0.13, 0.10 and 2.80 pM, a respectively in the presence of cyproheptadine, phentolamine and mianserin). Preliminary data suggest that idazoxan had an effect similar to cyproheptadine and phentolamine (data not shown). Overall, although coincubation with these antagonists increased the Ka for XAMI, it had no effect on the maximal attainable response (i.e. V an effect max) ’ typically observed in cases of competitive inhibition. Tissue and Species-Specific Effects of XAMI The relative affinity of XAMI in other insect tissues was investigated using cockroach brain (Figure 11), cockroach hemocytes (Figure 12) and tobacco hornworm nerve cords (Figure 13). In all three systems, XAMI is a potent stimulator of cAMP production. Comparative potencies (Table 51 Table 3. Effects of various potential antagonists on XAMI- mediated elevation of cyclic AMP in nerve-cord membrane preparations of Periplaneta americana. Antagonist Kia Mianserin 7.1 x 10'9b Phentolamine 3.1 x 10'7 Cyproheptadine 5.3 x 10‘7 Idazoxan 4.6 x 10'7 Butaclamol >10‘4 Spiperone >10'4 Propranolol >10"4 a - Ki a IC50/[1 + (S/Ka)], where ICSO - concentration of antagonist giving 50% inhibition of XAMI-mediated cyclic AMP production. 8 8 concentration of XAMI used (0.1 pM). b - Values are the mean of at least 3 separate experiments. SEM was less than 15% of the mean. 51 Table 3. Effects of various potential antagonists on XAMI- mediated elevation of cyclic AMP in nerve-cord membrane preparations of Periplaneta americana. Antagonist Kia Mianserin 7.1 x 10'9b Phentolamine 3.1 x 10'7 Cyproheptadine 5.3 x 10'7 Idazoxan 4.6 x 10'7 Butaclamol >10'4 Spiperone >10’4 Propranolol >10'4 a - Ki = ICSO/[l + (S/Ka)], where ICSO a concentration of antagonist giving 50% inhibition of XAMI-mediated cyclic AMP production. S a concentration of XAMI used (0.1 UM). b - Values are the mean of at least 3 separate experiments. SEM was less than 15% of the mean. 1200 OXAMI A O XAMI + CYPRO. M 51000" AXAMl-I-PHENT. <2 g AXAMI+MSN g a 800- 22.” E’ g- E 600- < E \ 8 g; 400- 0 — ). O o E 3 200. O "’ ' ' ""'I ii""*“[ ' ' """l ' """T fi'wvfi"! 10-1010-9 10'8 10-7 10-6 10-5 10-4 ‘0'3 XAMI CONCENTRATION (M) Figure 10. Effect of a fixed concentration of various antagonists on XAMI-mediated cyclic AMP production in cockroach nerve cord membranes. Values are the mean :1; SEM of 3 separate experiments, assayed in duplicate. Control cyclic AMP production was 170.3 1 17.5 mel/min/mg. 53 700 0 0A I 600 ‘ 500 ‘ 400 - 300 . 200 - CYCLIC AMP INCREASE (pmoles/min/mg protein) 100‘ O . O 1 ' ""'l T 'vrmvu - rvvvvvv; fwvnnw—v -.fi...‘ 10-1010-9 10-8 10-7 10-5 10-5 10-4 CONCENTRATION (M) Figure 11. Agonist-induced cyclic AMP production in cockroach brain membranes. Values indicate the mean :1: SEM of 4 experiments, assayed in duplicate. Control cyclic AMP production was 255.3 i 4.2 pmol/min/mg. All agonists were tested in the same preparation. 55 1000 .——c 750 - CYCLIC AMP lNCREASE (pmoles/IO min/mg protein) N m on o o o o g, - OW f *WVVVVV' v v 1vvvva v v vTrv—v-v—I f v v rv v' f v v "j'vj 10-8 10-7 10-5 10-5 10-4 10'3 CONCENTRATION (M) Figure 13. Agonist-induced cyclic AMP production in tobacco hornworm nerve cord homogenates. Values are the mean 1 SEM for 3 separate experiments assayed in duplicate. Control cyclic AMP production was 419.9 3 88.8 pmol/lo min/mg. Both agonists were tested in the same preparation. .oocaEuouoo o: I o: .ocaEmQOuoo mo “82> w mm commoumxo ma xme> I Q .coauooooum mz< owaoao Hosanna won mcamomo umficomm mo coflumuucoocoo I n& I o Hm o.m an mo.o em vo.o om no.0 H24x to o: ooa m.~ mm v.0 . om H.H muz o: o: no no no no: mm 5.0 Save 00H m.- 00H m.m ooa m.m ooH m.m ocfismoouoo KQE>W MK ”MEN/w QVm flak/w fig ng>w flmv~ ouoo o>uoz mou>UOEmmm camum ouoo o>noz moobcmz. Moonwamwhmm muocmqmwuom numcmqmwumm umwcom< .ommao>o oumaacoom o>fluamcomIoc«Emoouoo mo mumflcowm mo mmflocouom o>aumumoeoo .v magma 563 4) suggest that in the case of cockroach nerve cord membrane preparations, XAMI is 37 times more potent than NC5 and 185 times more potent than 0A. In this tissue, however, XAMI is = 80%) as is DCDM (V max = 56%). In a partial agonist (Vmax cockroach brain membranes, XAMI is 157 times more effective than 0A and only 10 times more potent than NC5. The Ra’s for XAMI in cockroach brain and nerve cord are similar, 0.04 and 0.03 uM, respectively. In the cockroach brain, however, XAMI appears to act as a full agonist (Vm - 94%) whereas ax NC5 has a reduced Vma 82%). In the third cockroach tissue X tested, haemocytes, XAMI is a partial agonist (vm = 71%) ax with a Ka similar to the two other cockroach tissues (i.e. 0.02 )JM) . In the hornworm nerve cord homogenates however, XAMI is a full agonist (V max 91%) and has a reduced affinity (Ka = 3 pM). DISCUSSION Identification of a Potent Agonist of Insect Adenylate Cyclase To date characterization of the OSAC suggests that in addition to 0A itself, the imidazoline NCS (Nathanson, 1985a; Nathanson, 1989), and the formamidine DCDM (Hollingworth and Johnstone, 1983; Orr g; 31., in press) are the most potent activators of this receptor-linked enzyme system. Since a large population of insect 0A receptors mediate their actions via a Gwprotein transduced elevation 57 in intracellular cAMP levels (i.e. via AC activation), pharmacological agents with high affinity for these AC- coupled receptors would provide a greatly needed probe for studying' this receptor» In ‘the present study, the imidazoline, XAMI, has been pharmacologically characterized and its affinity for the 0A receptor compared with those of 0A, DCDM and NC5. Of these compounds, in cockroach nerve cord membranes, XAMI exhibits the highest reported affinity for insect OSAC. In this tissue, based on the vmax values, DCDM and XAMI are partial agonists, with maximal stimulation of 56% and 80% of 0A while NC5 appears to be a full agonist. Once the effectiveness of XAMI as an activator of insect AC was established, it was necessary to confirm that the mechanism involved a typical receptor-G-protein interaction. The XAMI-mediated production of cAMP in cockroach nerve cord membranes is totally dependent on the presence of GTP, thus suggesting an essential role for a GTP-binding protein in this response (Levitzki, 1987; Birnbaumer, 1990). Subthreshold concentrations of forskolin also enhance XAMI-mediated production of cAMP in this tissue. These effects are typical of hormone-sensitive AC (Daly, 1984) and suggest that XAMI is activating AC via interaction with a receptor-G-protein complex. Since the cockroach nerve cord contains both a 5- hydroxytryptamine (5-HT)- and a dopamine (DA)-sensitive AC, as well as the OSAC, it was necessary to ascertain whether 58 XAMI was interacting with one or more of these receptor types to produce an increase in cAMP levels. Additivity studies suggest that coincubation with maximally stimulating concentrations of XAMI and DA or XAMI and 5-HT, results in an additive increase in cAMP production, whereas when XAMI is coincubated with 0A or the 0A agonist DCDM, the response is nonadditive. This data suggests that XAMI, at its maximally stimulating concentration is interacting solely with the 0A receptor and not the S-HT or DA sites and thus, is a selective agonist of the AC-coupled 0A receptor in this tissue. The high octopaminergic potency of XAMI is further confirmed in hemocyte studies since this tissue has been previously shown to contain only the OSAC (Orr e_t; gin 1985) . The reversible nature of binding of XAMI to the 0A receptor was investigated. Treatment of cockroach nerve cord membranes with 1 pM XAMI results in the production of approximately 820 pmol/min/mg. This effect can be reproduced if XAMI-pretreated tissue is washed a minimum of three times. The rinses did not have any significant effect on basal cAMP production, suggesting that the binding was reversible. Similar trends have been demonstrated for the interaction of DCDM (Nathanson and Hunnicutt, 1981) and NC5 (Nathanson, 1985a) with the OSAC. This indicates that the effects of these three compounds do not reflect a permanent change in the receptor-AC system, but are the result of a 59 typical, reversible agonist-receptor interaction. Pharmacological Characterization of the XAMI-Mediated Elevation in Cyclic AMP Levels Study of the ability of various previously known octopaminergic (mianserin, phentolamine, cyproheptadine) and cpl-adrenergic (idazoxan) antagonists to inhibit the XAMI- mediated production of cAMP demonstrated that mianserin is the most potent antagonist of this XAMI-mediated effect, followed by phentolamine and cyproheptadine, which are roughly equipotent. Idazoxan is a potent, selective antagonist of the vertebrate aé-adrenergic receptor (Langin and Iafontan, 1989). This is the first study showing the effectiveness of idazoxan in inhibiting an octopaminergic response. Although idazoxan has a relatively poor Ki of 4.6 ‘pM, the fact that both it and phentolamine are imidazolines demonstrates that imidazolines can act either as agonists (e.g. NC5, XAMI) or antagonists (e.g. phentolamine, idazoxan) at 0A receptors. The B-adrenergic antagonist, propranolol and the dopamine antagonists, (+)-butaclamol and spiperone, were poor blockers of this response. Since insects have not been shown to have any B-adrenergic-type receptors, propranolol is classically used as a negative control when. pharmacologically' characterizing’ the octopaminergic nature of a response. Overall, these results indicate that the pharmacological properties of the XAMI- induced response are very similar to those of the OA-induced 60 response observed in this tissue (Downer e; 11., 1985) and therefore, support the premise that XAMI is interacting specifically with the OA receptor. Furthermore, the rightward, parallel shift of the XAMI dose-response curve in the presence of mianserin, phentolamine, cyproheptadine and idazoxan (data not shown), suggest that these antagonists serve as competitive inhibitors of the binding site. The competitive nature of the inhibition of OSAC by phentolamine, mianserin and cyproheptadine has previously been shown in insect tissues (Orr gt a;., 1985). Comparative Effectiveness of XAMI in Several Insect Systems The effectiveness of XAMI in stimulating the production of cAMP was examined in other cockroach tissues (brain and hemocytes) and in the nerve cord of another insect species (tobacco hornworm) . In all cases, the dose-response curve for XAMI is notably flatter than that of OA and exhibits a plateau phase around 0.1 )m. This suggests the existence of multiple affinity sites (i.e. high and low) for XAMI, but clearly, confirmation will require binding studies using radiolabelled XAMI. However, both the effectiveness and degree of agonism of XAMI appears to vary with the tissue and species of insect tested. In the case of cockroach brain and hornworm nerve cord, the activity of XAMI approximates that of a full agonist, whereas in cockroach nerve cord and haemocytes it acts as a partial agonist. In contrast to NC5, XAMI has similar 61 affinity in all cockroach tissues examined, suggesting that it may have wider application as a ligand for studying different 0A receptors. However, the affinity of XAMI for the OA receptor varies depending on the species tested. In all three cockroach tissues, XAMI is the most potent agonist, with a Ka ranging from 157 to 185 times more effective than that of OA and 9 to 146 times more effective than that of NC5. In the case of the hornworm nerve cord, however, XAMI is only 7 times more effective than OA indicating a basic difference in this receptor's response to OA agonists. While there is limited pharmacological data to suggest that the cockroach and hornworm neural 0A receptors are different, formamidines generally exhibit greater affinity for the Lepidopteran 0A receptor whereas imidazolines tend to exhibit lower affinities in this insect (Hollingworth and Johnstone, 1983). I have been able to show some distinct pharmacological differences in the 0A receptors in these two orders (see Chapter 2). It is worth noting that the high potency of XAMI as an 0A receptor agonist, as shown by these in yitgg studies, is in agreement with its high in 2129 potency in eliciting OA- like behavioral patterns in crayfish (Hollingworth and Johnstone, 1983) and moths (Linn and Roelofs, 1987). Hence, the demonstrated affinity of XAMI for the 0A receptor in neural and non-neural insect tissues establishes the importance of XAMI and its analogues as candidates for 62 specific OA-receptor probes. We now have available a highly selective, high-affinity ligand with which to further characterize and explore the pharmacological variations evident in invertebrate OA receptors. CHAPTER 2 Identification and Pharmacological Characterization of an Insect Cell Line as a Model System for Studying Octopamine Receptors. 63 64 INTRODUCTION Octopamine (0A) is a major biogenic amine found in high concentrations in invertebrates. In insects, this monohydroxy analog of norepinephrine (NE) has been shown to function as a neurotransmitter, neuromodulator and neurohormone (Orchard, 1982) . Many of the physiological functions of 0A are mediated by the activation of an octopamine-sensitive adenylate cyclase (OSAC). Biochemical and pharmacological characterization of OSAC-coupled receptors in insects has been significantly hampered by the need for tedious dissection and large quantities of tissue. The establishment of an insect cell line for this purpose would provide a readily available and homogeneous source of these receptors. To date there has been only one such study (Gole gt a)“, 1987) which established the existence of an insect cell line expressing an OSAC; however, the OSAC was not typical, being insensitive to the adenylate cyclase (AC) activator, forskolin. This aberrant characteristic made this line suspect as a model system for studying these receptors. Octopamine receptors have been previously subclassified (Evans, 1981) into three types: OA-l, OA-2A and OA-ZB, based on the relative activities of selected agonists and antagonists on the locust extensor tibiae muscle preparation. There is mounting evidence, however to suggest that although this classification has some general applicability, there are significant interspecies and 65 intertissue differences in OA-receptor pharmacology. Although the biochemical events and structural components regulating the interaction of the 0A receptor with AC appear to be analogous to those described for B- adrenergic-receptor activation of AC (Bodnaryk, 1982; Orr g; ‘al., 1985; Orr and Hollingworth, 1990), the pharmacology of the OA-receptor is distinct from B-adrenergic receptors and has greater similarity with d-adrenergic receptors (Evans, 1980; David and Coulon, 1985; Orr g1; a_l., 1990). In the past, attempts have been made to characterize insect OA- receptors as AYZ-adrenergic-like (David and Coulon, 1985) . In order to test this hypothesis, compounds with known adrenergic activity were used to characterize the AC-coupled OA receptors in an insect cell line derived from pupal ovarian tissue of the Fall armyworm, Spggoptera frugiperdg (Sf9). This pharmacological profile was compared with that observed in the ventral nerve cord receptor of the American cockroach, Periplaneta americana L. The Sf9 cell line is readily available and easily cultured, making it an ideal source of the OSAC-coupled receptor complex to be used in the screening of putative octopaminergic compounds and investigating the biochemical nature and dynamics of this site. The present data suggest that the Sf9 cells are devoid of DA- or 5-HT-sensitive AC's, but do express OSAC. There also appear to be pharmacological differences between this Lepidopteran OA 66 receptor and the cockroach site, suggesting the importance of defining interspecies differences when proposing a receptor classification scheme for the 0A receptor in insects and for selective insect control agents based on OA- receptor ligands. MATERIALS .AND METHODS Cockroach Nerve Cord Preparation Adult male American cockroaches (Periplanetg americana L.) between 1 and 3 months after the final molt were taken from a stock colony maintained as described in Chapter 1 (Materials and Methods). Prior to dissection, the insects were held without food in individual petri dishes for 1 to 3 In to minimize any handling-induced stress effects (Downer, 1979). Ventral nerve cords were dissected as described in Chapter 1 (Materials and Methods) and immediately placed in ice-cold insect saline (0.17 M NaCl, 6.0 KCl, 2.0 mM NaHCO3, 17.0 mM glucose, 6.0 mM NaHzPO4.HZO, 2.0 mM CaC12.2820, 4.0 mM MgC12.6H20), at pH 7.0. The nerve cords were kept on ice for no longer than 30 min following which they were transferred to ice-cold Tris-EDTA/EGTA/DTT buffer (10.0 mM Tris, 1.0 mM EDTA, 2.0 mM EGTA and 1.0 mM DTT, pH 7.2). The tissue was homogenized with a glass-teflon homogenizer (15 strokes at 400 rpm) and the resulting pellet was resuspended in Tris-DTT buffer (10.0 mM Tris, 1.0 mM DTT, pH 7.2), homogenized and centrifuged as above. The pellet was 67 resuspended in Tris-DTT buffer for determination of cAMP content. Sf9 Tissue Culture A- cell line derived from pupal ovarian tissue of the Fall armyworm, ‘§EQQQQL§L§, frugiperda (Sf9) was jpurchased from American Type Culture Collection, Rockville, MD. These cells are readily available and are routinely the insect cell of choice for baculovirus expression. The cells were 2 tissue culture flasks grown as monolayer cultures in 75 cm in a 27°C incubator. Carbon dioxide was not required and antibiotics were not used for culturing. The Sf9 cells were supplied frozen and had been reared in TNM-FH medium which consists primarily of Grace's medium, supplemented with yeastolate, lactalbumin hydrolate and 10% fetal bovine serum. The frozen cells were thawed as recommended by the supplier but transferred to Ex-cell 400 medium (J.R. Scientific, Woodland, CA) which did not require preparation or filtration of medium and also did not contain fetal bovine serum. Transfer from a fetal bovine serum containing medium to one devoid of this supplement resulted in the cells initially (first 10 passages) attaching very firmly to the bottom of the culture flask. Consequently, as a routine procedure, the cells were subcultured at least 3 times a week and removed from the flask surface with the use of sterile cell scrappers. If more than 5% of the culture contained floating cells, the medium was replaced with fresh 68 medium prior to subculturing. Sterile techniques were observed during all aspects of cell culturing. Healthy, viable cells appear to be spherical and uniformly shaped with a diameter of 10 to 12‘pM. Cell viability was investigated by adding 0.1 ml of a 0.4% stock solution of trypan blue buffered with isotonic salt solution (pH 7.2-7.3) to 1.1m1 of cells and examining under an inverted microscope. Both bright- and dark-field was used for this purpose, depending on the density of the cell suspension. Using a hemocytometer, cell viability was calculated and cells which took up trypan blue were considered to be nonviable. Cell viability ranged from 85 to 95% (Figure 14). A doubling time of approximately 24 h was observed in Ex-cell 400 medium and the cells were used when confluent. In all cases, there was a 24 h lag phase during each subculturing cycle, during which time the free cells initially reattached to the flask prior to diViding. Sf9 Cell Preparation Intact Cells Ex-cell 400 medium was decanted from the culture flasks and 10 ml (27°C) of insect saline (0.17 M NaCl, 6.0 mM KCl, 2.0 mM NaHCO3, 17.0 mM glucose, 6.0 mM NaHzPO4.H20, 2.0 mM CaC12.2H20, 4.0 mM MgC12.6H20, pH 7.0) was gently added. The cells were suspended and centrifuged at 900 g for 5 min (4°C). The pellet was resuspended in 10 ml of 69 30.0 g 25.0- 89 gé 20.0- <0 50. 15.0- ‘58 av- 35 10.0“ 5 z 5.0« C 0.0 I I ' , r T 0 1 2 3 4- 5 DAYS Figure 14. Representative growth curve for Sf9 cells grown in Ex-cell 400 media. Cell viability ranged from 85 to 95%. 7O insect saline (27°C) and again centrifuged as above. The cells were diluted with insect saline and used for cAMP determinations. Examination of this cell suspension under a light microscope revealed the presence of primarily intact cells. Sf9 Membranes Intact cells were prepared as above and resuspended in ice-cold Tris-EDTA/EGTA/DTT buffer (10.0 mM Tris, 1.0 mM EDTA, 2.0 mM EGTA and 1.0 mM DTT, pH 7.2) and homogenized using a glass-teflon homogenizer (15 to 20 strokes at 400 rpm). The resulting homogenate was centrifuged at 27,000 g for 15 min (4°C) and the pellet was resuspended in cold Tris-DTT buffer (10.0 mM Tris, 1.0 mM DTT, pH 7.2) and centrifuged again at 27,000 g for 15 min (4°C). The pellet was resuspended in Tris-DTT buffer for cAMP determination. Light microscopic examination revealed less than 1 to 5% intact cells in this preparation. Analytical Procedures Chemicals The pharmacological classification and supplier of compounds used in this study are shown in the Appendix. XAMI was synthesized according to the method of Kristinsson and Traber (1981) and N-demethylchlordimeform (DCDM) was synthesized by the method of Hollingworth (1976). All other drugs and chemicals were purchased from Sigma Chemical Co., 71 St. Louis, MO. Protein Assay Protein concentration was estimated using the method of Lowry gt g1. (1951) with bovine serum albumin as the protein standard. Aliquots of intact cells were rapidly frozen in liquid nitrogen, then thawed and vortexed to form a homogeneous solution prior to protein determination. Cyclic AMP Radioimmunoassay Cyclic AMP measurements were performed using a radioimmunoassay (RIA) kit, purchased from New England Nuclear Research Products (NEN), MA. Cyclic AMP determinations for cockroach nerve cord membranes and Sf9 membrane samples was as described in Chapter 1 (Materials and Methods). In general, for all Sf9 preparations, incubations were carried out at 27°C (except for temperature experiments), and at 30°C for nerve cord tissue. In the case of intact Sf9 cells, the reaction medium consisted of 50 ul of tissue sample (or buffer for blanks), lo‘pl of 0.1 mM 3-isobutyl-1-methy1xanthine (IBMX) and 40‘pl of treatment compound (or buffer). The reaction was initiated by the addition of tissue and terminated by boiling the samples for 3 min in a boiling water bath. The samples were diluted with 0.05 M sodium acetate buffer (pH 6.2) and rapidly frozen by immersion in liquid nitrogen. Samples were allowed to thaw at room temperature, diluted and used for 72 cAMP determination. When checked under a light microscope, these samples contained no intact cells. Desensitization Experiments Sf9 cells were cultured until they were confluent. These cells were subcultured to generate clones of the same initial cell population. For each replicate, or experiment, all treatments were tested on clones derived from the same cell population. Control cells were treated in the same manner as treated cells, with the exception that instead of adding pharmacological agents to the medium, an equivalent aliquot of fresh medium was added. To maintain consistency, an effort was made to handle flasks containing treated and untreated cells in a similar manner. Each compound to be added was first dissolved in sterile Ex-cell 400 medium and diluted to the appropriate stock concentration under sterile conditions in a flow-hood. Stock solutions were filtered using a 0.2‘pM filter attached to a 5 ml disposable syringe and directly added to sterile medium. Once the required concentration of the compound was prepared, the existing medium was gently decanted from the cell-containing culture flasks and control or treatment medium was added without dislodging the cells. The flasks were capped and gently shaken to allow contact of the cells with the medium and kept in the incubator for 24 h. At the end of this incubation period, the cells were inspected with the aid of an inverted microscope to note any contamination or 73 morphological changes. Only if these were not observed, were the cells used for cAMP determination. Pharmacological Procedures Determination of V K max ' a' IC50 and Ki values were conducted as described in Chapter 1 (Material and Methods). Statistical Procedures All descriptive statistics were calculated using a Texas Instrument TI-60 calculator. Group means were compared using a two-tailed t-test (Zar, 1974) . Standard curves for cAMP quantification were calculated as described in Chapter 1 (Materials and Methods). RESULTS Identification of an Insect Cell Line Expressing Octopamine— Sensitive Adenylate Cyclase The ability of various agents to stimulate cAMP production in intact Sf9 cells was investigated (Table 5). These preliminary experiments employed confluent cells which were incubated at 27°C for 30 min. Basal cAMP production was 49.8 i 4.2 pmol/30 min/mg (n=4). Incubation with 100 pM octopamine (OA) resulted in a statistically significant increase in cAMP production (147.4 pmol/30 min/mg). Since insect tissues have been shown to contain DA- and 5-HT- sensitive adenylate cyclase (Lingle gt g1., 1982; Downer gt g1” 1985), these amines were investigated at 100 yM 74 Table 5. Effects of various agents on cyclic AMP production in whole-cell preparations of Sf9 cells. Cyclic AMP Increasea Treatment (pmol/BO min/mg) Octopamine (100 uM) 147.4 $8.9!”c Dopamine (100 uM) -10.8 i 2.6 S-HT (100 uM) 3.5 1.3.0 XAMI (10 NM) 68.5 .+_ 13.06 Forskolin (10 uM) 219.8 1 12.5c Octopamine + Phentolamine (10 MM) 10.2 1 4.8 XAMI + Phentolamine (10 MM) 19.9 i 4.1 a - Basal cyclic AMP production 3 49.8 i 4.2 pmol/30 min/mg. b - Values are the mean 1 SEM for 4 separate experiments. c - Statistically different from control (p < 0.05) 75 concentrations and found to have no significant effect. The octopamine agonist, XAMI, produced an increase over control of 68.5 pmol/30 min/mg. The diterpene forskolin (10 1114) produced a pronounced increase over control of 219.8 pmol/30 min/mg. Both the OA- and XAMI-stimulated cAMP production was blocked by coincubation with 10 uM of the octopamine antagonist, phentolamine. Characterization of the Adenylate Cyclase-Coupled Octopamine Receptors in Intact Sf9 Cells In order to determine the optimal range of protein concentrations which could be used to study cAMP production in intact cells, a protein-linearity study was undertaken (Figure 15). Using a maximum (100%) protein concentration in the range of 0.38 to 0.41 mg, basal and OA-mediated (0.1 mM) cAMP production was found to be linear with r2= 0.995 and 0.998, respectively. A time course was produced for the OA-mediated (0.1 mM) cAMP production in intact cells (Figure 17). The earliest time point that could be tested was 1 min. Cyclic AMP production is approximately linear in the range of 1 to 5 min (r2 = 0.979) and then slowly begins to stabilize and eventually decline. For all subsequent experiments with intact cells, an incubation period of 5 min was used. Characterization of the Adenylate Cyclase-Coupled Octopamine Receptor in Sf9 Membranes Since the Sf9 cells were grown at a temperature of 1 .5 O CONTROL . OCTOPAMINE Z 9 . g 1 g ......... o ? .0 . ...--°' ...... 8 g o. > ,a- to o if a? E. as” .x" - ..J ’.' #0.“! o .f’.’ 6 x... ”_a/ ,r ......... of .//Q'/.G/O 0.0oi~="""°"w . . . . E O 20 40 60 80 100 % PROTEIN CONCENTRATION Figure 15. Effect of Sf9 intact-cell protein concentration on basal and octopamine-mediated (0.1 mM) cyclic AMP production. Values are the mean of two experiments assayed in duplicate, with SEM of less than 14%. _ The values for 100% protein ranged from 0.38 to 0.41 mg. The correlation coefficients for basal and octopamine-mediated effects were 0.995 and 0.998, respectively. 77 14001 A D Basal Z 1200‘ -Octopomine (0.1 mM) 9,5 5 g 1000- D 8} m°§ 800‘ 0.6 §Q EOO« J— < a o 8% 400‘ ). U 200‘ - :09: m R F1 0 , Q , 1O 23 27 TEMPERATURE (0C) 5 400 8 '7- T o , :1 O 8 ‘ . 12:3 300« \1 . o.e C) a: 3 E 20 200‘ 0\ )- .. o- o .2: E 0. UV 10 O m ?——/"" :5 O o: 0 Z 0 r r . . r r 5 1O 15 20 25 30 35 TEMPERATURE (0C) Figure 16. Effect of temperature on cyclic AMP production in membrane preparations of Sf9 cells. (A) Histogram of control and octopamine-mediated (0.1 mM) cyclic AMP production. (8) Data from Figure 16A is expressed as an increase in cyclic AMP production.- Control cyclic AMP production at 10, 23, 27 and 35°C was 99.6 :1; 13.8, 130.8 :1: 15.6, 599.1 ;I-_ 39.0 and 966.9 _4; 29.4 pmol/30 min/mg, respectively. 78 _J 400 O E z 350- 0 1 0 fit 300. 0‘s” 123% 250« we Gio. 2v zoo. '— E g 150- Lu °' 100 . . . e , . . O 2 4 6 8 IO 12 14 16 “NEIOnNO Figure 17. Time-dependent production of octopamine-mediated (0.1 mM) cyclic AMP production in intact Sf9 cells. Values are the mean of 3 to 6 separate experiments. The correlation coefficient for the range of points between 1 and 5 min = 0.979. 79 27°C, it was necessary to establish the (optimal) temperature profile for the AC. Membranes were incubated for 30 min at 10, 23, 27 and 35°C and cAMP production was measured under basal and OA-mediated (0.1 mM) conditions. (Figures 16A and 16B). At all four temperatures, OA produced a greater response than under basal conditions (Figure 16A) . However, as the temperature was increased, there was a corresponding increase in both basal and OA- mediated cAMP production. To better demonstrate temperature-dependent increases over basal cAMP production, these data were replotted (Figure 16B). It is apparent that a maximal increase in cAMP production is seen at the cell- rearing temperature of 27°C. This effect exhibits a biphasic pattern, i.e. at temperatures lower or higher than 27°C, cAMP production is reduced. In order to further describe the membrane-associated AC in these cells, membrane preparations were used to generate a protein linearity curve (Figure 18). Using a control (100%) protein concentration in the range of 9.1 to 14.9 ug and incubating for 30 min, a correlation coefficient of 0.982 was calculated, suggesting a linear relationship between the membrane concentration used and the OA-mediated (1 mM) cAMP production (increase over control). Membrane-protein concentrations in the linear range (as determined from Figure 18) were used when characterizing the time course for OA-mediated (1mM) cAMP production in Sf9 5 o . 3 0°40 ‘ o ............. . 8 0.35 A , """ o- "w"... g? 0.30 « . ...... o g 0.25 ‘ /,r’/ O :1 T: 0’ g g 0.20 - 0v o-..” z 0.1 5 ‘ .’ “J 0.10 - -------- “’" 5 0.05 «D ”f... o E 0.00 s . . . A O 20 40 60 80 100 PROTEIN CONCENTRATION (% OF CONTROL) Figure 18. Octopamine-mediated (1 mM) cyclic AMP production in Sf9 membranes as a function of protein concentration. Values are the means of 2 experiments, with SEM of less than 20% (r2 8 0.982). The values for 100% protein were in the range of 9.1 to 14.9 mg. 81 membranes (Figure 19). Cyclic AMP production increases linearly between 5 to 45 min and is constant from 45 to 60 min. All subsequent experiments were performed with a 30 min incubation. The GTP dependence of OA-mediated cAMP production in Sf9 membranes was investigated (Figure 20), along with the effect of forskolin on this response. In the absence of GTP, there is limited production of cAMP with increasing OA concentrations. The addition of 0.05 mM GTP significantly enhances OA-mediated production, as does forskolin at 0.5 uM. In these two respective cases, maximal production (Vmax) represents a 50 and 70% increase over control. For these data, the Ka for 0A in the presence of GTP is 0.2 PM and in the presence of both GTP and forskolin, 0.3 )JM. Since the OA dose-response curve exhibited a large increase in cAMP production between 0.1 and 1.0 mM, the Ka was calculated assuming a Vmax at 0.1 mM. Basal cAMP production in the absence of GTP, presence of GTP and presence of GTP and forskolin was 14.1, 17.8 and 44.2 pmol/min/mg, respectively. Since there was a definite GTP-dependence, the effect of varying concentrations of GTP on control and OA-mediated (0.1 mM) cAMP production was investigated in membranes (Figure 21). Basal cAMP production was 13.8 and 16.7 pmol/min/mg, respectively for the control and OA-treated samples. In both cases, GTP had a biphasic effect on cAMP 00A (1 mM) I INCREASE IN CYCLIC AMP PRODUCTION (pmoI/mg) O \I I r r 0 1 0 20 3O 4O 50 60 F f T TIME (min) Figure 19. Time-dependent production of octopamine-mediated (1mM) cyclic AMP in membrane preparations of Sf9 cells. Values are the mean 1 SEM of 2 separate experiments. g 32.0 .: O -GTP g 28.0 - o +GTP 8 A +F'ORSK mA 24.0 " 0.. g, 1' O. . :\ 20.0 A < .S I 0 16.0 - 3% I A A 9 ° 12 O« ' A Q E ' T £3 . . 33, 8.0 - I ' E 4.0 - ‘ 'T _ T - 0 (Z) O O ("A :.:.::::::T f: :39 : : :32m ‘ - ”‘4‘“ - ~ - -8 -7 -6 -5 -4 -3 L00 OCTOPAMINE CONCENTRATION (M) Figure 20 . Dose-dependent octopamine-mediated cyclic AMP production in Sf9 membranes in the presence (0.05 mM) and absence of GTP and in the presence of GTP + 0.5 uM forskolin. Values are the mean -_I-_ SEM of 3 separate experiments. Control values, in the absence of GTP = 14.1 i 0.4 pmol/min/mg and in the presence of GTP and GTP + forskolin = 17.8 i 0.7 and 44.2 1; 2.8 pmol/min/mg, respectively. 22 OCONTROL OOCTOPAMINE I 5 20« I 5,. g E” O} 180 O: .. 0- E %> (E 16" 9.9- ..l 9 14- o . 12 --1 - - "I -- IO-7 10-5 10-5 10.4 IO-3 CTR CONCENTRATION (M) Figure 21. Cyclic AMP production as a function of the GTP concentration in control and octopamine-mediated (0.1 mM) Sf9 membranes. Values are the mean :1; SEM of 6 experiments. Basal cyclic AMP production (in the absence of GTP) for control and octopamine-treated samples was 13.8 1; 0.1 and 16.7 i 0.9 pmol/min/mg, respectively. 85 production, with maximal effects in the concentration range of 0.01 to 0.1 mM. Dose-response curves were also generated for other activators of AC, such as forskolin, NaF and GppNHp (Figure 22). At higher concentrations of forskolin (e.g. greater than 0.5 uM), basal production of cAMP was dramatically enhanced, making it difficult to quantify this response without diluting the samples. Both GppNHp and NaF exhibited dose-dependent elevations in cAMP production with respective maximal effects at concentrations of 0.1 to 1.0 mM and 1.0 to 10.0 mM. At a concentration of 100 mM, NaF was inhibitory and/or toxic, reducing cAMP production to basal levels. Pharmacology of the Sf9 Adenylate Cyclase Since the magnitude of the OA-mediated response was fairly small in the membrane studies, all future pharmacological studies were performed using intact cells. The ability of three OSAC agonists, the phenylethylamine, OA, the formamidine, DCDM and the imidazoline, XAMI, to increase cAMP production was tested in intact Sf9 cells (Figure 23). At a maximally stimulating concentration of 1 mM, OA, XAMI and DCDM, respectively increased cAMP levels by 2.8, 1.9 and 2.2 fold over control. Control values for these experiments were 3.0 i 0.2 pmol/min/mg, and for the entire study, the control values for cAMP production were relatively consistent (i.e. in the range of 2.3 to 3.2 150 I O FORSK . O NOF 125 AGppNI—Ip 100- 75- 50‘ CYCLIC AMP PRODUCTION (mel/mIn/mg) N 01 l v r v rvv v v O Vvvv' v v vvvvvw' f1 vrvvvv' v rv vvvw' r v 'vv'vv-l v v vv'vvv‘ r r m“ v v v vvvvv' vvv' -9 -a -7 —e -5. -4 -3 -2 -1 LOG CONCENTRATION (M) Figure 22. Dose-dependent effects of forskolin, sodium fluoride (NaF) and 5'-guanylylimidodiphosphate (GppNHp) on basal production of cyclic AMP in Sf9 membranes. Values are the mean 1; SEM of 3 separate experiments. Control cyclic AMP production - 15.6 i 1.3 pmol/min/mg. 87 5.2 0 0A - o XAMI 4-2 . A DCDM 3.2 - INCREASE OVER CONTROL (pmoI/mIn/mg) N N I 2« O 02- 0 ~08 -- LOG CONCENTRATION (M) Figure 23. Effects of octopamine agonists on cyclic AMP production in intact Sf9 cells. Values are the mean 1 SEM of 3 to 6 separate experiments. Control cyclic AMP production was 3.0 t. 0.2 pmol/min/mg. 88 pmol/min/mg). The respective Ka's for OA, XAMI and DCDM were 3.5, 0.7 and 0.3 )iM. It is evident from the Vmax values of 100, 53, and 74% of control, respectively, that both XAMI and DCDM are partial agonists of this receptor. The ability of various octopaminergic and adrenergic agonists to stimulate cAMP production was investigated in intact cells and expressed in terms of Ka and Vma values if x the compound resulted in at least a 40 percent increase over control at the highest concentration tested. For compounds which were not effective agonists, the percent increase over control at 100 )AM is indicated (Table 6). The basal cAMP production of these studies was 2.5 i 0.2 pmol/min/mg. Based on Ka values, DCDM and XAMI have the highest affinity for this OSAC (0.3 and 0.7 pM, respectively). D,L- octopamine and its N-methylated analog synephrine are 10- fold less effective with respective Ka values of 3.5 and 2.8 pM. Both naphazoline and NC5 are respectively 4.0 and 10.3 fold less effective than OA itself in stimulating cAMP production. Other imidazolines such as clonidine and tolazoline are poor activators, with Ka’s of greater than 100 uM. Of these 8 relatively effective compounds, only OA and NC5 are full agonists. Naphazoline and synephrine have reasonable Vmax values of 85 and 76%, respectively. DCDM and XAMI exhibit the lowest vmax of this series (74 and 53%, respectively). At a dose of 100 uM, the imidazolines, naphazoline, clonidine and tolazoline produce a 71 to 129% Table 6. Effects of various octopaminergic and adrenergic agonists on the production of cyclic AMP in whole- cell preparations of Sf9 cells. Agonist Ka (0M) ngx %Increase Over Control at 1000M DCDM 0 3a 74b 107 XAMI 0.7 53 90 Synephrine 2.8 76 127 D,L-Octopamine 3.5 100 149 Naphazoline 14.1 85 129 NC5 36.0 100 113 Clonidine >100 52c 75 Tolazoline >100 53c 71 Medetomidine -- --d 73 UK-14304 -- -- 68 Chloroethyl- Clonidine -- -- 50 Tyramine -- -- 47 BET-933 -- -- 47 N-Acetyl- Octopamine -- -- 46 2,4-XAMI -- -- 3 a - Values are the mean of 3 to 6 separate experiments with the SEM less than 15%. Basal cyclic AMP production = 2.5 i 0.2 pmol/min/mg. b - Maximal stimulation as a % of maximal octopamine stimulation. c - Stimulation at highest concentration tested (0.1 mM). d - Agonists tested at 0.1 mM. 90 increase over control. The remaining 7 compounds in this list are relatively poor activators of OSAC with values ranging from 3 to 73% increase over control. The major metabolite of 0A in insects, N-acetyl-OA and the 2,4- dimethl-substituted analog of XAMI, 2,4-XAMI, stimulate Sf9 OSAC 46 and 3%, respectively. Twenty four octopaminergic and adrenergic antagonists were also tested in intact Sf9 cells for their abilities to block OA-stimulated (50 uM) cAMP production (Table 7). Both the alkaloid, ergotamine and the OA antagonist, phentolamine were very effective in blocking this response, with respective Ki values of 0.18 and 0.21 1.1M. The (+) isomer of mianserin, a potent OA antagonist, as well as the idazoxan analog, RX821002A, were also very effective blockers, with respective Ki values of 0.41 and 0.44 PM' The (i) and (-) isomers of mianserin had significantly different affinities of 1.0 and 2.6 pM, respectively. Antagonists such as the z-adrenergic antagonist, MPV-295, the mianserin analogs C6811049A and CGSlS413A, chlorpromazine, idazoxan and guanfacine, had Ki values in the range of 1.38 to 2.46 1.1M. Atipamezole, metoclopramide, cyproheptadine and guanabenz had Ki's in the range of 5.05 to 9.75 pH. Of these 16 antagonists, the greatest percent inhibition at 100 uM was seen with ergotamine, phentolamine, (+)- and (3)-mianserin, ranging from 92 to 98%. The majority of the remaining 13 antagonists inhibited 60 to 80% at 100 uM. Although 91 Table 7. The effects of various antagonists on octopamine- mediated cyclic AMP production in intact Sf9 cells. Antagonist Ki“ (uM) % Inhibition at 100 MM Ergotamine 0.18b 95 Phentolamine 0.21 98 (+) Mianserin 0.41 93 RX821002A 0.44 88 (+/-) Mianserin 1.00 92 MPV-295 1.38 75 CGSllO49A 1.50 85 Chlorpromazine 1.80 70 Idazoxan 1.84 71 Guanfacine 1.90 83 CGSlS413A 2.46 72 (-) Mianserin 2.60 70 Atipamezole 5.05 60 Metoclopramide 6.50 50 Cyproheptadine 8.20 41 Guanabenz 9.75 39 Yohimbine ——° 44 Corynanthine -- 36 Rauwolscine -- 34 Spiperone -- 28 BF9290 -- 24 Propranolol -- 16 (+) Butaclamol -- 10 Prazosin -- 9 a - Ki - ICSO/[l + (S/Ka)], where ICSO - concentration of antagonist giving 50% inhibition of octopamine—mediated cyclic AMP production. S = concentration of octopamine used (50 uM). b - Values indicate mean of 3 to 6 separate experiments with SEM of less than 15%. Control cyclic AMP production = 2.5 i 0.2 pmol/min/mg. c - Antagonists used at 0.1 mM. 92 metoclopramide, cyproheptadine and guanabenz had Ki’s in the micromolar range, they were only able to inhibit 39 to 50% at 100 uM. Nine other antagonists were tested in this system, with the greatest inhibition observed at 100 uM with the 0(2- antagonist, yohimbine (44%). The q’l-antagonist, corynanthine and the ”é-antagonist produced a 34 and 36% inhibition, respectively. The Idopamine antagonists, spiperone and (+)-butaclamol, the B-antagonist, propranolol and the ai-antagonist, prazosin, all inhibited less than 28% of the OA-mediated cAMP production. The putative octopaminergic antagonist, BF9290 was relatively ineffective, with 24% inhibition at 100‘pM. Pharmacology of the Cockroach Octopamine-Sensitive Adenylate Cyclase For comparison with the Lepidopteran AC-coupled OA receptor seen in the Sf9 cells, a similar pharmacological profile was generated for the Orthopteran receptor seen in cockroach ventral nerve cord preparations. Table 8 shows the effects of various octopaminergic and adrenergic agonists on cAMP production in cockroach nerve cord membranes. In the cockroach, XAMI is the highest affinity agonist (also see Chapter 1) with a Ka of 0.03 pM. Synephrine and DCDM are the next most potent agonists with respective Ka's of 0.32 and 0.67 pM. Synephrine has a 17.5 fold higher affinity than OA itself. The other 93 Table 8. Effects of various octopaminergic and adrenergic agonists on cyclic AMP production in cockroach ventral nerve-cord membrane preparations. Agonist Ka“l (1.1M) Vmamb %Increase Over Control at 100 uM XAMI 0.03c 80 225 Synephrine 0.32 90 243 DCDM 0.67 56 163 NC5 1.10 90 252 Naphazoline 3.80 59 169 D,L—Octopamine 5.60 100 250 Clonidine 7.50 49 126 Tolazoline 18.30 42 112 Tyramine --d -- 122 Medetomidine -- —- 87 UK-14304 -- -- 21 N-Acetyl- Octopamine -- -- 6 Chloroethyl- Clonidine -- -- 0 BET-933 -- -- O 2, 4-XAMI -- -- O a - Concentration of agonist causing 50% maximal cyclic AMP production. b - Vmx is expressed as a % of the Vmx of octopamine. c - Values are the mean of 2 to 4 separate experiments with a SEM less than 15%. Control cyclic AMP production = 160.8 1 19.4 pmol/min/mg. Agonists tested at 0.1 mM. 0 I 94 imidazolines, NC5, naphazoline, clonidine and tolazoline have respective Ka's of 1.1, 3.8, 7.5 and 18.3 pM. Tyramine was very effective in the nerve cord (122% increase over control). In this tissue, essentially all the imidazolines tested are partial agonists, with Vhax’s in the range of 42 to 90%. The dZ-agonist, medetomidine significantly stimulates (87%) over control at 100 )iM, however a Ka was not determined. Based on single concentrations of 100 PM! the remainder of the 5 agonists are essentially ineffective in stimulating OSAC (0 to 21% increase over control), with N-acetyl-OA and 2,4-XAMI producing 6 and 0% increases over control. Table 9 shows the effects of various octopaminergic and adrenergic antagonists in the nerve cord. Octopamine was used at 10 pM. Based on the Ki value, the most effective antagonist of the nerve cord OSAC is the mianserin analog, CGSllO49A (Ki = 0.009 pM) followed closely by the three isomers of mianserin. The other mianserin analog, CG815413A, guanabenz and phentolamine have respective Ki's of 0.109, 0.219 and 0.35 )AM. In the nerve cord, guanfacine and cyproheptadine have respective Ki's of 0.489 and 0.820. Idazoxan and metoclopramide have similar Ki’s in the cockroach, in the range of 5.18 to 6.52 1.1M. RX821002A, yohimbine, ergotamine, MPV-295 and atipamezole all have Ki's of greater than 10 pM. Of these 17 compounds, the majority inhibit between 81 and 100% at 100 M. Compounds such as 95 Table 9. Effects of various antagonists on octopamine— mediated cyclic AMP production in cockroach ventral nerve-cord membrane preparations. Antagonist , Kia (uM) % Inhibition at 100 pM Antagonist CGSllO49A 0.009b 100 (-) Mianserin 0.013 100 (+/-) Mianserin 0.017 100 (+) Mianserin 0.052 100 CGS15413A 0.109 95 Guanabenz 0.219 99 Phentolamine 0.350 95 Guanfacine 0.489 100 Cyproheptadine 0.820 99 Chlorpromazine 2.750 97 Idazoxan 5.180 79 Metoclopramide 6.520 81 Yohimbine > 10 50 Ergotamine > 10 46 MPV-295 > 10 37 RX821002A > 10 22 Atipamezole > 10 15 (+) Butaclamol --c 72 Rauwolscine -- 28 Spiperone -- 21 Prazosin -- 19 Propranolol -- 17 Corynanthine -- 13 BF9290 -- 12 a - Ki = ICSO/[l + (S/Ka)], where IC50 = concentration of antagonist giving 50% inhibition of octopamine-mediated cyclic AMP production. S = concentration of octopamine used (lOIflM). b - Values are the mean of 2 to 4 separate experiments with SEM of less than 15%. Control cyclic AMP production = 160.8 + 19.4 pmol/min/mg. c - Antagonist tested at 0.1 mM. 96 yohimbine, ergotamine, MPV-295, RX821002A and atipamezole inhibit between 15 and 50% at this dose. Other antagonists tested at this dose included (+)-butaclamol, rauwolscine, spiperone, prazosin, propranolol, corynanthine and BF9290. They all inhibited between 12 and 37%, however, (+)- butaclamol inhibited 72% at 100 pM. Agonist-Induced Desensitization in Intact Sf9 Cells In order to determine the ability of octopaminergic agonists to desensitize OA-mediated cAMP production, the cells were exposed to either OA or XAMI for 24 hours as described in Material and Methods. The dose-dependent production of cAMP in the presence of OA was determined for control, 0A (0.1 mM) and XAMI (0.01 mM) pretreated cells (Figure 24). Pretreatment with OA and XAMI reduced the vmax (Table 10) of CA by 37 and 22% respectively. The Ka for CA however does not change and is 2.5, 2.8 and 2.7 PM for the control, OA- and XAMI-pretreated samples, respectively. Pretreatment with both 100 )3! GA and 10 1.1M phentolamine reduces the OA-induced desensitization by reducing the drop in Vma from 37 to 13%. The Ka for 0A (in the presence of x OA and phentolamine) is 3.2 PM' At higher doses (e.g. 100 1.1M), phentolamine itself has a desensitization-like effect of reducing the Vmax (unreported data). 5.0. OCONTROL OOA (0.1 mM) AXAMI (0.01 mM) .I .4 5.0 . >-—-o t 4.0 ~ . ‘ o . - .l 30 2/A l 2.0" /* J. INCREASE IN CYCLIC AMP PRODUCTION (meI/mIn/mg) 1.0- / / .1, "’6 l 0.0 m... . - 1 - - -7 -6 -5 -4 -3 OCTOPAMINE CONCENTRATION (LOG M) Figure 24. Agonist-induced desensitization of octopamine- mediated cyclic AMP production in intact Sf9 cells.* Values are the mean 1 SEM of 3 to 6 separate experiments. Denotes points which are significantly different from the equivalent control (p<0.05). Table 10. Effects of 24 hr pretreatment of Sf9 cells with octopaminergic compounds on octopamine-mediated cyclic AMP production. Pretreatment Octopamine Ka % Reduction in (uM) Octopamine Vhax Control 2.5a 0 Octopamine (100 uM) 2.8 37b XAMI (10 1.1M) 2 7 2210 Octopamine + Phentolamine (10 uM) 3.2 13 a - Values represent the mean of 3 to 6 separate experiments with SEM less than 15%. b - Significantly different from control (p < 0.05). 99 DISCUSSION Identification and Characterization of an Octopamine- Sensitive Adenylate Cyclase in Sf9 Cells To date, characterization of octopamine receptors has involved the use of electrophysiological preparations (Evans, 1981; Orchard and Lange, 1986), receptor binding assays (Dudai, 1982; Hashemzadeh gt a_l., 1985; Minhas gt 11., 1987; Nathanson, 1989) and assays to determine the activity of OSAC (Downer gt g1., 1985; Orr gt gl., 1990). Since most 0A receptors mediate their actions via a G- protein-transduced elevation in intracellular cAMP levels, the availability of an insect cell culture expressing OSAC and devoid of other amine-sensitive AC's, would provide a greatly needed, readily available source of these receptors. In the present study, a Lepidopteran cell line (Sf9) is identified as containing an OSAC. This enzyme-receptor system, is characterized and a pharmacological profile is developed and compared with that of the AC-coupled 0A receptor in the cockroach nerve cord. Sf9 cells contain a forskolin-sensitive AC-coupled 0A receptor and do not respond to other amines such as DA and 5-HT. This OA- mediated elevation in cAMP is optimal at 27°C, although there is a possibility that the temperature optimum lies in the untested range of 27 to 35°C. This seems unlikely however, based on the observations that cell viability is reduced at growing temperature of less than 25°C and greater 100 than 29°C (unreported data). Once the presence of OSAC in Sf9 cells was determined it was necessary to study some of its more basic biochemical parameters. Octopamine-mediated cAMP production in cell membranes is dependent on the presence of GTP and enhanced by threshold concentrations of forskolin. Therefore, in these cells OA appears to be directly activating a membrane- associated receptor-coupled AC complex which is regulated by GTP and contains a forskolin-sensitive C-subunit. These data are in agreement with other studies of OSAC (Bodnaryk, 1982) and those of vertebrate hormone-sensitive AC (Daly, 1984). The dose-dependent enhancement in cAMP levels in the presence of forskolin and the G-protein activators NaF and the nonhydrolyzeable GTP analog, GppNHp, indicates the existence of a functional membrane-associated AC with classical characteristics. These data suggest that Sf9 cells may be more useful as a model system than CF1 cells which have been previously shown to contain an OSAC not sensitive to forskolin (Gole gt g_., 1987). Agonist-Induced Desensitization of the Sf9 Adenylate Cyclase Receptor desensitization is a widespread biological phenomenon, which has been most extensively studied using the B-adrenergic receptor-coupled AC as a model (Harden, 1983; Sibley and Lefkowitz, 1985; Clark, 1986). Two categories of desensitization have been described for the B- adrenergic system: agonist-specific or homologous and 101 agonist-nonspecific or heterologous desensitization. Recently, the first report of OA-receptor dynamics was presented by Orr and Hollingworth (1990) in an extensive evaluation of OA-induced desensitization of the cockroach hemocyte OSAC. Consistent with this study, 24 h exposure of Sf9 cells to OA agonists results in up to a 37% reduction in subsequent maximal stimulation with little change in the affinity of the receptor. The occurrence of agonist-induced desensitization in Sf9 cells demonstrates the potential of these cells as an easily manipulated model systems with which to study this and other phenomena. The OA antagonist phentolamine was used to reverse the OA-induced desensitization seen in the cells. However, phentolamine appears to induce a loss of response at high concentrations, making it difficult to separate this effect from one due to agonist-induced desensitization. Similar experiments using antagonists in cockroach hemocytes (Orr and Hollingworth, 1990) and frog erythrocytes (Mickey gt 11., 1976) show unexpected alterations in subsequent agonist-induced effects. It is therefore possible that long term exposure to phentolamine may result in some unrelated membrane/receptor changes. This requires further investigation including the use of shorter time periods of exposure to antagonists and the use of a variety of other OA antagonists. Receptor desensitization in yiyo is of great 102 physiological and pharmacological importance in vertebrate systems (Perkins gt 11., 1982). The physiological significance of biogenic-amine receptor desensitization has yet to be determined in insects but as proposed by Orr and Hollingworth (1990), it may play a significant role in field-effectiveness of receptor-active insecticides. They explain that often insects are exposed to chronic levels of these chemicals which could result in desensitization of the target site in surviving individuals and thus, could provide a measure of short term resistance making subsequent treatments less effective. Although 3 h pretreatment of hemocytes (above study) with 10 pH of the formamidine insecticide, DCDM, resulted in a 46% reduction of V I max' was unable to study the effects of this compound in Sf9 cells because long-term exposure abolished cAMP production in response to subsequent exposure to 0A. There was, however, appropriate forskolin-mediated cAMP production in these cells. Since these cells did not exhibit any morphological changes, it suggests that the long term incubation with DCDM was detrimental to the receptor-enzyme complex and not to the ability of the C-site to catalyze cAMP production (unpublished data). Pharmacology of the Octopamine-Sensitive Adenylate Cyclase- Coupled Receptor Adrenergic receptors are a family of proteins that are coupled to their effector systems via G-proteins. The 0(2- 103 adrenergic receptor is coupled to a class of G-proteins (Gi-3' Go) which are sensitive to pertussis toxin; the best characterized biochemical effect of this subtype of the 0(- adrenergic receptors is inhibition of AC (Gilman, 1987). Alpha2 receptors recognize a number of molecules of diverse chemical structure, including the yohimban diastereoisomers, yohimbine and rauwolscine, catecholamines, guanidinium analogs and imidazolines such as clonidine (Parini gt 11., 1989). In the present study, a number of selective 0(2- receptor active compounds, along with other known octopaminergic and aminergic drugs have been investigated for their ability to stimulate OSAC in Sf9 cells and cockroach nerve cord, with the goal of defining the degree with which 0A receptors in these tissues exhibit dz-like pharmacology. In cockroach nerve cord and Sf9 cells, OA has a micromolar Ka which is consistent with previously reported values for OA (Gole gt g;., 1983). Interestingly, however, the N-methylated analog of OA, synephrine, is equipotent to 0A in the cells but 18 fold more effective than 0A in the cockroach. This is not surprising, since synephrine's affinity for the 0A receptor appears to be tissue- and species-dependent (Orr,1985) The formamidine, DCDM is the most effective agonist in Sf9 cells. Nathanson (1985) has demonstrated the efficacy of the demethylated analog of DCDM, DDCDM which is 8 fold 104 more effective than GA in the tobacco hornworm (Manducg ggxtg) nerve cord. DCDM itself has been previously reported to be more potent than 0A in activating 0A receptors in hornworm nerve cord (Hollingworth and Land, 1983), firefly light organs (Nathanson, 1979), cockroach nerve cord (Orr gt 11., 1990; Chapter 1) and locust brain (Cambridge, 1981). Relevant to these data are the physiological observations that the formamidines, in general, are potent pesticides against hornworm but are very weak against cockroach (Hollingworth and Lund, 1982) . These data suggest that there are apparent species differences in the efficacy of the formamidines at the 0A receptor as well as confirming the pharmacological similarity between the Sf9 system and another Lepidopteran tissue (e.g. hornworm nerve cord). Of the imidazolines tested (e.g. XAMI, clonidine, NC5, tolazoline), the rank order of potency (ROP) is clearly different between the cockroach nerve cord and Sf9 cells. In the Sf9 cells the following ROP is observed: XAMI >> naphazoline > NC5 >> clonidine = tolazoline whereas the ROP seen in the nerve cord is XAMI >> NC5 > naphazoline > clonidine > tolazoline. In all cases XAMI is the highest affinity agonist (also see Chapter 1). The ROP for both tissues suggests that naphazoline > clonidine; this fact along with the suggestion that OA-2 receptors mediate their action via AC (Evans, 1985) place the Sf9 and nerve cord receptors in the OA-z-like category (see Table 1). However, 105 clonidine is roughly equipotent to tolazoline in the Sf9 cells but is at least 3 fold more effective than tolazoline in the cockroach. Therefore, based on the agonist ROP we are unable to unequivocally define these two receptors in terms of the OA-2A or OA-2B classification. The potent imidazoline, NC5 was first reported- to exhibit potent OSAC stimulating activity in the firefly light organ (Nathanson, 1985a; 1985b) and its azido analog has since been radiolabelled and binding studies performed (Nathanson, 1989). In Sf9 cells, however, NC5 is 10 times less potent than OA but it is approximately 5 fold more potent than 0A in the nerve cord. This again demonstrates the species-specific variability observed in 0A receptors. The effectiveness of the most potent imidazoline, XAMI (Orr gt 11., 1990), in both tissues suggests that it may have broader use than NC5 as a ligand for the 0A receptor. The ability of the phenylethylamine, tyramine, to stimulate OSAC was also investigated. Tyramine is found in large quantities in insect ganglia (Robertson and Juorio, 1976) and recently, a putative tyramine receptor has been cloned and characterized in 21191111111 (Saudou gt 11., 1990). In Sf9 cells, tyramine is relatively ineffective whereas it is considerably more effective in the nerve cord, although a dose-response was not generated. In firefly light organs, the Ka of tyramine is 7 fold less effective than that of 0A and there is strong evidence, based on 106 additivity studies, that at 100 uM levels, tyramine interacts with the 0A receptor in this system (J.A. Nathanson, personal communication). Evidence for tyramine’s actions at the 0A receptor is also presented by Orr gt 11. (1985) in cockroach hemocytes. The z-adrenergic agonist, UK-14304 (Pazos gt 11., 1988), was found to be ineffective in both tissues. These data are similar to the compound's effects against OSAC in firefly light organ (Nathanson, 1985a; 1985b). Other 2- selective agonists such as BHT-933, medetomidine and MPV-295 (Howden gt 11., 1983) were also ineffective. The OA metabolite, N-acetyl-OA, and the inactive 2,4-dimethyl- substituted analog of XAMI, 2,4-XAMI, are poor activators of OSAC in the cockroach nerve cord and Sf9 cells. These data are consistent with their potency in stimulating firefly OSAC (R.M. Hollingworth, personal communication), and thus appear to be typical of AC-coupled 0A receptors. The dz-agonists clonidine, and its irreversible analog, N-chloroethyl clonidine exhibit some dl-adrenergic activity as well, and were found to be poor activators of OSAC. In terms of the relatively limited effectiveness of q’z-agonists and the fact that the AC-coupled 0A receptor mediates its actions via stimulation of AC and not inhibition of this enzyme, the 0A receptor is unlike theCYz- receptor of ‘vertebrates, although it does recognize imidazolines and phenylethylamines with high affinity. The 107 non-adrenergic nature of the 0A receptor is underscored by the fact that catecholamines are also poor activators of OSAC (Evans, 1980; Orr gt 11., 1985). The antagonist profile for the two tissues exhibits a number of novel antagonists which have not been previously shown to inhibit the OSAC. The most effective antagonist in the Sf9 cells was the ergot alkaloid, ergotamine which has never been tested as an antagonist of OSAC. It has however been tested as an agonist of this receptor in cockroach hemocytes and gotpus ggrdiacum, and was found to be a poor activator (Orr, 1985). The significant difference between Sf9 and nerve cord, again suggests differences in receptor recognition in the two systems. The imidazoline antagonist, phentolamine is routinely used to inhibit OSAC (Hollingworth and Johnstone, 1982) and is very effective in Sf9 cells and nerve cord. To date however, the most potent antagonist of OSAC has been (1)-mianserin (Evans, 1981; Orr gt 11., 1985). The two isomers of this tricyclic compound have not been previously tested against OSAC. The Sf9 receptor is able to differentiate between these isomers, with the (+) isomer being 6 times more effective than the (-) and the (3;), having an intermediate affinity. The nerve cord receptor shows an overall high affinity for all three isomers of mianserin and the mianserin analogues, CGSllO49A and CGSlS413A. Mianserin, however has also been shown to exhibit insect-specific affinity for this receptor 108 (Hollingworth and Johnstone, 1982). It is interesting to note that (+)-mianserin is more effective in Sf9 cells (similar to vertebrate 5-HT receptors) whereas the cockroach nerve cord OA-receptor has a greater affinity for the (-)- mianserin analogue. The imidazolines act both as agonists and antagonists of the 0A receptor (see Chapter 1) . This is the first report of the high affinity of idazoxan and its analog, RX821002A, in inhibiting OSAC. These antagonists have a better affinity for the Sf9 receptor, although idazoxan is more effective than RX821002A in the cockroach. This suggests that perhaps the Sf9 receptor has an overall slightly higher affinity for imidazoline antagonists. Selective antagonists of the dz-adrenergic receptor such as yohimbine and its diastereoisomer, rauwolscine are poor inhibitors in both insect systems, again suggesting that although the OA-receptor recognizes certain 0(2- compounds, it is not blocked by many ag-classical compounds. Selective q’I-antagonists such as corynanthine and prazosin are also poor OSAC inhibitors, as is the B-antagonist, propranolol, strongly suggesting against any pharmacological similarity of the 0A receptor with these two subtypes of the vertebrate adrenergic receptor. Overall, it does seem that of those antagonists that are most effective in each of these two insect systems, those used in the Sf9 cells appear to exhibit a shift in 109 affinity (i.e. up to 10 times better) compared to those in the nerve cord. In comparison with Evans classification (1981) in which he describes OA-2 receptors as having the following ROP: metoclopramide >> chlorpromazine, yohimbine, whereas OA-2A and OA-2B receptors respectively exhibit the following ROP: metoclopramide > (1)-mianserin > chlorpromazine >> cyproheptadine and metoclopramide > mianserin and chlorpromazine is roughly equipotent to cyproheptadine. The profile seen in the Sf9 and cockroach receptors, respectively is: (1)-mianserin > chlorpromazine > metoclopramide > cyproheptadine and (1)-mianserin >> cyproheptadine > chlorpromazine > metoclopramide. Neither the Sf9 nor the cockroach AC-coupled OA receptor precisely fit the OA-ZA or OA-2B subtypes. These studies point out the lack of universal applicability of the locust classification. Recent data implicates the nucleus reticularis lateralis (NRL) of the medulla oblongata as an important site for the hypotensive action of the a’Z-agonist, clonidine. Studies by Bousquet gt 11. (1984) found that 6Y2- selective catecholamines were not active in this region, however imidazolines were able to induce this hypotensive effect, thus suggesting the existence of imidazoline- preferring sites. Other studies (Boyajian gt 11., 1987 ; Bricca gt 11., 1989) have provided anatomical evidence that idazoxan labels a heterogeneous population of A’z-sites 110 (which are also labelled by clonidine). In fact, one population of these was selectively labelled by rauwolscine, a potent antagonist of pre- and post-synaptic a’Z-receptors (Timmermans gt 11., 1984), i.e. these rauwolscine-sensitive sites appeared to be a subset of the idazoxan sites. In rabbit fat cell membranes [3H]idazoxan labels non-0(2 sites. Binding to these sites is not blocked by epinephrine, NE, or yohimbine, but is inhibited by the imidazoline, naphazoline (Langin and Lafontan, 1989). In these fat cells, [3H]yohimbine did not label the 0(2- receptor, but the presence of these qg-sites was determined by [3H]UK-14304 binding. UK-14304 exhibits greater 42/41 selectivity than yohimbine (Doxey g 11., 1983). In the rabbit kidney, two separate binding sites have been solubilized and partially characterized as the ag-adrenergic receptor and the imidazoline/guanidinium receptive site (IGRS). This IGRS, also referred to as the imidazoline- preferring receptor (IPR) is rauwolscine-insensitive but labelled by [3H]idazoxan (Parini gt 11., 1989). Similar to the NRL sites, the IPR/IGRS does not recognize endogenous ligands for known membrane-receptor proteins, but does recognize the clonidine-displacing substance (CDS), a small non-catechol, non-protein isolated from bovine brain (see Atlas and Burstein, 1987) . Although there have been at least three IPR's suggested, in addition to the dz-receptor (Michel and Insel, 1989), the possible physiological 111 relevance of these IPRs has not been established. The existence of these IPRs in vertebrates has prompted me to re-evaluate OA pharmacology in insects. In general, ‘é-specific agonists such as medetomidine, UK-14304 and BHT- 933 are relatively poor activators of the cockroach nerve cord and Sf9 cell OSAC. The imidazoline series, clonidine, naphazoline and tolazoline are more effective than the latter series, however in vertebrates these compounds have greater ”S-activity than IPR-activity. In fact, the most potent OSAC-agonists are the imidazolines, XAMI and NC5. A similar trend is observed in the case of IPR-antagonists such as the imidazolines, phentolamine, idazoxan and RX821002A. These compounds are more generally effective in insects than classical high-affinity aa-antagonists such as rauwolscine, yohimbine and MPV-295. The IPRfiqa-adrenergic guanidinium agonists, guanfacine and guanabenz, also exhibit a relatively high (antagonist) affinity for the OSAC-coupled receptor. Although, there are differences in the ROP of these imidazolines (i.e. vertebrate IPR vs. insect OA- receptor), the 0A receptor appears to exhibit greater pharmacological similarity to the IPR than the qg-receptor. Recently, work done by R.B. Raffa (personal communication) clearly demonstrates the potent antinociceptive potential of the OA agonists, XAMI and DCDM in mice. These effects were mimicked by GA (Raffa gt 11., 1989), blocked by phentolamine and are naloxone-insensitive, leading to the speculation 112 that IPR’s are involved. Further evidence for the effectiveness of imidazolines in stimulating octopaminergic sites has been reported in the ventral eyes (B.A.Battelle, personal communication) and lateral eyes (R.B. Barlow Jr., personal communication) of L1mu1us, in which tissue-specific differences have been demonstrated in the ROP of clonidine, naphazoline, tolazoline and NC5. In both tissues however, XAMI is the most potent activator tested, both of OSAC and OA-mediated neural activity. Overall, these data provide convincing evidence for the pharmacological diversity of the insect 0A receptor, but at the same time define some similarities, particularly with regards to the effectiveness of imidazolines at this site. CHAPTER 3 Identification of a Novel Signal-Transduction Mechanism for Insect-Octopamine Receptors: Elevation of Inositol-1,4,5- Trisphosphate Levels 113 114 INTRODUCTION Activation of receptors for hormones and neurotransmitters which specifically hydrolyze membrane phosphoinositides (PI) results in an increase in intracellular calcium (Ca2+). This perturbation of membrane phospholipids represents a fundamental transduction mechanism that initiates a signal cascade resulting in the mobilization of Ca2+, the activation of protein kinase C (PKC) and the release of arachidonic acid (Berridge, 1984). The key reaction for this transducing mechanism involves a receptor-mediated. hydrolysis. of phosphatidylinositol 4,5- bisphosphate (PIPZ) to yield diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (1P3), both of which may function as second messengers to initiate the signal cascade. It is now generally agreed that a number of agonists (neurotransmitters) interact with receptors that are linked via a G-protein to a phosphoinositidase or phospholipase C (PLC) located on the inner plasma membrane which when activated, hydrolyze membrane phospholipids. Examples of such systems include noradrenaline (0(1), acetylcholine (M), serotonin (5-HT1C and S-HTZ), histamine(H1), glutamate (quisqualate), bradykinin (82), vasopressin (V1), neuropeptide Y and a variety of tachykinins, including substance P (Nahorski, 1988). The action of 1P3 is rapid (seconds) resulting in a 2+ rise in cytosolic Ca concentrations of up to 2 to 5 fold 115 that of resting levels (Eichberg and Berti-Mattera, 1986). The transient nature of this effect is due to the fact that IP3 is rapidly metabolized either by dephosphorylation to IP2 or by phosphorylation to inositol 1,3,4,5- tetrakisphosphate (IP4) . IP2 can then be hydrolyzed by specific phosphatases, ultimately to form D-myo-inositol which is recycled to produce more phosphatidylinositol. In some systems IP4 is believed to be involved in the entry of Ca2+ across the plasma membrane (Irvine and Moor, 1986). The ability of IP3 to mobilize intracellular Ca2+ is mediated by its binding to an endoplasmic reticulum- associated specific, high-affinity receptor which consists 2+ of a Ca -selective channel. The IP3-receptor has been purified from rat cerebellar membranes where it occurs in high concentrations (Supattapone gt 11., 1988; Bredt gt 11., 1989) . There is mounting evidence that the source of this IP3-regulated intracellular pool of Ca2+ is not mitochondrial but may be a component of the endoplasmic reticulum and may even be contained in a novel organelle termed a 'calciosome’ (see Exton, 1988). 2+ The internal Ca stores of most cells are limited and 2+ thus, sustained agonist effects on cytosolic Ca require alterations at the plasma membrane to allow Ca2+ regeneration from extracellular sources. Unfortunately, the mechanisms by which agonist-induced activation of these receptors results in opening of plasma membrane Ca2+- 116 channels or inhibition of the Ca2+-ATPase pump remains obscure. However, it is accepted that any build up of Ca2+ in the cytosol is remedied by both a sequestration in the mitochondria and by binding to calmodulin (Abdel-Latif, 1986). To date there have been a limited number of studies implicating PI turnover as a signal-transduction mechanism in insects, although the number is rapidly increasing. In fact, work in the blowfly salivary gland was instrumental in generating a great deal of interest in the field of PI turnover (see Lummis gt 11., 1990). More recently, evidence has been presented for the carbachol-mediated production of 1P3 in locust metathoracic ganglia (Trimmer and Berridge, 1985), proctolin-mediated effects in locust extensor tibiae (Worden and O'Shea, 1984), oviduct (Lange, 1988; A.B. Lange, personal communication) and mandibular closer muscle (Baines gt 11., 1990; R.G.H. Downer, personal communication) and hypertrehalosemic factor-mediated effects in cockroach dispersed fat body cells (J.E. Steele, personal communication). Most of these studies have made use of ion- exchange chromatography or HPLC techniques to detect the accumulation of labeled inositol monophosphates. Despite the widespread distribution of octopamine (0A) receptors in many insect tissues no direct evidence has been available to suggest the involvement of the PI cascade as a signal- transduction mechanism for this biogenic amine. 117 Consequently, the objective of the present study was to characterize and analyze the potential of a radioreceptor assay (RRA) for IP3 detection in insect tissues. Because these RRA kits were supplied free of charge for premarket testing, only a finite number of experiments could be performed. Brain slices from the American cockroach, £g11111§gt1 ameticana L. and intact cells from the Fall armyworm, Spodopter1 frug1pgtda (Sf9) were used to test these kits and the ability of OA and its agonist, XAMI (see Chapter 1), to stimulate 1P3 production was investigated. MATERIALS AND METHODS Insects Adult males between 1 and 3 months after the final molt were taken from a stock colony of American cockroaches, Egtip1a1et1 1meticana L., as described in Chapter 1 (Materials and Methods). Cell Culture Sf9 cells were reared at 27°C as described in Chapter 2 (Materials and Methods) and used no later than 2 days post- confluence. Preparatory Period Prior to dissection, male cockroaches were held without food, in individual plastic petri dishes for 1 to 3 hours. Adequate water was placed in each petri dish and the insects 118 were kept at room temperature until dissection. Tissue culture flasks containing Sf9 cells were removed from the 27°C incubator and kept on the bench top at room temperature until used. This period was no longer than 1 hour. Dissection of Cockroach Brain The head was removed and pinned, ventral side up, in a wax dish containing 1 ml ice-cold insect saline (0.17 M NaCl, 6.0 mM KCl, 2.0 mM NaHCO3, 17.0 mM glucose, 6.0 mM NaHZPO4.HZO, 2.0 mM CaC12,2H20, 4.0 mM MgC12.6H20) at pH 7.0. A longitudinal cut was made in the head capsule from the rostral to the caudal end of the head. Two perpendicular cuts were made and the cuticle was pinned back to expose the two cerebral lobes. Any adhering tissues were removed and the optic lobes were severed. The brains were gently removed from the head and quickly immersed in glass test tubes containing 0.5 ml of ice-cold insect saline and stored on ice. In the case of pretreatment studies, the brains were dissected and directly transferred to the pharmacological agent (on ice). For each replicate, generally two brains were used (except for protein linearity and dilution experiments). Sf9 Intact Cell Collection The Sf9 cell-containing flasks were decanted to remove as much of the media as possible. To each flask, 15 ml of insect saline (maintained at room temperature) was added, and the cells were gently scrapped. The cell mixture was 119 centrifuged at 900 9 (room temperature) for 5 min. The supernatant was decanted and a second wash performed. The cells were diluted in insect saline and stored at room temperature for no longer than 15 min prior to use. Crude Brain-Slice Preparation To slice the brains rapidly and at a cold temperature, all utensils used during the procedure were precooled by placing them on ice. Initially, a styrofoam container, with a vent cut out at the top lip, was filled with liquid nitrogen and a glass petri dish was placed upside-down on top of the styrofoam container such that the glass surface was cooled rapidly. This procedure resulted in parts of the brains becoming frozen to the petri dish and thus resulted in tissue damage. The method of choice was to use a square styrofoam box (1/2 inch thick walls) which was filled with crushed ice. A cleaned glass petri dish was placed (upside- down) directly on the ice, the box was covered with the styrofoam lid and placed in the refrigerator to cool for at least an hour. Since the box was large enough, cleaned (95% ethanol and distilled water) utensils such as blades and shaved wooden sticks (used as spatulas) were also stored in the box. The brains were gently placed on the petri dish and sectioned with a blade in 10 parallel cuts from anterior to posterior and then in 10 parallel cuts from left to right. This was possible with some degree of consistency since the brains measured approximately 1 x 2 mm. The 120 consistency of the brain tissue is such that despite these cuts, the brains can be transferred without much difficulty or loss of sections. The brains were immediately transferred to another set of glass test tubes (on ice) containing 0.5 ml of insect saline and kept on ice for 30 min. Trichloroacetic Acid Extraction of Inositol 1,4,5- Trisphosphate Brain slices or Sf9 intact cells were placed in 1.5 ml polypropylene eppendorf tubes containing 200 ul of buffer, or 200 pl of treatment and in the case of time '0’ blanks, 200 pl buffer and 1 ml of 1M trichloroacetic acid (TCA). The reactions were performed in a shaking water bath at 27°C or 30°C, for the Sf9 cells and brain slices, respectively. At the end of 10 min the reaction was terminated by the addition of 1 ml of ice-cold TCA (1 M) and the eppendorf tubes were placed on ice. Using a diamond knife, glass pasteur pipettes were out such that the bore size would allow uptake of the brain slices without any adherence to the sides of the pipette. The tissue and incubation medium were removed using these out pipettes and transferred for homogenization in a glass- teflon homogenizer (400 rpm, 15 strokes). The homogenized samples were returned to the 1.5 ml eppendorf tubes which were placed on ice and the homogenizer was washed with distilled water between each sample homogenization. The 121 samples were centrifuged at 2000 g for 15 min (4°C). The supernatant was transferred to 3 ml polypropylene test tubes and kept at room temperature for 15 min and then placed on ice. Removal of Trichloroacetic Acid from Extracts Based on the method of Challiss gt 11. (1988) a 3:1 solution of 1,1,2-trichloro-1,2,2-trifluoroethane (TCTFE) and trioctylamine was mixed just prior to use. To each sample 2 ml of this mixture was added and mixed by vigorous trituration for 15 sec using disposable polypropylene pipettes. The mixture was allowed to sit for 3 min at room temperature. In earlier experiments, this latter step was replaced by a 1000 9 spin at 4°C for 5 min, but was subsequently found to be unnecessary. At this point two layers become visible; a clear aqueous top layer which contains the IP3 and a cloudy bottom layer. The top layer was carefully removed by tipping the test tube at 45°. Extreme care was necessary to avoid drawing up any of the lower solvent layer as this greatly reduces the efficiency of the extraction. A blank sample containing insect saline and TCA was also extracted in every experiment. Once extracted, the samples were stored on ice while the RRA was prepared (no longer than 1 h). 122 Analytical Procedures Chemicals XAMI was synthesized according to the method of Kristinsson and Traber (1981). Scintillation cocktail (Safety-Solv), was obtained from Research Products Int., Mt. Prospect, IL. All other compounds and solvents were purchased from Sigma Chemical Co., St. Louis, MO. Protein Assay Protein concentrations were estimated using the method of Lowry gt _1. (1951) with bovine serum albumin as the protein standard. Tissue pellets produced after homogenization and centrifugation were preserved and diluted with 0.15 M sodium hydroxide and vortexed or sonicated until mixed thoroughly. Aliquots of the tissue suspension were then assayed for protein using this procedure (as described in Chapter 1: Materials and Methods), with the inclusion of 0.15 M sodium hydroxide in all the standard curve samples. Inositol-1,4,5-Trisphosphate Radioreceptor Assay 1P3 measurements were performed using a commercially available RRA kit obtained for premarket testing through the courtesy of New England Nuclear Research Products, MA. The basic principle of an RRA is competitive ligand binding where a radioactive ligand competes with a non-radioactive ligand for a fixed number of receptor binding sites. 123 The NEN-RRA kit uses a membrane preparation (supplied in lyophilized form) derived from calf cerebellum which contains the 1P3 receptor (Supattapone gt 1_., 1988). [3HJIP3 can be measured by centrifuging the membranes and counting the amount of radioactivity in the pellet. In this kit, the concentration of receptors and [3H]IP3 have been selected for optimal sensitivity and reproducibility. Unlabeled 1P3, added to the incubation medium as a standard or unknown sample, competes with [3H]IP3 for the receptor sites and thus the amount of 1P3 in the sample is inversely proportional to the amount of radioactivity in the pellet (i.e. [3H]IP3 bound). In this assay, non-specific binding is measured by saturating the receptor with excess unlabeled inositol hexaphosphate (IP6) rather than IP3. IP6 produces the same level of non-specific binding as high concentrations of 1P3 and is used because it is much more stable than 1P3. The RRA required the use of polypropylene minitubes (supplied with the kit) which were placed in specialized racks and kept on ice. In each tube 100 )11 of appropriate blanks (IP6 or distilled water), standard or sample were added. The reaction was initiated by the addition of the receptor/tracer (400 )11) and all samples were briefly vortexed. The samples were incubated on ice, in the refrigerator for 1 h, sealed in parafilm and placed in a tightly sealed styrofoam container. The reaction was 124 terminated by centrifuging the samples at 2500 g (4°C) for 10 min. The supernatant was decanted by placing the minitubes in tightly fitting foam holders and inverting on an absorbent surface. This procedure was repeated until the minitubes contained no more liquid. Fifty pl of 0.15 M sodium hydroxide was added and each sample was vortexed and incubated at room temperature for 10 min. The minitubes (containing the samples) were again vortexed and put in 20 ml glass scintillation vials containing 10 ml of scintillation cocktail. The vials were inverted until the contents were homogeneously mixed and counted in a beta- scintillation counter, Model 6895 (TM Analytical Inc., Elk Grove Village, IL). In earlier experiments, duplicate IP3 standard curves were generated, however, since these curves were reproducible (with an error of less than 5 % of the mean), only single curves were used for all future experiments. Due to the labile nature of the 1P3 standard, it was critical to use all 1P3 solutions as soon as possible (i.e. within 4 days). A typical example of average net counts per minute for each IP3 standard (0.12 to 12.0 pmol) as a percent of the average net counts per minute of the IP3 blank (i.e. B/Bo) for the RRA is shown in Figure 25. To demonstrate the labile nature of the solubilized IP3 standard,in three of the reported experiments the 1P3 standard was used within 4 H5 120 C3—C3 Expt. 1 new std. . -- Expt. 2 new std. 100- a A A—AExpt. 3 new std. §8\A A—AExpt. 4 old std.) 80- \\\\. ‘\‘\~‘k 0\\\ a? - §§\\\\\ . - E 6°' \‘\ . 3\ 20‘ Ii 0 f ' V ' *fi—T ' r ' T ' ' ' ' T ' l 0.1 1.0 ICLO IR3 PRODUCTION (pmol) Figure 25. Representative D-myo-inositol 1,4,5- trisphosphate standard curves (0.12 to 12.0 pmol) obtained with a tritiated radioreceptor assay. Each curve represents a separate experiment with new and old standard indicating the use of the standard (without refreezing) within 4 and by 6 days of solubilization, respectively. 126 days (i.e. new standard). The fourth standard curve is shifted to the right, indicating reduced sensitivity with standards which were 6 days or older. Statistical Procedures All descriptive statistics were calculated using a Texas Instrument TI-60 calculator and group means were compared with a two—tailed t-test (Zar, 1974). RESULTS Characterization of the IP3 Radioreceptor Assay for Use with Insect Tissues In order to characterize the RRA for its usefulness in studying insect tissues, the effect of protein concentration on basal IP3 production was monitored after 10 min of incubation. As seen in Figure 26A, 1P3 concentration was linear for 1, 2 and 4 cockroach brains per sample. Figure 26A is a representative experiment; the data points yield an r2 = 0.993. The protein concentration of these same brain samples was then determined using the Lowry protein assay (see Materials and Methods) and is presented in Figure 26B. Again, the data points exhibit a strong correlation of r2 = 0.999. Because of the fact that basal IP3 production was linear within this range, all subsequent experiments (except where indicated) employed 2 brain equivalents per sample. Although according to the manufacturer, the RRA exhibits high specificity for IP3 (100% competition), the 30 127 g; A o 25 - ._,...-O :> y, 3 JE 20. . z: , .9. . "' I— 1 5 . ,x" g Q o . 82 Q_ I O .. r: -w E: ',r’ 5 "'3... Q T I I I r r 1 2 3 4 BRAIN EQUIVALENTS PER SAMPLE 30 E B E ,,x 52 25- ,CFJ > 0 1w” 3, 20 ~ ' Z .- ..d”. .0- ..." " '- 15 « g 0 o 2% , Q, 10‘ P1 9.- 5 '"Or 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 PROTEIN CONCENTRATION (mg/scmpIe) Figure 26. Representative protein linearity curves for the basal production of inositol 1,4,5-trisphosphate in cockroach brain slices. (A) Protein line rity expressed in terms of brain equivalents per sample (r =- 0.993). (B) Data from Figure 30A expressed as protein concentration per sample (r2 = 0.999). 128 compatibility of this assay for use with insect tissues has not been examined previously. Tests of protein linearity and spike recovery were performed (Figure 27) using buffer (insect saline), buffer + spike (2 pmol of IP3 standard), Sf9 intact cells, Sf9 cells + spike, brain slices, brain slices + spike. Samples of each three type (i.e. buffer, Sf9 cells, brain slices) were diluted to varying concentrations using a 100% control of insect saline, 4 x 106 cells per sample and 6 brain equivalents per sample, respectively, and aliquots of each dilution were assayed for 1P3 content. A portion of these samples were "spiked" with a known amount of 1P3 standard (i.e. 2 pmol) and their levels were similarly assayed. In all cases, basal 1P3 levels were linear with respect to the protein concentration i.e., r2 = 0.97, 0.99, 0.99 and 0.99 respectively for the samples of brain tissue, brain tissue + spike, Sf9 tissue, and Sf9 tissue + spike. Respective levels of IP3 for buffer and buffer + spike samples were also linear (r2 = 1.0, 0.99) with respect to the concentration of buffer (diluted with distilled water). The recovery of IP3 in samples spiked with 2 pmol of 1P3 ranged from 95-100%, 87-90% and 85-90%, for buffer, brain slices and intact Sf9 cell samples, respectively. It is evident from the recovery ranges that addition of tissue, either brain slices or Sf9 cells, reduces the recovery compared to the buffer samples, thus suggesting some form of A 6'0 ‘ O SUPER .E O BUFFER + SPIKE E 5.0 . A Sf9 TISSUE O A Sf9 TISSUE + SPIKE { CI BRAIN TISSUE '5 4.0 - I BRAIN TISSUE + SPIKE ”if, E: Mg ''''' z 3 .0 - . ”Aw-T .0. #1-! I'- .-" ,,,,,, / O “I... ....... .-. D 2.0 d E"... «If,- f“ 1.5/J 8 f,w-:'//RD—-' /: a: .g3 #- .1—1- 0.. 1 ’0 . .,.-'".‘...-"".’ Aw!” I") .«"'..r'iw/ 0.0 --=—="“ '-’ ”:9, . 3 1 . 9 O 20 40 60 80 I 00 1 20 PROTEIN CONCENTRATION (% OF CONTROL) Figure 27. Inositol 1,4,5-trisphosphate production and spike (2 pmol 1P3) recovery as a function of protein concentration. IP recovery for insect saline, cockroach brain slices and intact Sf9 cells ranged from 95-100%, 87- 90% and 85-90%, respectively. Data are from a representative experiment with 100% protein =- 4 million cells/sample and 6 brains/sample, respectively for cockroach brain slices and Sf9 intact cells. Correlation coefficients for the six curves, buffer, buffer + spike, brain tissue, brain tissue + spike, Sf9 tissue and Sf9 tissue + spike were 1.00, 0.99, 0.97, 0.99, 0.99, respectively. 130 interference in those samples. Pharmacology of IP3 Production The pharmacological nature of IP3 production in cockroach brain slices was investigated (Table 11). Treatment of brain slices with 0.1 mM OA and 0.01 mM XAMI, significantly increased IP3 levels by 21% and 66%, respectively. Maximally stimulating doses of 0.1 mM dopamine (DA), serotonin (5-HT) and the muscarinic agonist, carbachol, had no significant effect of 1P3 levels. The OA- and XAMI-mediated IP3 production was blocked by coincubation with 0.1 mM phentolamine. Pretreatment of brain slices with 0.1 mM of the muscarinic antagonist, atropine, was accomplished by placing the brains directly into atropine rather than in insect saline, immediately following dissection. This treatment resulted in a significant reduction of basal IP:3 levels (35%). Incubation of brain slices in the presence of 1 pM fOrskolin, 0.5 mM dibutyryl cyclic AMP (dbcAMP) and mastoparan (75 pg/ml) had no significant effect on IP3 production. Two experiments using 50 mM potassium chloride (KCl) to stimulate IP3 production were undertaken using brain slices, but no change in basal IP3 production was observed (data not presented). In the case of intact Sf9 cells, basal production of IP3 was observed at 27°C (for 10 min) to be 9.0 to 12.0 pmol/ 10 min/mg. However, attempts to show stimulation by 0.1 mM OA and 0.01 mM XAMI failed. Ln Table 11. Effect of various compounds on 1P3 production in cockroach-brain slices. Treatment IP3 Production (% Control)a Octopamine (0.1 mM) Dopamine (0.1 mM) 5-HT (0.1 mM) XAMI (0.01 mM) Carbachol (0.1 mM) Octopamine + Phentolamine (0.1mM) XAMI + Phentolamine Atropine (pretreat. 0.1mM) Forskolin (1.0 MM) Dibutyryl Cyclic AMP (0.5mM) NaF (5.0 mM) Mastoparan (75 pg/ml) 121 i sbvc 109 i 10 91 i 6 166 i 3‘D 109 i 8 94 i 5 86 _+_ 9 65 1, 8b 118 _+_ 30 127 i 20 169 i 610 90 i 20 a - Control 1P3 production for 20 experiments = 75 i 9 pmol/10 min/mg. b - Significantly different from basal 1P3 production (p < 0.05). c - Values are the mean 1 SEM for 3 to 6 separate experiments. 132 DISCUSSION Characterization of an Inositol 1,4,5-trisphosphate Radioreceptor Assay in Insects Phosphoinositides constitute 2 to 8% of the total cell lipid in eukaryotic cells (Litosch, 1987) and several phosphoinositidases or phospholipases C (PLC) have been described that generate phosphoinositide-derived messenger molecules. More recently, a great deal of interest has been focused on the receptor-stimulated PIPz-specific PLC activity because it provides the link between occupation of many cell-surface receptors and formation of the intracellular messengers IP:3 and DAG, which respectively regulate intracellular Ca2+ and PKC activity (Lummis gt 11., 1990). The availability of a radioreceptor assay for measuring intracellular levels of 1P3 prompted the present investigation of this branch of the PI cascade in insects. Because a great deal of pharmacological data is available on the cockroach neural octopamine-receptor-coupled adenylate cyclase (e.g. Gole and Downer, 1979; Downer gt 11., 1985;), the cockroach was chosen as the model system. Furthermore, since a large number of vertebrate studies use brain slices to study receptor-mediated changes in PI levels (Berridge gt 11., 1982), a similar procedure was developed for cockroach brain tissue. This has proven to be an excellent preparation not only because of the ease with which it can 133 be prepared, but also because it appears to readily allow penetration of pharmacological agents to the appropriate receptor sites for subsequent generation of IP3. The IP3-RRA is a highly reproducible assay with a sensitivity of as low as 0.12 pmol per sample. One disadvantage of this procedure, however, is the labile nature of the 1P3 standard. Once dissolved, it must be used within 4 days or with time, the standard curve begins to shift to the right and thus sensitivity is reduced. The IP3 standard, however can be frozen once solubilized, and thawed up to 3 times without a significant loss of activity (unpublished observations). The major disadvantage of using this RRA is the tedious tissue preparation and IP3 extraction procedures, which need to be carefully performed for each individual sample. This is very time consuming and even once the TCA-IP3 extraction is complete, the samples are labile and should be used in the RRA immediately. However, the precision and reproducibility of this assay compensate for these disadvantages. This RRA has not been previously tested in insects and the initial objective of this study was to determine the suitability of this assay for insect tissues. The protein linearity observed for basal 1P3 production in cockroach brain slices suggests that 1P3 production can be expressed either in terms of brain equivalents or protein concentration. Because of the limited availability of these 134 kits, the investigative emphasis was placed on cockroach brain slices and hence, protein linearity was not determined for the Sf9 cells. In the case of both tissue types, a 10 min incubation period (exposure to pharmacological agents) was used. Unfortunately, a time course could not be determined, consequently all 1P3 levels are expressed per 10 min. It is possible that a 10 min period of exposure to agonist would result in an attenuated response due to receptor desensitization but similar experiments using this assay in vertebrates have used similar incubation times (Challiiss gt 11., 1988). In the blowfly salivary glands, IP3 accumulated within the first second after stimulation by S-HT (Berridge gt 11., 1984). This rapid time course of IP3 production is typical of that seen in many other systems (Berridge, 1984). Since most studies make use of the total incorporation of radiolabelled myo-inositol (Nahorski, 1988), Li2+ is often added to the incubation medium since it inhibits inositol 1- phosphatase activity (Sherman gt 11., 1981) and it can penetrate many different cell preparations and thus prevent inositol 1-phosphate (IPl) hydrolysis (Berridge gt 11., 1982). By inhibiting the conversion of IP1 to inositol and of inositol 1,4-bisphosphate (1P2) to IP, Li+ greatly amplifies the accumulation of inositol phosphates during agonist stimulation of tissues both it 1:13.132 and 11 111219 (Berridge gt 11., 1982). In the present study, Li+ was not 135 used because its use was not recommended by the manufacturer of the RRA kit and is in fact not necessary since the RRA only measures IP3 levels and cross-reactivity with the other PI hydrolysis products is negligible. Despite the specificity of the IP3-RRA, the maximal recovery of IP3 in any given sample can be affected by the presence of interfering compounds within the sample. In order to determine if maximal recovery was achievable, 1P3 production was assayed as a function of protein concentration and it was observed that the spiked and unspiked samples began to lose linearity with increased dilution. This may be due to two possible explanations: with increasing dilution, there is increased interference, and/or the accuracy of the 1P3 recovery is reduced. This interference can be better quantified in terms of the percent 1P3 recovery in the spiked samples and is in the range of 95-100%, 87-90% and 85-90%, respectively for the buffer, brain and Sf9 tissue. Therefore, there appears to also be some interference attributable to the tissue. This requires further investigation such as studying the effects of different buffers and tissue preparations, to wash out or reduce any interfering compounds. Again, because of the availability of a finite number of kits, this was not possible. The data does however indicate that this RRA can be used in cockroach neural tissue, as well as in an insect cell line with a limited compromise of sensitivity (BS-90% 136 recovery). Pharmacology of IP3 production in cockroach brain slices Once the suitability of the RRA was established, the next objective was to determine the existence of an OA receptor-coupled to PI hydrolysis in insect neural tissue. Cockroach brain slices and intact Sf9 cells had basal IP3 production of 75 i 9 pmol/10 min/mg and 9.1 to 12.0 pmol/10 min/mg (two unreported experiments), respectively. In rat cerebral cortex slices, basal IP3 levels of 20.4 i 0.8 pmol/6 min/mg were determined using this same RRA (Challiss gt 11., 1988). The low levels of basal 1P3 production seen in Sf9 cells could be due to a number of factors including a small density of PI hydrolysis-coupled receptors and non- optimal conditions such as composition of the incubation medium, time course and tissue preparation and concentration. Since the cockroach brain slices exhibited an easily measured basal IP3 level, the focus of the remainder of this study was on this tissue. The statistically significant IP:3 production seen in brain slices, in the presence of OA and its agonist, XAMI, suggests the involvement of an OA-like amine receptor which is coupled to PI hydrolysis. This case is further strengthened by the lack of stimulation seen when the tissue is treated with endogenously occurring amines such as 5-HT and DA. This OA- mediated effect can be blocked by the OA antagonist, phentolamine, suggesting the selective nature of this 137 response. The combined data, however, does not rule out the possible existence of other PI-coupled receptors in this system or the potential that the observed increase in 1P3 levels is not a direct OA-receptor-mediated effect. Definition of this response as purely "octopaminergic", would require a more extensive pharmacology and additivity studies. To date, there has been only one other study investigating PI hydrolysis in insect neural tissue. Trimmer and Berridge (1985) studied the uptake of [3H]inositol into crude slices of locust metathoracic ganglia. Anion-exchange chromatography was used to separate IPl, IP2 and IP3. However, these investigators failed to observe OA, S-HT, DA or carbachol stimulated production of IP3. Because they observed a dramatic decrease in levels of free inositol with corresponding increase in IPl, 1P2 and 1P3, suggesting that the turnover of the inositol phosphates may be linked to receptor function, they theorized that endogenous transmitters were being released by the tissue. Therefore, if the PI cycle were already turning at very high rates, this would explain the ineffectiveness of exogenously applied agonists. Also, when the tissue was pretreated with the muscarinic antagonist, atropine, there was a 30% reduction in basal IP3 levels, again supporting the theory of high basal levels of transmitter release. In cockroach brain slices carbachol is ineffective in 138 eliciting an 1P3 increase, however, similar to the aforementioned study, pretreatment with atropine reduced basal levels by 35%. This suggests that in this tissue there are probably elevated levels of acetylcholine which interact with muscarinic receptors and produce an elevated basal effect. In the presence of atropine pretreatment, the muscarinic receptors are blocked, reducing basal activity but blocking 1P3 production when the tissue is exposed to carbachol. This is a significant observation since it not only corroborates the Trimmer and Berridge study, but also suggests the applicability of this RRA for use in insect tissue. Little is known about neural muscarinic receptors in insects and recently I have been able to autoradiographically localize discrete, high density populations of these receptors in the cockroach calyces (Orr gt 11., in press). The function of the calyces is also not clearly understood, although these areas are known to receive sensory inputs from the antennal lobes (Schurmann, 1987) and it is possible that in the brain, stress such as dissection-induced trauma results in a massive release of acetylcholine and possibly other factors which increase IP3 levels. The diterpene forskolin is used in adenylate cyclase (AC) investigations to study the functionality of the catalytic subunit of the enzyme (Daly, 1984). Forskolin 139 does not produce a significant increase in 1P3 levels, suggesting that activation of AC does not indirectly increase IP3. To further examine the role of cAMP in PI turnover, the tissue was exposed to dbcAMP and again no significant effect on basal 1P3 levels was observed. These two observations suggest that within the 10 min incubation period, the agonist-induced elevation of 1P3 was not an indirect effect of elevated cAMP levels. Incubation with sodium fluoride (NaF) produced a significant increase of 69% over control. Sodium fluoride, and specifically aluminum fluoride (produced as a result of the interaction of NaF with aluminum from some glassware) acts. directly at the G-protein. Aluminum fluoride is thought to act by mimicking the effect of the erhosphate of GTP on the OI-subunits of G-proteins (Ross and Gilman, 1980; Kienast gt 11., 1987). Guillon gt 11. (1986) found that NaF increased IP3 accumulation with an ECso similar to that observed for AC-activation by NaF. These data along with the known ability of NaF to stimulate calcium mobilization in human neutrophils (see Blackmore and Exton, 1986) suggests a functional homology between the G8, G1 and the putative G-protein involved in receptor-mediated PIP2 hydrolysis. Therefore, the agonist-induced IP3 effects seen in cockroach brain, suggest the presence of a G-protein specific for this effect. Recently, the tetradecapeptide toxin, mastoparan, has 140 been isolated from wasp venom and shown to stimulate histamine release from mast cells (Okano gt 11., 1985) and enhance GTPase activity in several purified G-proteins, including Gi (Higashijima gt 11., 1988). In human polymorphonuclear leukocytes, mastoparan has been shown to induce a sustained activation of PLC, one dependent and one independent of a pertussis toxin-sensitive Gi (Perianin and Snyderman, 1989). Mastoparan has also been shown to enhance PI-kinase and PIP-kinase activities (Eng and Lo, 1990). In the cockroach brain, however, this effect is not observed. This result is perplexing but may have a number of causes. Since this is the first report of the use of mastoparan in insects, it is conceivable that the cockroach PLC is insensitive to mastoparan. It is also possible that the conditions for use were not optimal (i.e., concentration and time of Texposure to peptide; penetration problems). Unfortunately, these parameters could not be optimized. Wallace and Carter (1989) showed that mastoparan-induced activation of PLC purified from rabbit brain membranes required the presence of a higher concentration of Ca2+ than necessary for detection of enzyme activity. In the present study, the incubation medium contained 2 mM CaC12 (present in insect saline). It is possible that 2 mM Ca2+ may be inhibitory since there is precedence in the above mentioned 2+ study for the biphasic action of Ca on mastoparan-induced effects. Another basic problem may reside in the fact that 141 the present study uses intact tissue (brain slices). Wojcikiewicz and Nahorski (1989) found no effect of 20 pM mastoparan on [3H]inositol phosphate formation in intact human neuroblastoma SH-SY5Y cells. In contrast, [3H]inositol phosphate formation in electrically permeabilized cells stimulated with carbachol was inhibited by mastoparan. Inhibition of PIP2 hydrolysis was also observed (Wallace and Carter, 1989) at both high and low substrate concentrations if the molar ratio of mastoparan to PIP2 was greater than 1. Thus, these data suggest that the exact mechanism of mastoparan-induced elevation in IP levels is unclear. Although a limited pharmacology was generated, this study suggests the existence of a novel OA receptor(s) , coupled to PI turnover. However, because of the small response seen with OA, this is a difficult system to investigate. The effects seen with NaF, dbcAMP and forskolin suggest that there is a receptor-coupled PLC in the cockroach brain. It is also possible that this OA- receptor has a different pharmacological profile than the AC-coupled OA receptor. Indirect evidence for the existence of an OA-receptor coupled to PI hydrolysis has been previously presented in locust extensor tibiae muscle in which Evans (1984) has described a class of octopamine receptors (OA-l) which are not coupled to AC, but instead are involved in Ca2+ 142 mobilization. Jahagirdar gt 11. (1987), using an insect hemocyte cell line have been able to qualitatively show OA- induced changes in intracellular Ca2+ levels. This effect is blocked by the, OA antagonist, mianserin. Finally, a functional link between the AC system and inositol lipid hydrolysis has been demonstrated by Orr gt 11, (1988). Using tumor-promoting phorbol esters, which bind to and activate PKC, these investigators show enhanced basal and OA-stimulated cAMP production. This study suggests that OSAC can be modulated by agents stimulating the metabolism of membrane PI’s leading to the activation of PKC. It must be noted that there are still a number of parameters that would require optimization prior to further use of this RRA in insects. In fact, of a total of 4 RRA kits used for this study, experiments with one did not produce a measureable basal or OA-mediated IP3-elevation, although the standard curves could be reproduced. This and the limited access to the RRA has limited our ability to further investigate this system. There is however strong indication for the existence of PI-coupled octopamine receptors in cockroach brain. 143 CONCLUSIONS The present study describes a number of novel tools for studying the pharmacology and signal-transduction mechanisms of OA receptors. One of the greatest hurdles in studying these processes has been the lack of a specific, high- affinity ligand. Although ligands with high-affinity for the OSAC have been labelled and used for binding studies (e.g. [3H]OA, [3H]mianserin, [3HJNCSZ), they exhibit high levels of nonspecific binding. In the case of [3H]NCSZ, it has been recently shown (Kantham and Nathanson, 1990) that the ligand is primarily binding to a haemocyanin-like protein which is not the 0A receptor. The availability of an agonist such as XAMI provides a novel, high-affinity probe which exhibits an affinity higher than GA in a number of preparations and thus, may have broader use. Adenylate cyclase studies indicate that XAMI interacts primarily with the OA-receptor-coupled AC and its efficacy in stimulating cockroach nerve cord OSAC (Ka = 30 nM) suggests that it has a very high affinity for the 0A receptor itself. In radiolabelled form, XAMI could be used in a filtration binding assay, provided that nonspecific binding is at a sufficiently low level. Preliminary data suggest that XAMI can be tritiated with an acceptable level of specific activity. Pharmacological studies indicate that XAMI may be acting at the IPR in mice (R.B. Raffa, personal communication), thus suggesting the value of radiolabelled- 144 XAMI for studies of both vertebrate and invertebrate pharmacology. Another major problem in insect studies of OA receptor(s) by neurochemical methods involves the difficulty in obtaining sufficient quantities of tissue. The identification in this study of the Sf9 insect-cell line as a readily available source of OSAC overcomes this problem. This OSAC exhibits characteristics which are typical of other hormone-sensitive AC. The Sf9 cells are devoid of other amine-sensitive AC, making them a pharmacologically "clean" preparation with which to study the 0A receptor. Previous studies of the 0A receptor have also been limited by the availability of octopaminergic compounds. I have been able to identify a number of agonists and antagonists which are active at the OSAC, both in Sf9 cells and cockroach ventral nerve cord. This pharmacological profile presents evidence for the tissue- and species- differences observed in OA-receptor pharmacology. Based on the potencies of a number of selective “z-adrenergic compounds, the proposal that the insect 0A receptor is pharmacologically similar to the vertebrate aé-adrenoceptor is rejected. In general, octopamine receptors in Sf9 cells and cockroach nerve cord exhibit a greater preference for imidazoline-based compounds than for 0(2-receptor selective agents. To date, the existence of alternate signal-transduction 145 mechanisms has only been postulated for the OA receptor. The coupling of some 0A receptors to calcium mobilization has been suggested (Evans, 1984 ; Jahagirdar gt 11., 1987) and PI hydrolysis has been inferred from one study (Orr gt 11., 1988) . The present study demonstrates for the first time the existence of OA-mediated elevation in 1P3 levels in cockroach brain. The usefulness of the DuPont 1P3 radioreceptor assay in insect tissue is also demonstrated. However, a number of questions remain unanswered: Is this elevation of IP3 a direct effect of receptor occupation by 0A or is it modulated by some other messengers? Is the PI- coupled OA receptor pharmacologically distinct from the AC- coupled receptor? These questions can only be answered with further pharmacological and biochemical studies of this effect. unfortunately, the OA-mediated stimulation is quantitatively small and will make studying this process difficult. Overall, by providing us with novel tools, it is hoped that this study has significantly increased our ability to study octopamine receptors. In addition, the pharmacological characterization of the 0A receptor will help in developing receptor-active agents which can be used as pesticides and will also help in the understanding of the evolutionary relationship between invertebrate OA receptors and the vertebrate adrenergic receptors. Finally, the discovery of 0A receptors coupled to PI-hydrolysis provides the first 146 direct evidence for the association of 0A receptors with PI- hydrolysis and thus adds a further level of complexity to the understanding of 0A receptors in insects. APPENDIX Compounds Used in the Determination of the Pharmacological Properties of Octopamine-Sensitive Adenylate Cyclase in Sf9 Cells and Cockroach Nerve Cord Membranes. 147 148 APPENDIX Compounds used in the determination of the pharmacological properties of octopamine-sensitive adenylate cyclase in Sf9 cells and cockroach nerve cord membranes. COMPOUND CATEGORY SUPPLIER Atipamezole qa-antagonist Farmos Grp. Ltd., Finland BF9290 OA antagonist Bayer Fin, Germany BHT-933 aé-agonist Boehringer (+)-butaclamol CGSllO49A CGSlS413A Clonidine Chloroethyl clonidine Chlorpromazine Corynanthine Cyproheptadine Demethylchlor- dimeform (DCDM) DA-antagonist mianserin analogue mianserin analogue ahfixl-agonist irreversible clonidine analogue OA antagonist Da antagonist d-antagonist ai-antagonist OA antagonist 5-HT antagonist Histamine antagonist formamidine partial OA agonist Ingelheim, CT Research Biochem. Inc., MA Ciba-Geigy, NJ Ciba-Geigy, NJ Sigma Chem. Co., MO Research Biochem. Inc., MA Sigma Chem. Co., MO Sigma Chem. Co., MO Merck Sharp & Dohme Lab., NJ R.M. Hollingworth Dopamine (DA) Ergotamine Guanabenz Guanfacine S-hydroxy- tryptamine (S-HT) Idazoxan (RX781094) Medetomidine Metoclopramide (1)-Mianserin MPV-295 N-acetyl octopamine Naphazoline NC5 [2-(2,6-diethyl- phenylimino)- imidazolidine] D,L-octopamine ORG 4360 (+)-mianserin ORG 5859 (-)-mianserin 149 DA agonist ergot alkaloid aé-agonist ag-agonist 5-HT agonist d -antagonist 0A antagonist aa-agonist DA antagonist OA antagonist “-antagonist S-HT1 antagonist OA anEagonist az-agonist OA metabolite M/ l-agonist 0% partial agonist 0A agonist OA agonist mianserin isomer mianserin isomer Sigma Chem. Co., MO Sandoz Pharm., NJ Wyeth-Ayerst Res., NJ Boehringer Ingelheim, CT Sigma Chem. Co., MO Reckitt & Colman Pharm. Div., U.K. Farmos Grp Ltd., Finland A.H. Robins Co., VA Research Biochem. Inc., Farmos Grp Ltd., Finland Sigma Chem. Co., MO Ciba-Geigy Corp., NJ G. Shi Sigma Chem. 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