Pharmacology of the octopamine receptor from locust central nervous tissue (OAR3)

Transcription

1 BrRish Journal of Pharmaology (1995) B 1995 Stokton Press All rights reserved /95 $9. O Pharmaology of the otopamine reeptor from loust entral nervous tissue (OAR3) Thomas Roeder Universitdt Hamburg, Zoologishes nstitut, Dept. Neurophysiologie, Martin-Luther-King-Platz 3, D-2146 Hamburg, Germany 1 The present study haraterized highly effetive agonists from different lasses of ompounds for the neuronal otopamine reeptor (OAR3) of the migratory loust (Lousta migratoria L.). Biogeni amines and phenyliminoimidazolidines (Pls) were employed for the study of struture-ativity relationships. 2 The highest affinity Pls were predominantly those with substitutions at the positions 2 and 4 of the phenoli ring (e.g. NC 7, K, =.3 nm, NC 8, K, =.81 nm). Substitutions at these positions always had positive effets on the affinity of the respetive agonists. 3 Substitutions at the positions 3, 5 and 6, however, always had negative effets on the affinity. At the position one of the phenoli ring, heteroyli substituents are preferred. 4 Some PMs had a more than 3 times higher affinity for OARs than for a-adrenoeptors whih are the vertebrate homologues of the inset otopamine reeptors. 5 The only non-p with subnanomolar affinity was the aminooxazoline derivative AC 6 (K1 =.92 nm). 6 A variety of substanes with known insetiidal ativity suh as hlordimeform, demethylhlordimeform, amitraz or AC 6 had high affinity for the loust neuronal otopamine reeptor. Keywords: nset; nervous system; Lousta; phenyliminoimidazolidines; reeptor; otopamine ntrodution Sine its disovery in the salivary glands of Otopus (Erspamer & Boretti, 1951), the biogeni monoamine otopamine (p-hydroxyethanolamine) has been well established as a neurotransmitter, neurohormone, and neuromodulator in various invertebrate speies (Axelrod & Saavedra, 1977; Orhard, 1982; Evans, 1985; Saavedra, 1989). Espeially in insets, the modulatory role of otopamine in different peripheral organs has been studied in great detail (Evans, 1985) and also in the entral nervous system some physiologial effets of otopamine have been desribed (Nathanson & Greengard, 1973; Orhard, 1982). t is essential for the oordination of loomotor behaviour, suh as flying and running (Sombati & Hoyle, 1984) and even omplex funtions of the nervous system, suh as learning and memory (Biker & Menzel, 1989; Dudai et al., 1987) are under the ontrol of this neuroative substane. Reeptors for otopamine were studied in neuronal (Nathanson & Greengard, 1973; Roeder & Geweke, 199) as well as in non-neuronal tissues (Evans, 1981; Nathanson, 1985a,b). n the latter, three different reeptor lasses (OAR,, OAR2A and OAR2B) ould be distinguished (Evans, 1981). Most otopamine reeptors of non-neuronal tissues studied so far, have pharmaologial features omparable to the lass 2A reeptor (Evans, 1985). n the nervous tissue a peuliar reeptor lass is present (Roeder, 1992). ts pharmaology is (in ontrast to otopamine reeptors from non-neuronal tissues suh as the loust ext.-tibiae-musle or the firefly lantern), less well studied (Nathanson, 1985a,b). n nonneuronal tissues, ompounds suh as the formamidines, the phenyliminoimidazolidines (Nathanson, 1985a,b; Evans, 1987) or the aminooxazolines (Jennings et al., 1988) have high affinity properties. Loust neuronal otopamine reeptors have been studied using different methods. n addition to the study of otopamine-sensitive adenylate ylases, radioligand-reeptor assays with either [3H]-otopamine (Roeder & Geweke, ' Author for orrespondene. 199) or the P11-derivative, [3H]-NC-5Z (Nathanson & Kaugars, 1989; Roeder & Nathanson, 1993) were suessfully used. The identity of these binding sites with otopamine reeptors loated on isolated neuronal somata was reently onfirmed by eletrophysiologial methods (Kaufmann & Benson, 1991). As otopamine reeptors are the only non-peptide reeptors whose ourrene is restrited to invertebrates, they represent ideal targets for speifi insetiides. Therefore, the detailed pharmaologial haraterization of otopamine reeptors, espeially of those from neuronal tissues, ould result in the development of new leads for more speifi insetiides. Methods Migratory lousts (Lousta migratoria, L.) of either sex were reared in a light/dark yle of 12h/12h (35 C) under rowded onditions. They were fed with a diet of fresh wheat and bran. Two to 3 weeks after imaginal moult, nervous tissue (supraoesophageal-, suboesophageal-, and thorai ganglia) was disseted out and immediately stored on ie. The nervous tissue was homogenized in ie old inubation buffer (HEPES/NaOH 1 mm, 5 mm MgSO4, 1 jlm PMSF, ph 7.4) in a glass/teflon homogenizer followed by a glass/glass homogenizer (1 strokes eah). This homogenate was entrifuged (2, g, 3 min), the supernatant disarded, and the pellet resuspended in the original volume. This proedure was repeated twie to obtain washed pellets that were stored frozen until use (- 7C). The inubation was performed in a total volume of 1 pl with.3-.5 mg ml-' protein, and 1 nm [3H]-otopamine at room temperature. The inubation proeeded for approximately 1 h and was stopped by filtration through glass fibre filters (Whatmann GF/C) preinubated in.3% polyethyleneimine. After addition of the sample, the filters were washed with ie old buffer. All pharmaologial experiments were performed in the presene of indiated

2 onentrations of tested substanes. Eah substane was tested in 6-7 different onentrations at least 4-6 times in tripliate. Standard errors in the pharmaologial studies were usually below 1% of the respetive values. n order to inrease larity, error bars have been omitted. To determine non-speifi binding, 1WpM otopamine was added to the inubation medium. Radioativity on the filters was quantified with a sintillation ounter (app. 4% effiieny). K-values were determined using the LGAND programme (Munson & Rodbard, 198). A more detailed desription of the proedures was given previously (Roeder & Geweke, 199). Most of the Pls were from Boehringer ngelheim (Germany) or Shell Agriulture (ngelheim), St 92 and NC 5Z were gifts of Dr J.A. Nathanson. Demethylhlordimeform was from CBA-Geigy (Basel, Switzerland). AC 6 was a gift from Dr Jennings. Amitraz and BTS were from Cyanamid. Most of the other substanes were obtained from SGMA (Deisenhofen, Germany). Results Biogeni amines Although the biogeni amines are substanes with a struture related to that of otopamine, they vary substantially in their affinity for the otopamine reeptor, and minor strutural hanges lead to different affinities. The natural ourring p-isomer of otopamine has an affinity that is approximately 5 times higher if ompared with that of the m-isomer (Figure 1, Table 1). Comparison of the affinities of these two isomers with the non-hydroxylated analogue, phenylethanolamine, enabled quantifiation of the effets of hydroxylation at either position 4 or 3 of the phenoli ring. Whereas introdution of an hydroxyl-group at position 4 resulted in an approximately 15 fold inrease in affinity, this substitution at position 3 led to an approximately 5 times dereased affinity (Figure 1). This negative effet of hydroxylation at position 3 of the phenoli ring is one of the major strutural features of the otopamine reeptor that demarates it from ateholamine and espeially from vertebrate adrenoeptors, to whih otopamine reeptors are presumably homologous. %._ ) 1 - T. Roeder Pharmaology of inset otopamine reeptors The biogeni amine with highest affinity for the loust neuronal otopamine reeptor is the N-methylated analogue of otopamine, synephrine (K, = 3.38 nm). ts affinity is approximately twie as high as that of otopamine (K1 = 7.9 nm). This two fold inrease in affinity aused by N-methylation was also observed for noradrenaline and its N-methylated analogue, adrenaline (K1 = 416 and 98 nm respetively; Table 1). Tyramine, the biologial preursor of otopamine and itself a neurotransmitter andidate in the inset nervous system (Saudou et al., 199), has an affinity that is approximately 8 times lower than that of otopamine. The stereospeifiity of the binding was shown by the two fold inrease in affinity of the (-)-isomer of noradrenaline ompared with the (±)-raemate. Ahlquist (1948) used in his pioneering work (whih lassified adrenoeptors into a- and P-reeptors) the three biogeni amines adrenaline, noradrenaline and isoprenaline). nterestingly, the loust neuronal otopamine reeptor has the same rank order of affinities for these agonists as the vertebrate o-adrenoeptors. Phenyliminoimidazolidines 211 Currently, phenyliminoimidazolidines (Ps) are the most interesting agonists for otopamine reeptors. Among them are various ompounds with subnanomolar affinities. To quantify the effets of a given substitution, the affinities of those Pls were ompared that are different at only one position of the phenoli ring. As a simple example, the positive effet of bromination at position 4 of the phenoli ring was demonstrated by omparing the affinities of the 4-bromo-substituted analogue NC 12 (K1 = 14.9 nm) with the nonsubstituted P11, NC 1 (K, = 23.4 nm, Table 2). Sets of other P~s ould be used to quantify the effets of other substitutions. The hlorine substituted P11 derivatives, lonidine (2, 6-dihlor), NC 11 (2,4,6-trihlor), NC 3 (2,4,5-trihlor) and NC 8 (2,4- dihlor) vary substantially in their affinities. The 2,4-hor derivative (NC 8, K1 =.812 nm) has higher affinity than either the 2, 4, 5-trihlor (NC 3, K, = 2.27 nm) or the 2, 4, 6-trihlor derivative (NC 11, K1 = 18.7 nm). The negative effet of hlorination at position 6 is even greater than the negative effet of this substitution at position 5 (Table 2). The positive effet of a hlorine-group at position 4 is shown by the higher affinity of the 2, 4, 6-trihlor derivative NC 13 ompared with the 2, 6-dihlor P11 (lonidine, K1 = 47.4 nm). Regarding three different Pls it was possible to evaluate the effet of methylation at different positions of the phenoli ring. Whereas the inrease in affinity of the 2, 4, 6-trimethyl P11 (NC 13, K1 = 4.38 nm) ompared with the 2, 6-dimethyl derivative (NC 4, K1 = 19.8 nm) indiates the positive effet of para-methylation, methylation at position 6 results in a dereased affinity (the 2, 4-dimethyl PT, NC 9, K1 = 1.2 nm ompared with NC 13, K1 = 4.38 nm; Figure 2). The positive._ (3 E 5 - v Competitor onentration (log M) Figure 1 Displaement of speifi [3H]-otopamine binding from loust (Lousta migratoria L.) nervous tissue membranes by two otopamine isomers (meta- (-) and para- () otopamine) and phenylethanolamine (). Eah point represents the mean of at least 3-4 separate experiments performed in tripliate. Standard errors are not indiated, but they represent usually less than 1% of the respetive values. -2 Table 1 Affinity of various biogeni amines for the loust neuronal otopamine reeptor that share strutural similarities with otopamine Synephrine p-otopamine Tyramine Phenylethanolamine Adrenaline (-)-Noradrenaline (± )-Noradrenaline m-otopamine soprenaline K1 (nm) 3.38 ± ± ± ± ± ± ± ± ± K-values ± s.d. are given. Eah substane is tested in at least 5-7 different onentrations 3-4 times in tripliate. K1ozt means K1-value of otopamine, K1X means K1-value of the respetive substane.

3 212 effets of methylation or hlorination at position 4 are obvious. f the two derivatives NC 7 (4-hlor, 2-methyl-P, K1 =.3 nm) and NC 9 (2, 4-dimethyl derivative, K1 = 1.2) are ompared a ranking of these two substitutions is possible. The hlorine substitution is approximately 3 times more effiient than the methyl-substitution at the para position. The opposite is true for position 2, where methylation is more effetive than hloride substitution (NC 7 ompared with NC 8, the 2, 4-dihlor derivative, K1 =.81 nm). n the firefly lantern, 2, 6 di-alkyl substituted substanes have espeially high affinities (Nathanson, 1985b). For the loust neuronal otopamine reeptor, the affinity rises about 2 times from the 2, 6-dimethyl P, NC 4 (K1 = 19.8 nm) to the 2, 6-diethyl derivative, NC 5 (K =.87 nm). A further enlargement of the substituents at this position results in an approximately 15 fold dereased affinity as shown for the 2, 6-diisopropyl P, NC 2 (K1 = 132 nm) ompared with NC 5. The para-position of the phenoli ring appears to be the most interesting one. Even bulky substituents suh as the azido-group or the amino-group are tolerated without loss of affinity (p-azido- and p-amino-lonidine, K1 values 44.5 and 58 nm respetively ompared with lonidine K1 = 47.4 nm). All other substituents tested, although of different hemial struture (halogen substituents suh as bromide and hloride m T. Roeder Pharmaology of inset otopamine reeptors as well as alkyl substituents suh as methyl and ethyl) have positive effets on the affinity (Table 2). nsetiides A variety of substanes from different lasses of ompounds suh as the formamidines or the aminooxazolines ombine high affinity for the loust neuronal otopamine reeptor with insetiidal ativity. The two formamidine derivatives, hlordimeform and demethylhlordimeform, are espeially potent insetiides (Table 3). Although hlordimeform has a lower affinity (K = 137 nm), it is thought to be metabolized in the inset to the more potent demethylhlordimeform (K = 1.97 nm). The substane with highest affinity in this group, and the only non-p with a subnanomolar affinity, is the aminooxazoline derivative, AC-6 (K1 =.95 nm). Even substanes suh as BTS (K1 = 8.9 nm) and amitraz (K1 = 21.6 nm) whih are known to have a ertain insetiidal ativity, have relatively high affinity for the loust neuronal otopamine reeptor. t is suggested that at least some of these substanes exert their insetiidal ativity through interation with the neuronal otopamine reeptor. f the affinities of these substanes are ompared with eah other it is possible to quantify the effets of substitutions at position 1 of the phenoli ring. This is possible beause all, exept amitraz, have a 4-hlor, 2-methyl substitution at the phenoli ring (Figure 3). s with heteroyli substituents suh as the 2-aminooxazolines (AC-6) and phenyliminoimidazolidines (NC 7) have highest affinities (Table 3, Figure 3). Only slight strutural hanges suh as the N- methylation of demethylhlordimeform result in a strongly dereased affinity. nterestingly, the introdution of the o._ E o Competitor onentration (log M) Figure 2 Comparison of the relative potenies of different methylated phenyliminoimidazolidines (Ps) in ompeting with [3H]- otopamine for its speifi binding site. Eah point represents the mean of 3-4 experiments performed in tripliate: (U) 2, 4-dimethyl- P11; () 2, 4, 6-trimethyl-P; (@) 2, 6-dimethyl-P. -2 Table 3 Affinity of seleted insetiides derived from different lasses of ompounds for the loust neuronal otopamine reeptor Formamidines Demethylhlordimeform Chlordimeform Aminooxazolines AC-6 (4-hlor, 2-methyl-) Misellaneous BTS Amitraz K1 (nm) Klot/Khx 1.97 ± ± ± ± ±5.37 K1-values ± s.d. of substanes from three different lasses of ompounds are given (see Table 1). Table 2 Affinity of various phenyliminoimidazolidines for the loust neuronal otopamine reeptor NC 7 St 92 NC 8 NC 5 NC 9 NC-SZ NC 3 NC 13 NC 12 NC 11 NC 4 NC 1 p-azidolonidine Clonidine p-aminolonidine NC 2 Substitution 4-hlor, 2-methyl- 2, 4, 6-triethyl 2, 4-dihlor 2, 6-diethyl 2, 4-dimethyl- 4-azido, 2, 6-dimethyl- 2, 4, 5-trihlor- 2, 4, 6-trimethyl- 4-bromo- 2, 4, 6-trihlor- 2, 6-dimethyl- 4-azido, 2, 6-dihlor- 2, 6-dihlor- 4-amino, 2, 6-dihlor- 2, 6-diisopropyl- The K1-values ± s.d. of the different substanes are given (see Table 1). K1 (nm).3±.4.56 ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4 methyl-group at this position has a more negative effet than the extremely bulky side hain of BTS (Figure 3). Misellaneous As shown for some of the insetiides, this heterogeneous group of ompounds enables further quantifiation of the effet of a given substitution at position 1 of the phenoli ring (Figure 4). 2-midazoline substituents are again the most effetive substituents (tolazoline, K1 = 18.5 nm; phenyliminoimidazoline, K1 = 23.4 nm). These two agonists together with 2-phenyl-2-imidazolidine enabled us to study the effets of different bridging groups between the phenoli ring and the imidazolidine group. Whereas the methyl and imino group are approximately equipotent, the affinity drops drastially about three orders of magnitude if a spaing group is omitted (2-phenyl-2-iminoimidazolidine, K1 = 162 nm; Figure 4). Most of the high affinity agonists have substitutions at the position 2 and 4 of the phenoli ring. An exeption is the imidazoline derivative, naphazoline (Table 4; Figure 5; K1 = 3.3 nm). The five fold inrease in affinity between naphazoline and tolazoline ould be attributed to the strutural differene between both substanes. An additional benzol ring is added, attahed to positions 2 and 3 of the phenoli ring of tolazoline. T. Roeder Pharmaology of inset otopamine reeptors2 Reeptor speifiity Some of the tested agonists have a high degree of speifiity for the loust neuronal otopamine reeptor. The affinity obtained in the urrent study is ompared with the one obtained from [3H]-lonidine binding ((2-adrenoeptor) and [3H]-WB-411 (a,-adrenoeptor) binding to vertebrate tissues (DeJong & Soudijn, 1981; U'Prihard et al., 1977; Jarrott et al., 198; Rouot & Snyder, 1979). As seen in Table 5, the biogeni amines otopamine and tyramine are the substanes with maximal speifiity for the loust neuronal otopamine reeptor. Their affinities for the loust reeptor are up to 2 times greater than their affinities for vertebrate 12- or aladrenoeptors, to whih otopamine reeptors are presumable homologous. n ontrast, the vertebrate adrenergi transmitter, noradrenaline has an affinity that is approximately 3 times lower for the loust neuronal otopamine reeptor than for M2-adrenoeptors. Among the other substanes tested, the phenyliminoimidazolidines NC 5, NC 9 and NC 1 have the highest degree of speifiity although a 213 a O O3 NH- N=] 'H N NH<)2 O3 N4H H- * CH-CH2-NH2 UN lq A N=CHN7H b N,H- * N=CH-N-CH=N Cl b % O N=CH-N- 1 D m C D E _ a) 4-. a Q %4..- o. O C._ CDt o6 C o o E Z_ - 2: C. 4- NH- * CH2-] N{ Competitor onentration (log M) Figure 4 Differential affinity of otherwise nonsubstituted substanes for the loust neuronal otopamine reeptor. The struture of the different substanes is given in (a). n (b): (-) tolazoline; () phenyliminoimidazolidine; (-) phenylethanolamine; (A) 2-phenyl-2- imidazolidine. -2 v Competitor onentration (log M) Figure 3 Effet of different substitutions at position one of the phenoli ring of agonists that share otherwise the same strutural motif (4-hlor, 2-methyl-). n (a) the different substitutions at position one of the phenoli ring are shown. n (b) eah point represents the mean of 3-4 experiments performed in tripliate: (U) NC7; () AC-6; (A) demethylhlordimeform; () BTS 23376; () hlordimeform. Table 4 Affinity for the loust neuronal otopamine reeptor of misellaneous ompounds K1 (nm) KlOtl/Khx Naphazoline Tolazoline midazol-yl-phenol 2-Phenyl-2-imidazolidine 3.3 ± ± ± ± 47 K1 values ± s.d. are given (see Table 1)

5 214 they never reah the speifiity of otopamine (Table 5). On the other hand, Ps suh as the well known adrenoeptor ligands, lonidine and p-aminolonidine, have a high degree of speifiity for vertebrate adrenoeptors. Disussion Otopamine reeptors are thought to represent ideal targets for highly speifi insetiides beause they are the only known non-peptide ligand reeptors whose ourrene is restrited to invertebrates (Evans, 1985). Muh effort has been made to haraterize the pharmaology of otopamine reeptors from non-neuronal tissues (Axelrod & Saavedra, 1977; Harmar & Horn, 1977; Evans, 1981; 1985; Nathanson, 1985a,b). Although, a lass of highly speifi antagonists was haraterized for loust neuronal otopamine reeptors (Roeder, 199), these reeptors have been less well studied. A variety of highly speifi agonists for otopamine reeptors suh as the formamidines (Harmar & Horn, 1977; Nathanson & Hunniutt, 1981), the phenyliminoimidazolidines (Nathanson, 1985a,b) or the 2-aminooxazolines (Jennings et al., 1988) are available. n the urrent study the affinities of these ompounds for a different type of otopamine reeptor, the T. Roeder Pharmaology of inset otopamine reeptors neuronal lass 3 reeptor (Roeder, 1992) are evaluated, and struture-affinity evaluations have been made. Biogeni amines The most interesting pharmaologial feature obtained from the study of biogeni amines is their rank order of affinities that makes a simple type of lassifiation possible. With only three amines, otopamine, tyramine and noradrenaline is it possible to distinguish otopamine reeptors from their losest relatives, the vertebrate a-adrenoeptors and the inset tyramine reeptors (Saudou et al., 1992; Hiripi et al., 1994). Otopamine has the highest affinity, followed by tyramine and noradrenaline for the otopamine reeptor, but otopamine and tyramine hange plaes in their affinity for the inset tyramine reeptor. Noradrenaline is the substane with the highest affnity for adrenoeptors. nterestingly, otopamine and tyramine are the substanes with maximal speifiity for inset neuronal otopamine reeptors (Table 5). Otopamine reeptors and tyramine reeptors of insets are both thought to be homologues of vertebrate adrenoeptors reeptors. While otopamine reeptors appear to be homologous with vertebrate a-adrenoeptors, tyramine reeptors are more losely related to vertebrate P-adrenoeptors. Clas- H ++ NH24 ~~~H H27 N + NH K + N=CH- NH -OH -C - Cl sc2h5 5_6 < o GCH-CH,-NHz -OHH UO +C33 CH-CH2-NH2 + * C2H5 * Br O o N3 onh2 -OH 2- CH2-_CH2-NH2.e. + - N==CH-N-CH=N K) C -- N==CH-N- H Figure 5 Struture affinity relationship of agonists for the loust neuronal otopamine reeptor. The substitutions of eah position of the phenoli ring are listed, in the order of their effiay, at the respetive position. + + indiates an inrease in the affinity aused by the substitution of more than 1 times when ompared with the nonsubstituted situation (positions 2-6). For position one, omparison is made with the affinity of the natural agonist otopamine; + indiates an inrease in affinity between 2 and 1 times; o indiates no hange in affinity (.5-2 times the affinity); - indiates a derease in affinity (.5-.1), and -- indiates a marked derease in affinity (more than 1 times less affinity).

6 T. Roeder Pharmaology T.-fof inset otoparnine reeptors Table 5 Relative potenies of otopamine agonists in omparison with mammalian a-adrenoeptors Biogeni amines Otopamine Tyramine Phenylethanolamine Adrenaline Noradrenaline soprenaline Phenyliminoimidazolidines NC 5 NC 8 NC 9 NC 1 Clonidine p-aminolonidine 2-Benzylimidazolines Tolazoline Naphazoline Kl(a2)/K(OA loust) K(alA)/K(OA3 K1-values for a2-binding ([3H]-lonidine) were obtained from DeJong & Soudijn (1981), from U'Prihard et al. (1977), Jarrott et al. (198) and from Rouot & Snyder (1979). For a,-binding ([3H]-WB-411), the data were obtained from U'Prihard et al. (1977) loust) sial otopamine reeptor agonists, suh as otopamine itself, demethylhlordimeform, naphazoline and synephrine are relatively ineffetive at the loust tyramine reeptor (Hiripi et al., 1994). To distinguish the otopamine and tyramine reeptor in the loust nervous tissue, even better antagonists are available. Yohimbine, the antagonist with subnanomolar affinity for the inset tyramine reeptor (Saudou et al., 1992; Hiripi et al., 1994) has very low affinity for the neuronal otopamine reeptor of lousts (K = 82 gm). On the other hand, high affinity antagonists of the loust neuronal otopamine reeptor suh as mianserin and phentolamine (Roeder & Geweke, 199) have only low affinity for the loust tyramine reeptor (Hiripi et al., 1994). Phenyliminoimidazolidines The other lasses of ompounds tested allowed more sophistiated pharmaologial questions to be asked. The large number of high affinity phenyliminoimidazolidines used was neessary to obtain quantitative data about struture-ativity relationships. Ps suh as NC 5 have, as do otopamine and tyramine, high speifiity for otopamine reeptors if ompared with vertebrate a-adrenoeptors (NC 5 has a more than 3 times higher affinity for neuronal otopamine reeptors than for vertebrate a-adrenoeptors, Table 5). Substitutions at only two positions of the phenoli ring have a positive effet on the affinity. These are positions 2 and 4 of the phenoli ring (Figure 5). This is in ontrast to the otopamine reeptor of the firefly lantern, where Ps substituted at positions 2 and 6 are most effetive (Nathanson, 1985a,b) The neuronal otopamine-sensitive adenylate ylase of the tobao hawkmoth Mandua sexta has a pharmaologial profile that appears to be more similar to that of the loust nervous tissue (Hollingworth & Johnstone, 1983). n both preparations, substanes that are substituted at positions 2 and 4 of the phenoli ring are most effetive. n addition, the ability to stimulate the otopamine-sensitive tissue, adenylate ylase is orrelated with the insetiidal ativity of these ompounds (Hollingworth & Johnstone, 1983; Hollingworth et al., 1984). Another lass of ompounds, the phenylaminomethylimidazolidines were found to have very high affinity for otopamine reeptors from okroahes, whereas their affinity for Mandua otopamine reeptors is muh lower (Orr et al., 1991). nterestingly, the phenylaminomethylimidazolines have a different substitution preferene at the phenoli ring. Substitutions at positions 2 and 3 seems to be optimal for this lass of ompounds (Hollingworth et al., 1984; Orr et al., 1991). nsetiides and misellaneous t is likely that the insetiidal ativity of substanes suh as the formamidines, amitraz, BTS 23376, or AC-6 result from interation with otopamine reeptors. The oinidene of insetiidal ativity with high affinity for the otopamine reeptor supports the assumption that the neuronal otopamine reeptor is the target of these insetiides. Appliation at sublethal onentrations of the well-studied formamidines result in behavioural hanges, suh as hyperativity, that ould be attributed to the interation with neuronal type otopamine reeptors (Dadai et al., 1987). Therefore, it seems likely that only the neuronal otopamine reeptor is the target of otopamine insetiides, as these behavioural hanges ould not result from interation with otopamine reeptors from non-neuronal tissues. As the most interesting strutural feature of this study, various substituents at position 1 of the phenoli ring were tested. Heteroyli substitutions suh as the 2- iminoimidazolidines or the 2-benzylimidazidines are the most effetive. The spaing group between these two ringsystems is of great importane. Redution of the spaing leads to a drasti derease in affinity. The effet of further inreasing the spaing by one group was not studied beause the respetive substanes were not available. A further inrease of the affinity should result beause it has been shown for the okroah nerve ord that a substane with this kind of struture also has very high affinity (XAM; Orr et al., 1991). Further studies are needed to larify the pharmaologial harateristis of otopamine reeptors. t is inappropriate to ombine pharmaologial data from different otopamine reeptor subtypes suh as non-neuronal and neuronal otopamine reeptors (Roeder, 1992; Nathanson, 1993) or from studies performed with otopamine reeptor preparations from different inset speies. Therefore, well-defined otopamine reeptor preparations suh as the loust neuronal otopamine reeptor should be used to obtain quantitative pharmaologial data of a variety of different lasses of ompounds. Studies like these might result in the development of substanes with even higher affinity and speifiity for otopamine reeptors that ould serve as new leads for better insetiides as well as better probes to study otopamine reeptors in more detail. would like to thank Prof. Geweke for ontinuous support. This work was sponsored by a grant of the Deutshe Forshungsgemeinshaft (DFG Ge 249/12-1). Most Ps were kindly provided by Shell

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