High-affinity octopamine receptors revealed in Drosophila by binding of [3H]octopamine

163 ishers Ltd,

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The study descries; for:the: first:time, detection of a putative,: high,affinity octopamine r eceNor by direct binding,studies with aradiolabeled oligand. Crude membranes prepared from heads of Dros~_~,.~hi!a melanogaster bind [3H]octopamine at a level of 0.4 pmol per mg protein with an apparent Ka of 5 nM, mazine, but not of proprmmlot Hnding. The pH]octopamine ,aminergic ligands.

Octopamine h a s b e e n suggested to serve as a neurotransmitter and a neuromoduiator in invertebrates, including insects [1, 4]. Studies of octopaminergic mechanisms may shed light on the structure and fuction of insect nervous system and may also be o f practical vNue since octopamiae receptors have been recently implicated in the action of novel insecticides [6]. The effect of octopamine oe insect nerve cells has been studied by electrophysiological techniques a~d v>. ~ . . ~ , ~ ~,~,~-~ of adenylate ~sy~;~a~ - -~'~-- activation (reviewed in ref. 4). R should be of imeres~ t-~ exteed ..... nsect tiss~-,~eby investiga~.~ag direct bending studies with e detection, in Deosop]~i]a octopamine, as revealed by ~cepters are discussed in the ~ors in various organisms, iuaNe as a convenient probe ~s, were used Experiments s° The heads ~ag through a rmed in ',s dual homo-glassoTeflon : hemogenate 03~:3~)'82/~10-~/$

02.75 © Elsevier/North-Holland ~ientific Publishers Ltd.

164 was then centrifuged at 599 g for i0 mix. The pellet was discarded and the supernatant was further centrifuged at 305.K~0x g for 60 rain. The su~rnatant was discarded and the pellet was d~spersed and rehomogenized in a glass-Teflon homogenizer in 0.6 of the original volume, to y~eld the crude membrane preparation which was immediately frozen in liquid N~ and stored in I m t aliquots a't - 80 0C until use. D,L-[3,5-3H|Octopamine hydrochloride, 37 Ci/mmol, was fl:om the Radiochemical Centre, Amersham. The following drugs were from Sigma (St. Louis, MO): dihydroergotamine tartrate, dopamine hydrochloride, (-)-epinephrine bitartrate, nicotine, (-)-norepinephrine bitartarate, D,L.OCtopamhae hydrochloride, phentolamine hydrochloride, D,L-propranolol hydrochloride, seromnin creatinine sulfate, D,,.-synephrhae, n-tubocurarine chloride and yohimbine hydrochloride. Chlorpromazine was from Bayer (Leverkussen, F.R.G.) and methysergide dimaleate from Sandoz (Hanover, N3). All other chemicals were of analytical grade. [3H]Octopamine binding assay was conducted as follows: aiiquots of homogenate were incubated at 26°C in the presence of Buffer 1 containing 0.02% ascorbate in a final volume of 0.4, 0.5 or 1.0 ml, in the presence or in the absence of the appropriate non-labeled displacing ligand. The reaction was started by a~btion of PH]octopamine (final concentration 5 nM, unless otherwise indicated), ann terminated after 60 rain (unless otherwise indicated) by dilutirg with 4 ml of ice cold Buffer 1, followed immediately by vacuum filtration over a wet glass-fiber fire1 {GF/C, purchased from "lamar, Israel). The filter was then immediately rinsed twice with 4 ml portions of ice-cold Buffer 1, placed in vials containing tolueneTriiton scintillation liquid, and counted in a ~-counter. cpm Obtained were corrected fo~' quenching caused by the pigments present in Drosophila head homogenate, by counting in parallel standards of [3H~octopamlne on filters with the appropriate amount of homogenate. The amount of protein in the homogenate was determined according to Lowry et al. [9], employing BSA as a standard. ]Employing the assay method described above, the binding observed with a given concentration of [3H]c,ctopamine was linearly proportional to the amount of fly homogenate present, up to about 350 ~g protein per assay. Specific binding of [31qloctopamine, defined a total binding mimas the binding observed in the presence of 10 ~ 5 M phentolam~ne, was saturable (Fig. 1L Scatchard anafysis of the data revealed a single class of binding sites in the range of 0.1-20 nM, with an apparent K a of 4.9 ± 0.7 × 10 -9 M. The I( d a~ 0°C was found to be 2.8 x 10 -9 M, excluding the possibility that a high-affinRy active uptake mechanism [3] is involved. A Hill plot y~elded a slope of approximately 1.0 ~,Fig. ~). The amximal concentration of binding sites in tl~e crude membrane preparation was fom d to beO.4 ± 0.1 pmol per mg protein, The co~centratkm of [:'H]octopamine-binding ~ites in Drosophila head under the condition, s employed was high compared to other tissues tested by us, When.assa~yed i,~ the presence of I0 nM [3H[ocmpamine, aliquots of a crude m~;,mbrane fraction prepared from the isotate~ nervous system (t~ead gangtia plus nerve cord) of

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oc', ,MJNE. [VI Fig, 1. [3HlOctopamine binding ~o membrane fraction prepared from Drosophila head, after incubation for I h at 26°C at various [3Hloetopamine concentrations; o ~ o , specific binding; o ~ ; , non-specific binding, i.e., binding observed in the presence of 10" s M phentolamine. Insert: Hill pl,ot, Le. pio~ of log (Y/(I Y)) vs. log pH]octopamine concentration, where Y is occupancy.

Periplaneta americana, from the isolated nervous system (as above) of Locusta migrator&, f r o m whole mouse brain, and from Drosoph#a heads, bound [3H]octapamine specifically at levels of about 0.05, 0.05, < 9.01 and 0.17 pmol/mg protein, respectively. At 26 °C, in the presence of 5 nM [3H]octopamine, binding to Drosophila crude membrane fractions reached maximal values after about 15 rnin. The on-rate constant for the reaction receptor-ligand, k~, was found to be 1.9 _+9.5 x 197 M-1 rain--2 The half-life of the receptor-ligand comple×, under the same conditions, was found to be about 5 rain by cold-chase experiments. The off-rate constant was calculated to be k_~ = 0.14 + 0.03 rain-1. The apparent dissociation constant was calculated, on the basis of the kinetic constants, to be K d = 7 x 19 -9 M, in good agreement with the value obtained from Scatchard analysis of bindh~g isotherms. The binding of [3H]octepamine to DrosopM& crude membrane fractiol~a was found to be ion dependent. NaCl at 120 mM reduced the specific binding ~o 40-60% o f the binding observed in a control aliquot assayed in the presence of 50 mM Tris,Cl alone, In contrast, MgC12 increased binding, already at I raM,, with optimal results (more than twice the controb being obtained at 5-10 raM. CaC~2 at 5 mM had a similar effect to MgC12. 5 m ~ MgCIz were therefore roefinely ad6ed to the assay to obtain optimal binding levels. The specificity of [3Hloctopamine binding was examined by testing the ability of

various i!igands to displace the labeled octopamh~e from its biading sites. The results ergot alkaloid dihydroergotamine was the most potent a'adrenerg[c Iigand phent.o~amineand the c~l~rpro~azine were highly pote.t' NOrepi.ephrine" epinepn~iae

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and the for~iamidine inseetide chlor~meform were -~so effective, The ~:adrenergie ligm~d propranolol, serotonin = d the ~rotonergie ligand [es,~ eff~fiveo The choi~nergie |igan~ ~-tu~urafine and nicotine had little effect at high concentrations. .... ..... ............. O c t o p ~ n e was report,~ to a @ysiologieal fi~ cular preparation at the nM range [4i5]' Fhe ~topamin6 response was mLmi~k¢~ 6~!~: 3-adrenergic ligand, [4, 5]. H ~ # affinity oct expected in ~nsects. No eie::trophvsioi~ieal ~ata oa responsiveness *o oetopamine has yet Been ,courted in Drosop~i!a, Ht~wever, the frtdt fl~i as severa| other insects studied to date, conv~ins ader~ylate cyeiase which ~s responsive ~0 oetopamine [12]. The stimulation of the cydase by the biogenic amine is aga~a affected by some ~x-adrer~ergic iigand~ rather u~,ar~#-adrenergi¢ tigands, However, the stimulation o f adenytate cyclase by octc, pmrdne in Drosophiia [t2], as well as in the cockroach [7~ If ], in the firefly [10]and in the moth [21 is very small at I0~ ZM and half m~ima! s, imulafion is observed only at the ~M range, The affinity ofoctopamine ~nd other tigands for the Drosophila [~Hloetopamine-binding sites reported here is thus in the range reported for a physiological response in another msec~ but h~gl~er lhan that reported for cye~ase-couNed octopamine receptors *,n vitro i n several insects,

TABLE I iNHIBITION

OF

[3H]OC"fOPAM! NE

BtND~NG

TO

DRGS(9;L~.!*LA P A R T I C U L A T E

PREPARATION BY VARIOUS L I G A N D S Li~an,d

ICs~, M

Dihydrc~rg:J~amh~e

1 . 5 × l 0 --9

D,L-Oct~paraine Phentolamine

!.5 × 10 "~ 1.5 × i ~ - ~ 3 . 0 × 10 - a

od.oSynepM'ihe

ChIorpromazme ( - i}-Norepinephrine t - )-E~inephdrie Chiordimeform

8.0 × 10- 7 io- 6

Ychb~bine

4 . 0 ~ 20 ''6

Do, amine

5,0 × 10 - 6

Methysergide

2.5 × l e - 7

2 . 0 r" t~, - ~

i O- 5

Sem~o~

1.5 x i6 - 5

~},t,.Propra~olol

8.0 x tO - s

o-Iubocuraf~r~e

N~cothae

> 10" 4 > 10-4 . . . . . . . . . . . . . . . . . .

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~i~d~m3~f PHloo~opaminewa~ as~aye~as ~ i b e d L~the r~It~ se:ti~,~ in the i~,eac, e of ~ nM of the ~ab.~led ~ B g a n d . [C~o is the c o ~ c e ~ t r ~ f i o n o f figand that ~caused ~ 5 0 % decrease in. the N ~ d i n g o f

i,~H|oetopami.ne under the assa~ conditions.

167

iv,eluding the fruit fly. Two main possibilities should be considered with respect to thiSthatobservation: ( a ) dmediate o ~he [3H]octopamine,binding sites in Drosophila are receptors not their physiolo#cal effect via adenylate cyctase. It should be noted that 3 different classes of oetopamine receptors have been identified in the :yclase in vivo other .~rt the or a~Iternately, convert the cyclase-coup~ed -

_ _ dological evidence is required to corroborate the assumption flint [3H]octopamine-binding sites in Drosophila represent recep~.,~rs for octopamine. As was mentioned above, such evidence is still lacking. Genetic methods are feasible in Drosophila which, when combined with the [3H]octopamine-bind,~ng assay, may yield valuable information on the structure and function 9f the octopamine receptors. In addition, the [3H~octopamine-binding assay ~ay prove a convenient biochemical screening method to assess the potency of nove~ octopami,nergic iigands. The excellent technical assistance of Shoshana ~w is gratefully acknowledged. This work was supported by a gram from the United States-Israel Binadonat Science Foundation, Jerusalem. ,-tp

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i A×elrod, J. and Saavedra, J.~4., Octopamine, Nature (Lond.), 265 ~1977) 501-504. 2 Bodnaryk, R.P., Identification of specific dopamine- and octopamine°sensitive adenylate cyclases in the brain of Mamestra ccafigurata Wlk., Insect Biochem., 9 (1979) 155-162. 3 Evans, P.D., Octopamine: a high-affinity uptake mechanism in the nervous system of the cockroach, J. Neurochem., 30 (1978) 1015-f022. 4 Evans, P.D., Octopamine receptors in insects. In D.B. Sattelle et al. (Eds.), Recepv,rs for Neurotransrmtters, Hormones and Pheromones m Insects, Elsevier/North-Holland, Amsterdam, • ~0, pp. 245-258. 5 Evans, P.D,, Multiple receptors types for octopamine in the locust, J. Physiol. (Lond.L in press. 6 Evans, P.D. and Gee, J.D., Action of formamidine pesticides ~,n oetopamine receptors, Na~,ure (Lond.), 287 (1980) 60-62. 7 Harmar, A.J. and Horn, A.S., Octopamine-sensitive adenylate cyclase in cockroach brain: effects of agonists, antagonists and guanylyl nucleotides, Molec. Pharmacol., t3 (1977) 512-520. 8 Lewis, E.G., A new standard food medium, Dros. Inf. Set., 34 (1960) 117-118. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.3., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 10 Nathanson, J.A., Octopamine receptors, adenosine, 3',5'-monophosphate, and neural contro~ of firefly flashing, Science, 203 (1979) 65-68. tl Nathanson, J.A. a n d Greengard, P., Octopamine-sensitive adenylate cyclase: evidence for a bioicgical:~ole o f oct,)pamine ~n nervous tissue, Science, 180 (1973) 308-310. !2 ~Umn) A. and Dudai, Y., Aminergic receptors inDrosophita: responsiveness o; adenylate cyclase to putative neuro~ransmitter~;, submitted to J. Neur~:~chem. '

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