The d -octopamine content of the haemolymph of the locust, Schistocerca Americana gregaria and its elevation during flight

Insect B w c h e m , Vol 10, pp 393 to 397 '~@Pergamon Pres,s Ltd 1980 Printed m Great Brttam

0020-1700/80/0801-0393 $02 00/0

THE D-OCTOPAMINE CONTENT OF THE HAEMOLYMPH OF THE LOCUST, S C H I S T O C E R C A A M E R I C A N A GREGARIA A N D ITS ELEVATION D U R I N G FLIGHT MICHAEL W GOOSEY a n d DAVID J CANDY Department of Bmchemlstry, Umvers~ty of B~rmmgham, P O B 363, B~rmmgham BI5 2TT U K (ReceJved 28 September 1979, rewsed 21 December 1979)

Abstraet--Octopamme is present m the haemolymph of adult locusts at a concentration of 33 + 3 nM When locusts fly there is a rap~d increase in the concentratmn of octopamlne m the haemolymph until it reaches a maximum value of 173 + 8 nM after 10 mm Thereafter the concentration decreases untd, after 60 mm flight, it is close to the resting value It is suggested that octopamme could have a physmloglcal role m stimulating oxidation of substrates m the flight muscle during the early period of flight Evidence is presented that it Is the D-isomer of octopamme which is present m haemolymph A mo&fied assay procedure for octopamme is described in which the octopamlne is first enzymlcally methylated using labelled S-adenosyl methlonme and the ra&oactwe synephrme produced is selectively isolated by co-crystalhzatmn with carrier synephrme Key Word Index Schzstocerca amerwana gregarta, octopamme, synephrme, haemolymph, flight

INTRODUCTION OCTOPAMINE 1S a m o n o h y d r o x y analogue o f n o r a d r e n a h n e , a n d has been f o u n d in the nervous systems of every a n i m a l so far examined (see ROBERTSON a n d JURIO, 1976) In the locust it is present in various parts of the nervous system in higher c o n c e n t r a t i o n s t h a n chemically related amines such as d o p a m i n e a n d n o r a d r e n a h n e (ROBERTSON, 1976, EVANS, 1978). The physiological role of o c t o p a m l n e in invertebrates is not yet fully u n d e r s t o o d . It has been suggested t h a t it could act as a n e u r o t r a n s m l t t e r , a n d there is evidence for this in the light o r g a n o f the firefly, P h o t u r t s verstcolor (ROBERTSONa n d CARLSON,1976). A neurosecretory role for o c t o p a m i n e was suggested by HOYLE (1975) w h o f o u n d t h a t o c t o p a m m e caused an inhibition of the intrmstc r h y t h m of c o n t r a c t i o n o f the extensor tibia muscle of the locust. A specific n e u r o n e ( ' D U M E T f ) supplying this muscle was s h o w n by EVANS a n d O'SHEA (1978) to c o n t a i n o c t o p a m i n e in b o t h soma a n d axon. Spikes in this n e u r o n e p r o d u c e d effects on muscle tension which could be mimicked by superfusion o f DL-OCtopamIne (O'SHEAa n d EVANS, 1979) EVANS et al (1976) showed that o c t o p a m i n e is released from peripheral octopamlnerglc n e u r o n e s of the lobster into the circulatory system. They also f o u n d t h a t low c o n c e n t r a t i o n s of D-OCtopamine would increase the strength of c o n t r a c t i o n s elicited by nervous stimulation of the opener muscle of the dactyl of lobster walking leg These effects of o c t o p a m l n e on invertebrate muscles resemble in some ways the effects of catecholamines on vertebrate muscles. The possibility of m e t a b o h c effects of o c t o p a m i n e In locusts was suggested by previous experiments (CANDY, 1978) in which a d d e d DL-OCtopamine was f o u n d to stimulate the oxidation of c a r b o h y d r a t e s a n d hpids in isolated p r e p a r a t i o n s of locust flight muscle

However, the physiological relevance of these observations was not established because it was not k n o w n whether such muscles are ever exposed to o c t o p a m l n e tn vtvo We have therefore m e a s u r e d the o c t o p a m l n e c o n t e n t of h a e m o l y m p h from unflown locusts a n d locusts flown for different times. The results reported here show that D-OCtopamlne is present in locust h a e m o l y m p h a n d increases during flight to a c o n c e n t r a t i o n which would affect the m e t a b o l i s m o f flight muscle tn vttro. This p a p e r also reports a modified estimation procedure for o c t o p a m l n e It is based on the original m e t h o d ofMOLINOFFet al (1969) m which o c t o p a m i n e is enzymlcally methylated using radioactively labelled S-adenosyl methionlne, a n d the labelled synephrlne produced is extracted into isoamyl alcohol a n d its radioactivity is m e a s u r e d The original m e t h o d was f o u n d to be insufficiently specific for application to h a e m o l y m p h and was modified by including a further purification step involving co-crystallization of the labelled synephrine with unlabelled carrier.

MATERIALS AND METHODS Chemwals S-Adenosyl-L-[methyl-3H]-methlonme (15 C/mmole) was obtained from the Ra&ochemxcal Centre (Amersham, England) S-Adenosyl L-methlonme, DL-OCtopamme and OLsynephrme were obtained from the Sigma Chemical Co Ltd (Kingston-upon-Thames, England) DL-Octopamme was resolved into its two Isomers by fractional crystalhzatmn of the salt with D-camphosulphomc acid (KAVVE and ARMSTRONG, 1964) The D-octopamlne (free base) obtained by this procedure had [~]D--56 1° and the L-octopamme [ct]D + 56.8 ° Phenylethanolamme-N-methyl transferase (PNMT) was prepared using a mo&ficahon of the procedure introduced by AXELROO (1962) Bovine adrenal glands (60 g) were homogenized in 0 17 M KCI and centrifuged at 36,000 g for

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MICHAEL W

GOOSEY AND DAVID J CANDY

30 mln The supernatant was fractmnated with (NH,)2SO,, and the fraction precipitating between 30~ and 5 0 ~ saturation was dlalysed against 0 02 M Trls buffer pH 8 0 The protein was absorbed on a DEAE cellulose column (Whatman DE32, 5 c m x 2 7 cm l d ) which had been equdibrated with 0.02 M Trts buffer, pH 8.0 The column was then eluted using a hnear gradient between 200 ml 0 02 M Tris buffer, pH 8 0 and 200 ml of 0 3 M NaCI in 0 02 M Tms buffer, pH 8 0 The eluate was collected m 4 ml fractions which were then assayed for P N M T activity by incubatmn with 100 ,ul Tris buffer, pH 8 6 containing 0 03 pmole DLoctopamine and 20 #1 [3H]-S-adenosyl methmnine (0 05 pC, 0 8 nmole) for 15 mm at 37°C Radioactive synephrme was then extracted and measured as descmbed below The fractions with high P N M T activity were pooled, sohd (NH4) 2SO,Lwas added to 100~o saturation and the precipitate was collected by centrlfugauon and stored at - 20°C Samples were diluted with equal volumes of water and used &rectly as ' P N M T ' for octopamlne estimations Insects Locusts (Schistocerca americana gregaria) were fed on a mixture of equal parts by volume of bran, dried grassmeal and dried milk, supplemented with fresh barley seedlings Insects of both sexes were used for experiments 20--32 days after the final ecdysls Octopamme assay Haemolymph was removed from the neck of locusts with a fine capillary tube, was cooled at 0°C until the whole sample had been accumulated and was then heated at 100°C for 5 rain Haemolymph from 3-5 locusts was pooled for each sample, and the extraction up to heating was performed in less than 5 mm In prehmmary studies the monoamme oxldase inhibitor, pargyhne, was added to haemolymph extracts, but no s~gnificant effect on the measured octopamme concentrations was found Pargyhne was therefore omitted m subsequent experiments After heating haemolymph samples were weighed, mixed with 0 4 ml water and centrifuged at 1600 g for 5 min at 20°C Samples (100 pl) of the supernatant, with and without 4 ng of added D-octopamlne as internal standard, were dried m vacuo and assayed by a modMcation of the method of MOLINOFFet al (1969) A typical reaction mixture consisted of 200 ~1Tris buffer, pH 9 0, 2 pl PNMT, and 18 #1S-adenosyl L-[methyl3H]-methionme (0 25 #C, 0 135 nmole) Controls were also included m which substrate was omitted Reaction mixtures were incubated for 60 mm at 37°C and the reacUon was stopped by addition of 1 ml of 0 5 M sodium borate buffer, pH 10 0 saturated with NaCI Isoamyl alcohol (2 0 ml) was added, the mixture was shaken for 1 min and centrifuged at 1600 g for 5 mm at 20°C A sample (1 ml) of the lsoamyl alcohol fraction was dmed m vacuo and a known amount of carrier OL-synephrine-HCl was added The synephrlne was then crystalhzed from solution (0 2 ml) in methanol by addition of ethyl acetate (1 nal) The sample was then recrystalhzed twice Radioactwity was determined by hqmd scmtdlatxon counting using a Phdhps hquid scintillation analyser PW 4510/01 Values were corrected to dpm using the external standard procedure The identity of the radioactive product formed was checked by chromatography of the thrice crystalhzed product on thin layer plates of silica gel (Kieselgel G, Merck, Darmstadt) The solvents used were (1) butan-l-ol saturated with 1 M HCI, (2) propan-2-ol-ammoma-water (80 19 10 by vol), (3) butan-l-ol-acetic acid-water (4 1 1 by vol ) (Ew, Ns, 1978) The product from haemolymph showed a single radioactive peak which corresponded to that of authentic synephrlne m all three solvent systems Determination of the naturally occurring isomer oJ octopamme The relative rates of methylation of the D- and L-isomers of octopamme in the PNMT assay are considerably different

This property of the assay system was used to determine the Isomeric form of octopamtne in haemolymph The octopamme assay procedure described above was used except that the incubation times were vaned between 0 and 180 mln to give a rime course of methylatlon The rate of methylauon of t~ssue octopamine was compared to the rate of methylauon of the authentic D- and L-isomers Statistical treatment o/re~u[t,s Results are expressed as means ± S E M ' s with the number of rephcate experiments given m parentheses D~fferences between control and experimental results were analysed by an mdependcnt t-test RESULTS EJ]ect o f eo-crystalhzatton on the apparent values o f octopamme Figure 1 s h o w s the effect o f the n u m b e r o f recrystalhzatlons on the apparent octopamine content o f the h a e m o l y m p h f r o m b o t h c o n t r o l a n d flown locusts. T h e results s h o w t h a t the a p p a r e n t value d e c r e a s e d f r o m the first to the s e c o n d crystallization b u t r e m a i n e d c o n s t a n t t h r o u g h f u r t h e r crystalhzatlons. F o r s u b s e q u e n t e x p e r i m e n t s all s a m p l e s were routinely c r y s t a l h z e d t h r e e times to ensure t h a t all the m e a s u r e d r a d l o a c t l w t y c o - c r y s t a l h z e d with syn e p h r l n e I f the c r y s t a l h z a t l o n p r o c e d u r e was o m i t t e d values o b t a i n e d were high a n d erratic, a n d it was clear t h a t t h e s~mple e x t r a c U o n p r o c e d u r e using ~soamyl a l c o h o l was Insufficiently specific for the m e a s u r e m e n t o f o c t o p a m l n e in h a e m o l y m p h P r e s u m a b l y the P N M T catalysed m e t h y l a t l o n o f o t h e r a m i n e s p r e s e n t in h a e m o l y m p h a n d these are partly e x t r a c t e d by the 250

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Fig 1 The effect of number of recrystalhzatlons on the apparent concentration of D-octopamine in locust haemolymph Details of the estimation procedure are given in the Methods section Error bars indicate S E M s and the numbers of experiments are given m parentheses The variation m apparent concentration is due to biological variation between locusts as well as variation due to errors m the estimation procedure Haemolymph was from locusts flown for l0 mm (upper line) or from unflown locusts (lower line)

Octopamlne of locust haemolymph

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Fig 2 The effect of flight on the concentration of D-octopamme m locust haemolymph Detads of the esttmat]on procedure are gwen m the Methods section Error bars re&care S E M s and the number of experiments ~s given m parentheses

isoamyl alcohol and contribute to the radioactivity of the extract When unboded samples (1 ng) of various amines were tested m the assay procedure, the relative proportions of ra&oactlvlty (over control) extracted into lsoamyl alcohol were o-octopamine, 100, OLphenylethanolamine, 88, t-noradrenahne, 14, DLnormetanephrme, 49. After co-crystallization the proportions were octopamme, 100, phenylethanolamme, < 1 0; noradrenahne 1 0, normetanephrine < 1 0 When applied to authentic D-octopamine the co-crystallization assay procedure gave a hnear relationship between ra&oactwe incorporation and octopamine content up to 30 pmole per sample, with a twice background sensRiv]ty of 1 pmole

Octopamme concentrations m haemolymph during fltght Figure 2 shows the time course of changes in octopamme concentrations m haemolymph during locust flight. The results show that there is an increase in octopamme in the haemolymph after 2 mm flight (P < 0.05) which reaches a maximum after 10 min. The values at 5, 10 and 15 min are all very significantly higher than the control ( P < 0 . 0 0 1 ) When flight is extended beyond 10 mln the octopamine concentration falls until by 60 mm it is close to the unflown control value. The control values found m this experiment are somewhat lower than those shown in Fig. 1 The experiments relating to Fig. 2 were carried out some weeks later than those relating to Fig. 1 Different batches of locust were used, and the insects were handled more carefully for the second group.

lsomertc form of octopamme tn haemolymph Table 1 shows a comparison of the time course of methylation for authentic D- and L-Octopamme and for haemolymph extract. The results are expressed as the t~me taken to reach half maximal methylatmn (T, of methylation). The results show that within experimental error the T~ of methylation for the haemolymph product is the same as that of D-octo-

Table 1 Half time of enzymic methylatlon of authentic o- and L-octopamme and of the product from haemolymph of flown locusts The methylatlon was carried out as described m the Methods section and the time taken to reach half maximum methylatlon was calculated from the time course Haemolymph extract from unflown locusts was added to the o- and L-octopamme incubations so that all incubations contained a similar amount of haemolymph to compensate for any effect of haemolymph on the rate of methylahon Half-t~me of methylat~on + SEM (n)

Substrate D-OCtopamme L-OCtopamlne Product from flown locust haemolymph

10 8_+ 3 6 (4) 50 3+ 1 3 (3) 9 3_+0 5 (3)

pamme but clearly different from that of L-octop a m m e F r o m these results it is concluded that the isomer of octopamine in haemolymph is the D-form. DISCUSSION The results of the experiments reported here establish the presence of D-octopamme xn the haemolymph of resting locusts at a concentratton + SEM of 33 + 3 nM. During flight the concentration increased to reach a value of 173 + 7 nM after 10 min and thereafter decreased towards resting values. These results suggest the possibility that octopamme may be involved m hormonal regulation of metabolism during the early period of flight. O c t o p a m m e is known to stimulate the oxidation of substrates by Isolated working locust flight muscle preparations (CANDY, 1978). Significant effects on glucose oxidation were obtained at DL-OCtopamme concentrations of 300 nM. M o r e recent results (M. W. GOOSEY, unpublished) have shown that the D-isomer of octopamine at 150 n M concentration also gives a significant increase m the rate of glucose oxidatmn. Thus the concentrations found m haemolymph during flight are sufficient to

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MICHAEL W GOOSEYAND DAVID J CANDY

have a physiological effect on muscle It IS possible L-OCtopamlne in activating adenylate cyclase in that octopamlne may have a similar role in the locust cockroach brain The most c o m m o n method for the estimation of to that of adrenaline in mammals in acting as a hormonal signal to stimulate energy metabolism in the octopamlne in biological systems has been by the use of phenylethanolamine N-methyl transferase Most muscle methods reported in the literature are based on that The elevated octopamlne levels in haemolymph could have other effects related to flight physiology. described by MOLINOFF et al. (1969) and rely on the Thus the strength of contractions of locust flight selective extraction of octopamlne from borate buffer muscle was increased by octopamlne under some into isoamyl alcohol or lsoamyl alcohol/toluene conditions (CANDY, 1978), an effect which also mixtures. When this method was applied to resembles the lnotropic effect of adrenaline on haemolymph samples it proved insufficiently specific m a m m a h a n heart muscle It is also possible that to measure octopamine reliably and gave erroneously octopamme could affect the mobilization of fuel high values It is clear therefore that values for reserves from the fat body during flight DOWNER octopamlne contents of tissues reported in the (1979) has shown that octopamlne stimulates literature must be regarded with suspicion unless it has trehalose release from cockroach fat body, although been established by some additional procedure (e g the concentration of octopamine used (100/~M) was chromatography or electrophoresls of the product) greatly in excess of the haemolymph concentrations that methylated octopamlne constitutes the major reported here for the locust. ROBERTSON and STEELE radioactive component of the lsoamyl alcohol layer (1973) have shown that low concentrations (500 nM) The method reported here of co-crystallization with of octopamlne will activate phosphorylase of unlabelled carrier, although rather tedious to perform, cockroach nerve cord IS sufficiently selective to give reliable values for The origin of the octopamine found in haemolymph octopamlne. is not known A number of insect nervous tissues Including the thoracic gangha, brain and corpora Acknowledgement--We wish to thank the Science Research Council for a studentshlp to M W G and Miss S cardlaca all contain octopamine in significant NICKLIN for preparation of the PNMT concentrations (ROBERTSON, 1976, EVANS, 1978). Possible sites of release Include the neurohaemal organs such as the corpora cardlaca. Thus the corpora cardlaca are known to be involved in release of adipoklnetic hormone during flight (MAYER and REFERENCES CANDY, 1969) However, it can be calculated that the AXELROD J (1962) Purification and propernes of amount of octopamlne stored in the corpora cardaaca phenylethanolamlne-N-methyl transferase J btol Chem (EvAns, 1978) is insufficient to account for the 237, 1657-1660 observed elevation of haemolymph octopamlne CANDY D J (1978) The regulation of locust flight muscle Therefore, either octopamlne must be rapidly metabolism by octopamlne and other compounds Insect synthesized during flight or other tissues such as the Btochem 8, 177-181 brain and ganglia may also be involved in octopamxne DAVIS N T and ALANIS J (1979) Morphological and electrophyslologlcal characteristics of a dorsal unpaired release Th~s problem is presently under investigation median neuron of the cricket, Acheta dome3twuv Comp A role for octopamlne acting as a local factor Btochem Physlol 62A, 777-788 modulating contractions of the locust extensor tibiae DOWNERR G H (1979) Trehaloseproducnon in isolated fat muscle has been suggested by HOYLE (1975) This body of the American cockroach, Perlplaneta amerltana muscle is innervated by an octopaminerglc neurone Comp Btochem Physwl 62C, 31-34 ' D U M E T I ' (EVANS and O'SHEA, 1978) A similarlyEVANS P D (1978) Octopamlne distribution in the insect staining neurone ' D U M D L ' from the same group of nervous system J Neurochem 30, 1009-1013 cells in the metathoraclc ganglion has been reported to EVANS P D , KRAVITZ E A and TALAMO B R (1976) be associated with the mnervation of the dorsal Octopamlne release at two points along lobster nerve trunks J Phywol 262, 71-89 longitudinal flight muscle of newly moulted locusts (HovLE, 1978) and of crickets (Davis and ALANIS, EVANSP D and O'SHEA M (1978)The identification of an octopamlnerglc neurone and the modulation of a 1979), although no neurosecretory endings have been myogenic rhythm m the locust J erp Btol 73, 235-260 reported in flight muscles If this neurone is also HARMERA J and HORN A S (1977) Octopamlne-sensltlVe octopamlnergIc (and there IS no direct evidence for this adenylate cyclase in cockroach brain effects of agomsts, at present), local release of octopamlne could also antagonists and guanyl nucleotldes Molet Pharmac 13, affect flight muscle 512-520 Comparison of the relative rates of enzymeHOYLE G (1975) Evidence that insect dorsal unpaired catalyzed methylation of octopamlne from haemomedian (DUM) neurones are octopammerglc J evp Zool lymph with authentic D- and e-octopamlne provides 193, 425-431 good evidence that the o-isomer of octopamlne is HOYLEG (1978) The dorsal, unpaired, medmn neurones of the locust metathoraclc ganglion J Neurobtol 9, 43-57 present in locust haemolymph To our knowledge this KAPPE T and ARMSTRONGM D (1964) Preparation of is the first direct demonstration of the isomeric form of D-and L-octopamlne J reed Chem 7, 569-571 octopamlne in insects There is, however, indirect MAYERR J and CANDVD J (1969) Control ofhaemolymph evidence that the D-isomer is the naturally-occurring lipid concentration during locust flight an adlpokmetlc form In that where tested it is more active on hormone from the corpora cardlaca J ln~e~t Phv~tol 15, octopamlne-sensltlVe systems than L-OCtopamlne. 611--620 Thus HARMER and HORn (1977) showed that MOLINO~FP B, LANDSBLRGL and AXrLROD J (1969) An D-Octopamine was 200-fold more active than enzymatic assay for octopamme and other fl-hydroxy

Octoparnme of locust haemolymph phenylethylammes J Pharm exp Therapeutics 170, 253-261 O'SHEA M and EVANS P D (1979) Potentiation of neuromuscular transmission by an octopamlnerglc neurone in the locust. J ex# Btol. 79, 169-190 ROBERTSON H A (1976) Octopamme, dopamme and noradrenahne content of the brain of the locust, Schtstocerca gregarta Expertentla, 32, 552-554. ROBERTSON H A. and CARLSONA. D (1976) Octopamme

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presence in firefly lantern suggests a transmitter role J exp Zool 195, 159-164 ROBERXSONH A and JUORIOA V (1976) Octopamme and related non-catechohc arnlnes m invertebrate nervous systems Int Rev Neurobtol 19, 173-224 ROBFRTSOr4 H A and ST~L~ J E (1973) Effect of monophenohc amines on glycogen metabohsm m the nerve cord of the American cockroach, Perlplaneta americana Insect Bzochem 3, 53-59