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Role of acetylcholine in organophosphate-induced release of adipokinetic hormone in the locust Schistocerca gregaria
PESTICIDE
BIOCHEMISTRY
Role
AND
7, 283-288
PHYSIOLOGY
(1977)
of Acetylcholine in Organophosphate-Induced of Adipokinetic Hormone in the Locust Schistocerca gregaria
of
Department
Zoology,
Received
Downing
March
Street,
29, 1976;
Cambridge
accepted
June
CB2
SEJ,
Release
England
14, 1976
The release of adipokinetic hormone at the paralytic stage of poisoning is probably a result of the excitant effects of insecticides on the central nervous system. When poisoned with organophosphorus compounds, the synapt’ic buildup of acetylcholine acts as a transmitter for the release of hormone. Muscarinic and nicotinic antagonists prevent organophosphate-induced release of this hormone. INTRODUCTION
The nervous system is the primary site of action of many insecticides. Treatment with insecticide chemicals leads to multiple neurohormone release in insects (1, 2). The glandular lobes of the corpus cardiacurn of the desert locust Schistocerca gregaria contain the adipokinetic hormone, which on release elevates the level of lipid in the haernolymph (3). Insecticide poisoning, when it reaches the paralytic stage, brings about the abnormal release of this hormone from its natural release site (2). The demonstration of cholinergic synapses in the insect nervous system has established the role of acetylcholine (ACh)2 as a transmitter (4). The mechanism of the reaction between ACh and its enzyme, acetylcholinesterase, has been studied by Hellenbrand and Krupka (5). Poisons like nereistoxin act on the ACh receptor itself and carba6-2, while organophosphates 1 Present address : Strangeways Research Labor* tory, Worts Causeway, Cambridge CBl 4RN, England. 2 Abbreviations used: ACh, acetylcholine; NCCl and NCCP, nervi corporis cardiaci 1 and 2, respectively; RH, relative humidity.
mates are anticholinesterases (7). Poisoning with anticholinesterases results in the persistence of ACh in the synapse ; this prolongs the excitatory postsynaptic potential and then causes a block in nervous transmission (8). These latter insecticides are termed “cxcitat.ory” (8) in contrast to nereistoxin and its derivatives which do not depolarise the postsynaptic membrane and hence cause no excitatory symptoms (6). However, it does not follow that all “excitatory” insecticides are inhibitors of acetylcholinesterase ; insecticides with other modes of action are also included in this category (S). It is interesting to determine if adipokinetic hormone release is a general feature of insecticide poisoning or whether it is brought about by the excitant effects some poisons, for example, organophosphorus compounds, have on the nervous system. The action of anticholinesterases results in the accumulation of the cholinergic Anticholinesterases bring transmitter. about the release of adipokinetic hormone (2, and this investigation) ; the question arises as to whether the synaptic build up of ACh acts as a transmitter for the release 283
Copyright All rights
@ 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN 00453575
2s4
MASTHRI
tiAYARASATAKA
of adipoliinctic hormone in &hiSkJceJTU. If so, where do such synapses exist in t,hc nervous system? This paper dcscrihos sonl( cxperimcnts designed to assess the importance of t,hc cholincrgic system in the release of this hormone, and a tentat,ivc conclusion about, the participation of ACh in organophosphate-induced hormone release> is reached. MATERIALS
AND
METHOD8
Adult male Schistocerca yreyaria were taken from laboratory cultures maintained at 35’C and 30-35% RH under crowded conditions and fed on wheat and bran. Locusts were treated with the organophosphate Baythion, an emulsifiable conccntrate containing 500 pg/liter of phoxim (Bayer Agrochemicals), as previously described (2) or were injected wit’h the nereistoxin derivative cartap (May and Baker), which was dissolved in locust saline (9) at a concentration of 250 pg. g-l.5 &I. Haemolymph lipid was d(%cLrmined just before poisoning and at prostration according to the method of Goldsworthy et al. (3). ACh antagonists, d-tubocurarinc chloride (curare) and atropine sulphate (both from Sigma), were dissolved in saline, and 10 ~1 was injected into Xchistocerca. Locusts were poisoned with Baythion immediately after this injection, and a second inject,ion was given 15-30 min later. Haemolymph lipid was sampled just before poisoning and at, prostration and lipids were estimated as before (3). 111vitro preparatiom. These kvere made to determine if the cholinergic synapses existed in the corpus cardiacum. The corpus cardiacum was dissected (lo), and the glands mere transferred into saline wit’h minimum handling and kept in this medium for 2 hr during which the saline was changed two or three times. Such trcat,mcnt ensurcad that, the glands were unstimulated at the time of testing, a necessary precaution, sinct: it was obscrvcid t,hat soon aftctr rc-
moval from t,llt: ins& tlic,g were sufic*icWlJ cbxcitcbd tc.) release assayable a~ll~JUIltS of hormontl. hwps of sahic wtitaining nwtylcholine chloride (Sigma), physostigminc sulphatc (eserine, BDH), Bayfhion, or modifird locust saline containing SO mU potassium were held under liquid paraffin at a volume of 75 ~1 of solution pclr gland pair, several glands being pooled. Glands were incubated for 1.5-20 min, and 50 ~1 of this medium was injected ink) lOcUdS, and blood lipid was measured (3) after 2 hr. RESULTS
When poisoned with cartap, Schistocerca failed to show any significant elevation in the level of haemolymph lipid, although they became prostrate. As results in Table 1 show, cartap-poisoned insects maintained a constant level of haemolymph lipid in contrast t’o t’hose poisoned with Baythion, when lipid levels were elevated by as much as 2OOoj,. When Xchistocerca was injected with 10 ~1 of 1O-2 111acetylcholinc chloride, the haemolymph lipid content was elevated only by 4.3 f 3.157,. 150-
140130120‘ IIOIOOPO< 9
80
701 Ia 60.5 $ 50
E 40* 3020IOo-
FIG.
poisoning atropine.
1. Change
in haemolymph lipid after Baythion of Schistorerca which WPW pretrrakd with Dose of Baythion: 600 pg. g-‘. 5 plkI.
ROLE
OF ACETYLCHOLINE
IN
TABLE C’omparison
Insecticide
Cartap Baythion
(12)b (8)
HORMONE
1
between Excitatory and Nonexcitatory Ability to Elevate Haemolymph Dose
250 pg. g-r10 500 pg. g-l.5
Before
,u-’ pl-’
treatment”
5.77 f 0.48 4.23 rk 0.47
(r The values given are for milligrams of lipid per cubic the mean f SE. b The number of determinations is given in parentheses.
Figures 1 and 2 show the change in haemolymph lipids on Baythion poisoning of Schistocerca which were pretreated with either atropine or curare. Lipid levels in the haemolymph of such insects are increased by relatively smaller amounts when compared to those which were treated with Baythion alone (Table 1). Atropine and curare block ACh receptors, thereby preventing the buildup of acetylcholine at the synapse ; since the cholinergic antagonist were capable of preventing hormone release (Figs. 1 and 2), it can be inferred that cholinergic components participate in the release of adipokinetic hormone. However, 10-l M atropine did not prevent lipid release (Fig. 1) ; at high concent’rations, atropine may have a direct pharmacological action or other unspecified effect.
28,5
RELEASE
centimeter
Insecticidrs Lipid After
on the
treatment”
5.91 f 12.18 f
Percentage change
0.59 0.82
of haemolymph
+2.4 $202.89 and
are expressed
as
significant release of adipokinetic hormone when this medium was injected into Schistocerca (Table 2). DISCUSSION
On treating Schistocerca with the “nonexcitatory” insecticide cartap, haemolymph lipid levels were elevated by only 2.4% as compared to an increase of 202.89% observed in insects which were poisoned with the “excitatory” insecticide, Baythion (Table 1). This suggests that the release of neurohormone is probably a result of the excitant effects of insecticides on the central nervous system. Anticholinesterases bring about the release of neurohormones at paralysis in Rhodnius prolixus, but when treated with cartap they become paralysed without accompanying neurohormone release (11). In Vitro Studies A substance which mediates synaptic When the corpus cardiacum of Schisto- transmission, such as ACh, should fulfil certain criteria if it is to be characterised cerca was incubated in saline containing ACh, adipokinetic hormone was released as a transmitter (la), for example in the into the incubation medium. This was events leading to adipokinetic hormone shown by an increase in haemolymph lipid release. The ability of ACh antagonists to of locusts that were injected with this reduce insecticide-induced hormone release (incubation) medium. Results of such significantly (Figs. 1 and 2) supports the assays are set out in Table 2. In a similar hypothesis tha.t acetylcholine is required to manner, eserine and high-potassium saline mediate the release of this hormone. Howreleased adipokinetic hormone from iso- ever, the insect central nervous system is insensitive to extrinsic ACh (13-13), due lated corpora cardiaca, while Baythion either to the presence of a diffusion barrier produced a much smaller effect. In control (16) or to the hydrolysis of ACh (17). It is experiments, corpora cardiaca were incubated in normal saline, in the absence of therefore not surprising that ACh injected drugs or high potassium, and there was no into Schistocercawas without an appreciable
2sc,
MANTHRI
SAMARAXAYAKA
in(~r(~;b8(~ i11 t~;tc~molymph lipitl, :m(i (11’tl~ts t’wo, the> musearinic~ :mt:lgonist \V:IS perhaps more pot.cnt. That both should hc eflectivc is not unexpected; the ,t(lh receptor in t.he brain of 11fUSCCG, for example, shows both nicotinic and muscarinic characteristics (21). However, this system suffers a drawback becauseit is not- possible to establish where ACh synapses, particular to this pathway of hormone release, exist in Schistocerca. The corpus cardiacum of Schistocerca receives a double innervation from the brain (22). Goldsworthy et al. (23), studying the release of adipokinetic hormone in vivo, demonstrated that this release was prevented if t,he nerves from the brain to the corpus cardiacum, the NCCl and NCC2, were severed. From this it follows that information from the brain was necessary for normal release of hormone. We may speculate, therefore, that the ACh synapse participating in organophosphateinducted hormone release is located in the brain (corpus cardiacum complex); it is also known that the head of Schistocerca contains acetylcholine (24). Membrane depolarisation brings about the release of adipokinetic hormone from the isolated corpus cardiacum (Table 2), and potassium is a more effective depolarising agent than ,4Ch. Elevated potassium concentrations are known to relea.sediuretic hormones from their neurohaemal areas in
FIG. 2. Change in haemolymph lipid after Baythion poisoning of Schistocerca which were pretreated with curare. Dose of Baythion: 600 rg.g-’ .5 pl-I.
effect on lipid release. Injection of the AChlike drug pilocarpine also faiIed to elicit a response. Several lines of investigation have shown the presence of acetylcholine receptors in insects. Results of the experiments in vivo (Figs. 1 and 2) are in many respects similar to those observed with isolated preparations by other workers (18-20). Low concentrations of both the nicotinic antagonist, curare, and the muscarinic antagonist, atropine, were effective in limiting the TABLE The Effectiveness
of Various
2
Substances in Promoting
Release of Adipokinetic
Compound
in Vitro
Hormone
Concentrationa
ACh
+G9.1
i (4)
19.2
+71.2
Eserine -
B&him
* 21.2 (7) -
+ci9.9
* 20.3
+86.3
(6) +229.2
zt 51.9
-+ 136.2
(6)
-
i
17.7
f
49.0
-
(4)
(6) +7.1
f
6.3
+2.3
(61 Saline Increase
a The number
Normal-30 in blood
values given of observations
lipid
are
(%I
the percentage change is given in parentheses.
--G.l
in harmolymph
mM
I<+
80 1nM
* 4.0 (6) lipid
+224.2
i (6)
4.7
K’ * 37.1
(10) conrcntmtim
and
are cxpr~as~d
RS thr
mean
+ SE.
The
ROLE
OF ACETYLCHOLINE
Rhodnius and Glossina austerli (25). ACh induces considerable exocytotic activity in vivo in neurosecretory axons of the corpus cardiacum of Calliphora (26), but here it is not clear whether the effect is a direct one upon nerve endings or whether it is mediated through an intermediate nervous link. Of the anticholinesterases, eserine wa.s able to release the hormone from isolated glands, while Baythion was without much effect (Table 2). Eserine-like compounds possess cholinomimetic activity of the muscarinic type and as a consequence may well trigger activity at the nerve endings in a manner similar to ACh, therefore releasing the hormone from the isolated glands. Structurally, Baythion differs from eserine. Steric factors could well explain why Baythion seemsnot to be cholinomimetic; it is also possible that higher concentrations of Baythion are required to produce an effect similar to that of eserine. In these in vitro studies on the corpus cardiacum, the incubation medium injected into locusts contained ACh and eserine; it is very likely tha.t these suhstances themselves could act in vivo to produce an adipokinetic response. However, this is not so, for 10e2 M acetylcholine chloride and 10m3M eserine, when injected into Schistocerca, elevated blood lipid by 4.3 f 3.15 and 3.4 & 2.457,, respectively, and did not produce any symptoms of poisoning. Evidence for ACh mediation in hormone release in insects is scarce (26, 27). In the adrenal medulla of mammals, however, the role of ACh is well established (28). Cholinergic transmitters may not be directly involved in insecticide-induced release of adipokinetic hormone. However, cholinergic neurons may play an important role in the link between sensory information concerning the level of blood lipid and the subsequent release of adipokinetic hormone. Agents which mimic acetylcholine stimulate the nervous loop causing hormone release, whereas agents such as atropine and curare, which prevent transmission of ACh,
IN
HORMONE
RELEASE
257
will block secretion. Although ACh alone was capable of stimulating the release of hormone, this does not necessarily mean that it reacts directly with the intrinsic cells containing the adipokinetic hormone. Other components may well participate in such release i.e., the ACh loop involved, even within the corpus cardiacum, may not be acting by itself. This possibility is the subject of another publication (29). ACKNOWLEDGMENTS
I wish to thank my supervisor, Dr. S. H. P. Maddrell, and Drs. M. J. Berridge and H. A. Robertson for advice and helpful discussion. I acknowledge a Commonwealth Scholarship for financial support.
REFERENCES
1. 8. H. P. Maddrell and S. E. Reynolds, Release of hormones in insects after poisoning with insecticides, NatzLre (London) 236, 404 (1972). 2. M. Samaranayaka, Insecticide-induced release of hyperglycaemic and adipokinetic hormones of Schistocerca gregaria, Gen. Comp. Endocrinol. 24, 424 (1974). 3. G. J. Goldsworthy, W. Mordue, and J. Guthkelch, Studies on insect adipokinetic hormones, Gen. Comp. Endocrinol. 18,545 (1972). 4, R. M. Pitman, Transmitter substances in insects: A review, Comp. Gen. Pharmacol. 2, 347 (1971). 5. K. Hellenbrand and R. M. Krupka, Kinetic studies on the mechanism of insect acetylcholinesterase, Biochemistq 9, 4665 (1970’). 6. M. Sakai, Nereistoxin and its derivatives: Their ganglionic blocking and insecticidal activity, in “Biochemical Toxicology of Insecticides” (R. D. O’Brien and I. Yamamoto, Eds.), 13, Academic Press, New York, 1970. 7. W. N. Aldridge, The nature of the reaction of organophosphorus compounds and carbamates with esterases, in “Alternative Insecticides for Vector Control,” BuU. WHO 44, 25 1971. 8. T. Narahashi, Effects of insecticides on ex“Advances in Insect citable tissues, in Physiology” (J. W. L. Beament, J. E. Treherne, and V. B. Wigglesworth, Eds.), Academic Press, New York, 1971. 9. S. H. P. Maddrell and S. Klunsuwan, Fluid secretion by in vitro preparations of the Malpighian tubules of the desert locust Sch.istocerca gregaria, J. Insect Physiol. 19, 1369 (1973).
2S8
MANTHRI
SAMARAXAYATU
10. n1. S:tnl:tr:tll:ty:;tk:l, “Sludieh 011 lhc I.:lt’rcalh of Insecticides 011 r’ic:his/occ~~r~c~ y~yc~irr,” Ph. I ). Thesis, Ulliversity of (‘:tmbridge, (~:tn~bridg(~, 1975. 11. S. E. Reynolds, “The Plasticization of the Abdominal Cuticle in Rhodnius,” Ph.D. Thesis, Unive&y of Cambridge, Cambridge, 1973. 12. Y. Pichon, The pharmacology of the insect nervous system, in “The Physiology of Insecta” (M. Rockestein Ed.), Vol. 4, p. 102, Academic Press, New York, 1974. 13. B. M. Twarog and R. D. Roeder, Properties of the connective tissue sheath of the cockroach abdominal nerve cord, Riol. Bull. Woods Hole 111, 278 (1956). 14. T. Yamasaki and T. Narahashi, Synaptic transmission in the last abdominal ganglion of the cockroach, J. Insect Physiol. 4, 1 (1960). 15. J. E. Treherne, Some effects of the ionic composition of the ext,racellular fluid on the electrical activit#y of the cockroach abdominal nerve cord, J. Ezp. Riol. 39, 631 (1962). 16. R. D. O’Brien and R. W. Fisher, The relation between localization and toxicity to insects for some neuropharmacological compollnds, J. &on. Entomol. 51, 169 (1958). 17. J. E. Treherne and D. S. Smith, The metabolism of acetylcholine in the intact central nervous system of an insect (Per$anetn an~rirana L.), J. Exp. Biol. 43, 441 (196.5). 18. J. J. Callec and J. Boistel, Further evidence for ACh transmission in the cockroach central nervous system studied at the unitary level, in “Proceedings. XXV Int,ernational Congress of the IUPS, Munich,” Vol. 9, p. !15, 1971. 19. D. L. Shankland, J. A. Rose, and D. Donninger, The cholinergic nature of the cereal nervegiant fibre synapse in the sixth adbominal ganglion of the American cockroach, I’eriplaneta americana L., J. Neurobiol. 2, 247 (1971).
21.
22.
23.
24.
2;i.
26.
27.
28. 29.
il.
Toppazada I*%lerfrawi atld R. I ). O’Brien, Binding of nlwxm~o~~~ by Pxt*ra(.t‘: of housefly brain : Relationship t,o receptors for xcetylcholine, J. Newochem. 17, 1287 11970). K. C. Highnam, The hislology of the neurosecretory system of the adldt fenlale desert, locust, Schistocerca gregaria, Quart. .I. Microsc. fki. 102, 27 (1961). C:. J. (;oldswort,hy, R. A. Johlrsolt, and I$-. Mordue, In viva studies on the release of hormones from the corpora cardiaca of locI1sts, J. Camp. Physiol. 79, 85 (1972). D. Bellamy, The structure and metabolic properties of tissue preparations from Schistocerca gregariu (desert locust), Hi&em J. 70, 580 (1958). S. IT. P. Maddrell and J. 1). (ice, Potassiuminduced release of the dinretic hormones of Rhodnius prolixus and Glossina accsteni: Ca dependence, time course and localization of neurohaemal areas. J. Exp. Miol. 61, 1.55 (1974). T. C. Normann, The mechanism of hormone release from neurosecretory axon endings in the insect Calliphora erythrowphala, in “Aspects of Neuroendocrinology” 0V. Bergman and B. Scharrer, Eds.), p. 30, SpringerVerlag, Berlin and New York, 1970. H. I>. Rounds and W. A. St,uder, The effects of cholinergic and adrenergic blocking agents on the cardioacceleratory activity in extracts from actjive and inactive cockroaches, Gm. Camp. PharmacoL 1, 93 (1970). “The Hormones-Endocrine C. T. Sawin, Physiology,” Little, Brown, Boston, 1969. M. Samaranayaka, The possible involvement of monoamines in the release of adipokinetic hormone in the locrtst Schistocerca gregaria, J. Exp. Biol., in press.
BIOCHEMISTRY
Role
AND
7, 283-288
PHYSIOLOGY
(1977)
of Acetylcholine in Organophosphate-Induced of Adipokinetic Hormone in the Locust Schistocerca gregaria
of
Department
Zoology,
Received
Downing
March
Street,
29, 1976;
Cambridge
accepted
June
CB2
SEJ,
Release
England
14, 1976
The release of adipokinetic hormone at the paralytic stage of poisoning is probably a result of the excitant effects of insecticides on the central nervous system. When poisoned with organophosphorus compounds, the synapt’ic buildup of acetylcholine acts as a transmitter for the release of hormone. Muscarinic and nicotinic antagonists prevent organophosphate-induced release of this hormone. INTRODUCTION
The nervous system is the primary site of action of many insecticides. Treatment with insecticide chemicals leads to multiple neurohormone release in insects (1, 2). The glandular lobes of the corpus cardiacurn of the desert locust Schistocerca gregaria contain the adipokinetic hormone, which on release elevates the level of lipid in the haernolymph (3). Insecticide poisoning, when it reaches the paralytic stage, brings about the abnormal release of this hormone from its natural release site (2). The demonstration of cholinergic synapses in the insect nervous system has established the role of acetylcholine (ACh)2 as a transmitter (4). The mechanism of the reaction between ACh and its enzyme, acetylcholinesterase, has been studied by Hellenbrand and Krupka (5). Poisons like nereistoxin act on the ACh receptor itself and carba6-2, while organophosphates 1 Present address : Strangeways Research Labor* tory, Worts Causeway, Cambridge CBl 4RN, England. 2 Abbreviations used: ACh, acetylcholine; NCCl and NCCP, nervi corporis cardiaci 1 and 2, respectively; RH, relative humidity.
mates are anticholinesterases (7). Poisoning with anticholinesterases results in the persistence of ACh in the synapse ; this prolongs the excitatory postsynaptic potential and then causes a block in nervous transmission (8). These latter insecticides are termed “cxcitat.ory” (8) in contrast to nereistoxin and its derivatives which do not depolarise the postsynaptic membrane and hence cause no excitatory symptoms (6). However, it does not follow that all “excitatory” insecticides are inhibitors of acetylcholinesterase ; insecticides with other modes of action are also included in this category (S). It is interesting to determine if adipokinetic hormone release is a general feature of insecticide poisoning or whether it is brought about by the excitant effects some poisons, for example, organophosphorus compounds, have on the nervous system. The action of anticholinesterases results in the accumulation of the cholinergic Anticholinesterases bring transmitter. about the release of adipokinetic hormone (2, and this investigation) ; the question arises as to whether the synaptic build up of ACh acts as a transmitter for the release 283
Copyright All rights
@ 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN 00453575
2s4
MASTHRI
tiAYARASATAKA
of adipoliinctic hormone in &hiSkJceJTU. If so, where do such synapses exist in t,hc nervous system? This paper dcscrihos sonl( cxperimcnts designed to assess the importance of t,hc cholincrgic system in the release of this hormone, and a tentat,ivc conclusion about, the participation of ACh in organophosphate-induced hormone release> is reached. MATERIALS
AND
METHOD8
Adult male Schistocerca yreyaria were taken from laboratory cultures maintained at 35’C and 30-35% RH under crowded conditions and fed on wheat and bran. Locusts were treated with the organophosphate Baythion, an emulsifiable conccntrate containing 500 pg/liter of phoxim (Bayer Agrochemicals), as previously described (2) or were injected wit’h the nereistoxin derivative cartap (May and Baker), which was dissolved in locust saline (9) at a concentration of 250 pg. g-l.5 &I. Haemolymph lipid was d(%cLrmined just before poisoning and at prostration according to the method of Goldsworthy et al. (3). ACh antagonists, d-tubocurarinc chloride (curare) and atropine sulphate (both from Sigma), were dissolved in saline, and 10 ~1 was injected into Xchistocerca. Locusts were poisoned with Baythion immediately after this injection, and a second inject,ion was given 15-30 min later. Haemolymph lipid was sampled just before poisoning and at, prostration and lipids were estimated as before (3). 111vitro preparatiom. These kvere made to determine if the cholinergic synapses existed in the corpus cardiacum. The corpus cardiacum was dissected (lo), and the glands mere transferred into saline wit’h minimum handling and kept in this medium for 2 hr during which the saline was changed two or three times. Such trcat,mcnt ensurcad that, the glands were unstimulated at the time of testing, a necessary precaution, sinct: it was obscrvcid t,hat soon aftctr rc-
moval from t,llt: ins& tlic,g were sufic*icWlJ cbxcitcbd tc.) release assayable a~ll~JUIltS of hormontl. hwps of sahic wtitaining nwtylcholine chloride (Sigma), physostigminc sulphatc (eserine, BDH), Bayfhion, or modifird locust saline containing SO mU potassium were held under liquid paraffin at a volume of 75 ~1 of solution pclr gland pair, several glands being pooled. Glands were incubated for 1.5-20 min, and 50 ~1 of this medium was injected ink) lOcUdS, and blood lipid was measured (3) after 2 hr. RESULTS
When poisoned with cartap, Schistocerca failed to show any significant elevation in the level of haemolymph lipid, although they became prostrate. As results in Table 1 show, cartap-poisoned insects maintained a constant level of haemolymph lipid in contrast t’o t’hose poisoned with Baythion, when lipid levels were elevated by as much as 2OOoj,. When Xchistocerca was injected with 10 ~1 of 1O-2 111acetylcholinc chloride, the haemolymph lipid content was elevated only by 4.3 f 3.157,. 150-
140130120‘ IIOIOOPO< 9
80
701 Ia 60.5 $ 50
E 40* 3020IOo-
FIG.
poisoning atropine.
1. Change
in haemolymph lipid after Baythion of Schistorerca which WPW pretrrakd with Dose of Baythion: 600 pg. g-‘. 5 plkI.
ROLE
OF ACETYLCHOLINE
IN
TABLE C’omparison
Insecticide
Cartap Baythion
(12)b (8)
HORMONE
1
between Excitatory and Nonexcitatory Ability to Elevate Haemolymph Dose
250 pg. g-r10 500 pg. g-l.5
Before
,u-’ pl-’
treatment”
5.77 f 0.48 4.23 rk 0.47
(r The values given are for milligrams of lipid per cubic the mean f SE. b The number of determinations is given in parentheses.
Figures 1 and 2 show the change in haemolymph lipids on Baythion poisoning of Schistocerca which were pretreated with either atropine or curare. Lipid levels in the haemolymph of such insects are increased by relatively smaller amounts when compared to those which were treated with Baythion alone (Table 1). Atropine and curare block ACh receptors, thereby preventing the buildup of acetylcholine at the synapse ; since the cholinergic antagonist were capable of preventing hormone release (Figs. 1 and 2), it can be inferred that cholinergic components participate in the release of adipokinetic hormone. However, 10-l M atropine did not prevent lipid release (Fig. 1) ; at high concent’rations, atropine may have a direct pharmacological action or other unspecified effect.
28,5
RELEASE
centimeter
Insecticidrs Lipid After
on the
treatment”
5.91 f 12.18 f
Percentage change
0.59 0.82
of haemolymph
+2.4 $202.89 and
are expressed
as
significant release of adipokinetic hormone when this medium was injected into Schistocerca (Table 2). DISCUSSION
On treating Schistocerca with the “nonexcitatory” insecticide cartap, haemolymph lipid levels were elevated by only 2.4% as compared to an increase of 202.89% observed in insects which were poisoned with the “excitatory” insecticide, Baythion (Table 1). This suggests that the release of neurohormone is probably a result of the excitant effects of insecticides on the central nervous system. Anticholinesterases bring about the release of neurohormones at paralysis in Rhodnius prolixus, but when treated with cartap they become paralysed without accompanying neurohormone release (11). In Vitro Studies A substance which mediates synaptic When the corpus cardiacum of Schisto- transmission, such as ACh, should fulfil certain criteria if it is to be characterised cerca was incubated in saline containing ACh, adipokinetic hormone was released as a transmitter (la), for example in the into the incubation medium. This was events leading to adipokinetic hormone shown by an increase in haemolymph lipid release. The ability of ACh antagonists to of locusts that were injected with this reduce insecticide-induced hormone release (incubation) medium. Results of such significantly (Figs. 1 and 2) supports the assays are set out in Table 2. In a similar hypothesis tha.t acetylcholine is required to manner, eserine and high-potassium saline mediate the release of this hormone. Howreleased adipokinetic hormone from iso- ever, the insect central nervous system is insensitive to extrinsic ACh (13-13), due lated corpora cardiaca, while Baythion either to the presence of a diffusion barrier produced a much smaller effect. In control (16) or to the hydrolysis of ACh (17). It is experiments, corpora cardiaca were incubated in normal saline, in the absence of therefore not surprising that ACh injected drugs or high potassium, and there was no into Schistocercawas without an appreciable
2sc,
MANTHRI
SAMARAXAYAKA
in(~r(~;b8(~ i11 t~;tc~molymph lipitl, :m(i (11’tl~ts t’wo, the> musearinic~ :mt:lgonist \V:IS perhaps more pot.cnt. That both should hc eflectivc is not unexpected; the ,t(lh receptor in t.he brain of 11fUSCCG, for example, shows both nicotinic and muscarinic characteristics (21). However, this system suffers a drawback becauseit is not- possible to establish where ACh synapses, particular to this pathway of hormone release, exist in Schistocerca. The corpus cardiacum of Schistocerca receives a double innervation from the brain (22). Goldsworthy et al. (23), studying the release of adipokinetic hormone in vivo, demonstrated that this release was prevented if t,he nerves from the brain to the corpus cardiacum, the NCCl and NCC2, were severed. From this it follows that information from the brain was necessary for normal release of hormone. We may speculate, therefore, that the ACh synapse participating in organophosphateinducted hormone release is located in the brain (corpus cardiacum complex); it is also known that the head of Schistocerca contains acetylcholine (24). Membrane depolarisation brings about the release of adipokinetic hormone from the isolated corpus cardiacum (Table 2), and potassium is a more effective depolarising agent than ,4Ch. Elevated potassium concentrations are known to relea.sediuretic hormones from their neurohaemal areas in
FIG. 2. Change in haemolymph lipid after Baythion poisoning of Schistocerca which were pretreated with curare. Dose of Baythion: 600 rg.g-’ .5 pl-I.
effect on lipid release. Injection of the AChlike drug pilocarpine also faiIed to elicit a response. Several lines of investigation have shown the presence of acetylcholine receptors in insects. Results of the experiments in vivo (Figs. 1 and 2) are in many respects similar to those observed with isolated preparations by other workers (18-20). Low concentrations of both the nicotinic antagonist, curare, and the muscarinic antagonist, atropine, were effective in limiting the TABLE The Effectiveness
of Various
2
Substances in Promoting
Release of Adipokinetic
Compound
in Vitro
Hormone
Concentrationa
ACh
+G9.1
i (4)
19.2
+71.2
Eserine -
B&him
* 21.2 (7) -
+ci9.9
* 20.3
+86.3
(6) +229.2
zt 51.9
-+ 136.2
(6)
-
i
17.7
f
49.0
-
(4)
(6) +7.1
f
6.3
+2.3
(61 Saline Increase
a The number
Normal-30 in blood
values given of observations
lipid
are
(%I
the percentage change is given in parentheses.
--G.l
in harmolymph
mM
I<+
80 1nM
* 4.0 (6) lipid
+224.2
i (6)
4.7
K’ * 37.1
(10) conrcntmtim
and
are cxpr~as~d
RS thr
mean
+ SE.
The
ROLE
OF ACETYLCHOLINE
Rhodnius and Glossina austerli (25). ACh induces considerable exocytotic activity in vivo in neurosecretory axons of the corpus cardiacum of Calliphora (26), but here it is not clear whether the effect is a direct one upon nerve endings or whether it is mediated through an intermediate nervous link. Of the anticholinesterases, eserine wa.s able to release the hormone from isolated glands, while Baythion was without much effect (Table 2). Eserine-like compounds possess cholinomimetic activity of the muscarinic type and as a consequence may well trigger activity at the nerve endings in a manner similar to ACh, therefore releasing the hormone from the isolated glands. Structurally, Baythion differs from eserine. Steric factors could well explain why Baythion seemsnot to be cholinomimetic; it is also possible that higher concentrations of Baythion are required to produce an effect similar to that of eserine. In these in vitro studies on the corpus cardiacum, the incubation medium injected into locusts contained ACh and eserine; it is very likely tha.t these suhstances themselves could act in vivo to produce an adipokinetic response. However, this is not so, for 10e2 M acetylcholine chloride and 10m3M eserine, when injected into Schistocerca, elevated blood lipid by 4.3 f 3.15 and 3.4 & 2.457,, respectively, and did not produce any symptoms of poisoning. Evidence for ACh mediation in hormone release in insects is scarce (26, 27). In the adrenal medulla of mammals, however, the role of ACh is well established (28). Cholinergic transmitters may not be directly involved in insecticide-induced release of adipokinetic hormone. However, cholinergic neurons may play an important role in the link between sensory information concerning the level of blood lipid and the subsequent release of adipokinetic hormone. Agents which mimic acetylcholine stimulate the nervous loop causing hormone release, whereas agents such as atropine and curare, which prevent transmission of ACh,
IN
HORMONE
RELEASE
257
will block secretion. Although ACh alone was capable of stimulating the release of hormone, this does not necessarily mean that it reacts directly with the intrinsic cells containing the adipokinetic hormone. Other components may well participate in such release i.e., the ACh loop involved, even within the corpus cardiacum, may not be acting by itself. This possibility is the subject of another publication (29). ACKNOWLEDGMENTS
I wish to thank my supervisor, Dr. S. H. P. Maddrell, and Drs. M. J. Berridge and H. A. Robertson for advice and helpful discussion. I acknowledge a Commonwealth Scholarship for financial support.
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2S8
MANTHRI
SAMARAXAYATU
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