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Pharmacological profile of the excitatory neuromuscular synapses of the insect retractor unguis muscle
Comp. gen. Pharmac., i972, 3, 327-338- [Scientechnica (Publishers) Ltd.]
327
PHARMACOLOGICAL PROFILE OF THE E X C I T A T O R Y N E U R O M U S C U L A R SYNAPSES OF THE INSECT R E T R A C T O R U N G U I S MUSCLE T. J. M c D O N A L D , R. D. FARLEY, AND R. B. M A R C H Departments of Entomology and Biology, University of California, Riverside, California 92502
(Received 3° May, I972 ) ABSTRACT i. A series of 3o chemicals was tested for their ability to alter the neurally evoked contractions of the retractor unguis muscle isolated from the grasshopper, Romalea microptera, and the locusts, Locusta migratoria, Schistocercagregaria, and S. vaga. 2. L-Glutamate was one of the most active compounds tested, supporting evidence from previous studies that it may be the insect neuromuscular transmitter at excitatory junctions. Adrenergic and cholinergic drugs and carbamate and organophosphorus esters (anticholinesterase agents) had little or no effect on the responses of the preparation. 3. N-Ethylmaleimide, two phenothiazines, and a dinitrosalicylanilide at io-~M completely abolished the contractile response. 4- Neurally evoked contractions occurred in solutions withpH values between 6 and io. Contractions were reversibly abolished in pH 5 saline but irreversibly abolished in p H I I saline.
THIS study compares L-glutamate with other because acetylcholine, curare, prostigmine, synaptic drugs in their ability to alter the and most anticholinesterase agents were force of the neurally evoked contractions of ineffective. Negative results for cholinergic the insect retractor muscle. As previous transmission were also reported by other studies on this muscle have been limited to the workers (Roeder and Weiant, x95o ; Iyatomi action of L-glutamate and its analogues and Kaneshina, x958 ). The question of cholinergic transmission (Usherwood and Machili, x966, I968; Usherwood, I967 a, b, I969; Usherwood, was reopened when it was found that injected Cochrane, and Rees, x968; Usherwood, curare caused paralysis in insects (Larsen, Machili, and Leaf, 1968), it seemed reasonable Miller, and Yamamoto, 1966). It was further to establish for this preparation whether or reported that injected curare (McCann, not other transmitter substances might be just I966 ) or acetylcholine (McCann and Reece, as effective as L-glutamate. I f the synapses of 1967) resulted, respectively, in blockade or the retractor unguis muscle were glutanergic, depolarization of the neuromuscular juncadrenergic or cholinergic drugs might be tions of the fly, Sarcophaga bullata. Cholinergic transmission further seemed a possibility expected to have little or no action. Previous studies with whole insects and because of recent observations on an organism large complex nerve-muscle preparations related to insects by Futamachi (i972), who have yielded conflicting results on the cholin- suggested that the tonic flexor muscles of a ergic pharmacology of insect neuromuscular crustacean, Procambarus clarkii, were innerjunctions. Harlow (I 958) found that perfused vated by neurons whose transmitter may be solutions of atropine, eserine, nicotine, and acetylcholine. Furthermore, there are difficulties in various choline esters often induced muscular contractions. However, it was considered studying the action of cholinergic compounds unlikely that the synapses were cholinergic, on tissues, because many are ionized and do
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McDONALD AND OTHERS
n o t r e a d i l y p e n e t r a t e m e m b r a n e barriers (Hoyle, i 9 5 3 ; T w a r o g a n d R o e d e r , i956; O ' B r i e n a n d Fisher, i958; O ' B r i e n , 1959; Eldefrawi a n d O ' B r i e n , 1967). Consequently, one m i g h t w o n d e r if the n e g a t i v e results of F a e d e r , O ' B r i e n , a n d S a l p e t e r (i97o) a n d F r i e d m a n a n d C a r l s o n (I97O) for a cholinergic m e c h a n i s m at insect n e u r o m u s c u l a r j u n c t i o n s were d u e to tests o f only c h a r g e d molecules on large a n d c o m p l e x n e r v e muscle p r e p a r a t i o n s . T h i s difficulty was r e d u c e d in the p r e s e n t e x p e r i m e n t s , w h e r e a small n e r v e - m u s c l e p r e p a r a t i o n (i 50-350 gin. dia.) was used, a n d w h e r e the threshold c o n c e n t r a t i o n s o f b o t h ionized a n d u n - i o n i z e d cholinergic drugs were c o m p a r e d to t h a t o f L-glutamate. T o give a m o r e c o m p l e t e p h a r m a c o l o g i c a l profile o f the r e t r a c t o r unguis muscle, the p H sensitivity was d e t e r m i n e d . This was because o t h e r e x p e r i m e n t s w i t h isolated nerve a n d m u s c l e tissues have e m p h a s i z e d the i m p o r t a n c e o f e x t r a c e l l u l a r p H on tissue responses. F o r e x a m p l e , Brooks a n d H u t t e r (I962 , i963) f o u n d a n o p t i m a l p H r a n g e o f 5 - 1 o for the electrical a c t i v i t y of frog skeletal muscle. I n a n o t h e r study, a c t i o n potentials of lobster g i a n t fibres were reversibly b l o c k e d b y low p H a n d irreversibly blocked b y high p H ( H a f e m a n n , i969). M A T E R I A L S AND M E T H O D S The retractor unguis muscle was dissected from the metathoracic femurs of the insects and mounted in a Plexiglas chamber (o'25 ml.). A Harvard 975 infusion pump with two ioo-ml. syringes was used to provide a i-o ml. per minute flow of saline or drug'solution through the chamber. The saline (pH 6. 9) was prepared according to the formula of Usherwood and Machili (I968), but the calcium was added just before use to minimize the precipitation of calcium, presumably as calcium phosphate (McDonald, I972). In some instances a Tris-buffered modification of the saline was made to avoid the calcium precipitation problem. The Tris-buffered saline (pH 7'3) contained: x3o m M NaC1, Io m M KC1, 2 m M CaC12, 20 m M glucose, and Io m M tris-(hydroxymethyl)-aminomethane. Since only low concentrations ( i o - s_ i o - ~M) of drugs were used, they were dissolved in saline without compensating for the minute increase in osmotic pressure. The contractile force of the muscle was monitored with a Grass .o 3 FT force displacement transducer. A Grass 5PI D.C. preamplifier
Comp. gen. Pharmac.
excited the strain gauges of the transducer and amplified their output which was displayed on chart paper by a Grass polygraph. Retractor unguis preparations were taken from adult orthopterans obtained from several sources. The grasshopper, Romalea microptera, was collected in Florida (H. Williams, Dunnellon, Florida); Locusta migratoria and Schistocerca gregaria were supplied by the Anti-Locust Research Centre, London, England; and specimens of Schistocerca vaga were reared (Dr. W. Loher, Berkeley, California). The chemicals used in these experiments are listed by number in Table L They were obtained from the sources indicated in the table and were used without further purification. RESULTS BIOOENIC AMINES Solutions o f these amines a n d o t h e r drugs of Table I were a d m i t t e d into the tissue c h a m b e r o n l y after the n e u r a l l y evoked contractions were u n i f o r m in a m p l i t u d e for I o - 2 o minutes in U s h e r w o o d ' s saline. After a u n i f o r m a m p l i t u d e was a t t a i n e d in the test solution, it was d e l i v e r e d for a n o t h e r 5 minutes to allow a d d i t i o n a l time for c o m p l e t e d r u g action. N o r e p i n e p h r i n e (I), e p i n e p h r i n e ( I I ) , 3-hydroxytyramine (III), and 5-hydroxytrypta m i n e (IV) are biogenic a m i n e s t h o u g h t to be n e u r o t r a n s m i t t e r substances in various o r g a n isms ( K e r k u t , S h a p i r a , a n d W a l k e r , i965; A n d e r s o n a n d S h i b u y a , 1966; Elofsson, K a u r i , Nielsen, a n d S t r o m b e r g , I966; Florey, 1966). E x c e p t at high c o n c e n t r a t i o n s , these c o m p o u n d s h a v e very little effect on the n e u r a l l y evoked responses o f the r e t r a c t o r unguis muscle from L. migratoria a n d R. microptera (Table I). T h e y were ineffective at io .3 M ; a n d , as shown in the m y o g r a m s of Fig. I, concentrations o f lO .2 M caused only a slight r e d u c t i o n in c o n t r a c t i o n force. This lack o f sensitivity i n d i c a t e d t h a t these drugs a r e unlikely to be n e u r o h u m o r s at the neurom u s c u l a r j u n c t i o n s of this p r e p a r a t i o n . This conclusion was further s u p p o r t e d b y perfusion tests w i t h D L - a m p h e t a m i n e (V), a d r u g w h i c h is t h o u g h t to cause release o f c a t e c h o l a m i n e s from their storage sites in nerveendings (Carlsson, Fuxe, H a m b e r g e r , a n d Lindqvist, 1966; C a r r a n d M o o r e , I969). A t I o - 4 M it h a d no effect on the c o n t r a c t i l e
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response of the retractor unguis preparation, other preparations, that insect neuromuscular although I o - 3 M and l o - 2 M solutions, junctions are insensitive to cholinergic drugs respectively, decreased and totally abolished (Harlow, 1958; O'Conner, O'Brien, and the response. Because relatively high con- Salpeter, I965; Faeder and others, 197o ). centrations of the drug failed to potentiate the response, it was considered unlikely that it ANTICHOLINESTERASE AGENTS Acetylcholine is inactivated at cholinergic caused release of a catecholamine neurojunctions by acetylcholinesterase, an enzyme humor at the junctions.
FIG. I.--Effect of I o-2M solutions of norepinephrine, epinephrine, and 5-hydroxytryptamine on the
neurally evoked contractions of the retractor unguis muscle of Locusta migratoria.
QUATERNARY AMMONIUM COMPOUNDS
Compounds V l - l X act at cholinergic synapses in vertebrate tissue (Triggle, 1965; Cutting, 1967). Acetylcholine (VI) is a common transmitter substance in vertebrates and invertebrates (Triggle, 1965 ; Treherne, 1966); carbamylcholine (VII) is a cholinomimetic (Furchgott and Bursztyn, 1967) , and decamethonium (VIII) and succinylcholine (IX) block cholinergic transmission (Bowman, Hemsworth, and Rand, 1967). These drugs had essentially no effect on the contractile response (Table I and Fig. 2A, B), suggesting that cholinergic receptors are not involved in the synaptic transmission of this tissue. Other quaternary ammonium compounds ( X - X I I ) also had little or no influence on the tissue. These results confirm previous reports for
inhibited by certain carbamate and organophosphorus esters (Heath, 1961 ; Cutting, I967; O'Brien, i967). Since both ionized and un-ionized carbamate and organophosphate esters may be obtained, they are particularly well suited to test whether or not the inactivity of ionized cholinergic drugs is due to their inability to penetrate an ionic barrier. Although results with cholinergic drugs ( V I - X I V , X X I I - X X I V ) indicated that the neuromuscular junctions of the retractor unguis preparation are not likely to be cholinergic, it is possible that the failure of these compounds to affect the tissue was due to their inability to reach the receptor site. As a further test of possible cholinergic activity in this preparation, the tissue was
33 °
Co?tip. gen. Pharmac.
McDONALD AND OTHERS
exposed to b o t h i o n i z e d (XIII-XIV) a n d un-ionized (XVI-XX) anticholinesterase agents. T h e u n - i o n i z e d cholinesterase in-
Table L--CoMPARISON
hibitors,
Baygon ® (XVI) and aldicarb c o m m e r c i a l c a r b a m a t e insecticides (Weiden, Mo o r ef i el d , an d Payne,
(XVII) ar e
OF THE ABILITY OF VARIOUS COMPOUNDS TO CHANGE THE NEURALLY EVOKED CONTRACTIONS OF THE RETRACTOR UNGUIS MUSCLE
The normal response in saline (Usherwood and Machili, i968 ) is designated as ioo per cent. The values shown are typical of those obtained from two or more nerve-muscle preparations taken from the species indicated after each compound: Romalea mieroptera (Rm), Schistocerca gregaria (Sg), S. vaga (Sv), and Locusta migratoria (Lm).
No.
NAME
SPECIES
PERCENTAGE RESPONSE AT MOLAR CONCENTRATIONS i0-5
14 II 4
III 4 IV 4 VIa
Vp ~ VII ix VIII 15 X:6 Xla
XI 4 XII15
XIII 4 XIV19 XV 9 XVI 5 XVI116
XVIII a XIX 9 XX ~ (XI a
KXIP a XXlII* K X I V 15 XXV 1 XXVP a KXVII 7 K X V I I I 1. ~ , . . X I X le KXX a
Norepinephrine Epinephrine 3-Hydroxytyramine 5-Hydroxytryptamine creatinine sulphate DL-Amphetamine hydrochloride Acetylcholine iodide Carbamylcholine chloride Decamethonium bromide Succinylcholine chloride Choline chloride Betaine hydrochloride DL-Carnitine hydroehloride Physostigmine Prostigmine 2-Methyl-acetophenone oximine, W-methyl-carbamate Baygon*, o-isopropoxyphenyl-.N'-methylcarbamate Aldicarb, Temik ®, 2-methyl-2-(methylthio)propionaldehyde-O- (methylcarbamoyl) oxime Paraoxon, diethyl-4-nitrophenyl phosphate Sumioxon *, dimethyl-3-methyl-4-nitrophenyl phosphate Monitor ®, O-methyl-S-methyl phosphoramidothioate Ethyl 4-nitrophenyl-tert-butylphosphonate Nicotine Atropine sulphate D-Tubocurarine chloride L-Glutamate, disodium
Lm Lm Lm Lm, Rm Rm, Sv Sg Lm Lm Lm
10-4
xoo
i0-2
ioo lOO lOO IOO
75 9° 9° 60
IOO
85
O
IOO
lOO
95 lOO IOO IOO
I00
I00
Sg Rm Rm Rm, Sg Lm Rm
i0-3
8o
6o
Ioo
S~
IO0
IO0
IOO
IOO
Rm, Sg
I00
Sg Rm
ioo
ioo
ioo
ioo
Sv
ioo
ioo
I oo lOO
I oo IOO
IOO ioo
ioo 80
lOO
95
9o
o lOO o
IO0
IO
0
3°
o
Rm Rm, Sg Rm, Lm Rm, Sg Lm, Rm, Sg, Sv Rm aV-Ethylmaleimide Iodoacetic acid Rm Chlorpromazine hydrochloride Rm Phenothiazine, lO- (3-dimethylamino-2-methyl- Lm propyl,-, ( + ) - , tartrate 3"Nitr°salicyl'4-nitr°anilide Rm
95 lOO o Ioo
ioo ioo lOO* +~oo* lOO +2oo o o
o o
*Results for normal rather than molar solutions. Sources of Chemicals: 1Aldrich Chemical Co., aAmerican Cyanamid Co., aBen Venue Laboratories, 4Calbiochem., 5Chemagro Corp., eChevron Chemical Corp., ~Eastman Organic Chemicals, SDr. T. R. Fukuto, Dept of Entomology, University of California, Riverside, California, 9Hoffman La-Roche Inc., 1°Merck, Sharp, & Dohme, nNutritional Biochemicals Corp., 1~Sigma Chemical Co., xaSmith, Kline, & French Laboratories, x4Squibb Institute for Medical Research, 15The UpJohn Co., 16Union Carbide Corp.
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331
I965; O'Brien, i967). Paraoxon ( X V I I I ) , Io-4 M, since they have limited water soluSumioxon ® ( X I X ) , and Monitor ® (XX) bility. are un-ionized organophosphorus esters with Although the un-ionized anticholinesterase insecticidal action (Heath, i96x ; Ouistad, agents did not affect the retractor unguis Fukuto, and Metcalf, i97o ). preparation, they were very toxic to intact Physostigmine (XIV) was the only anti- insects at the concentrations tested in Table L cholinesterase which had a significant effect Locusts were dipped for about 3° seconds into on the preparation. At i o -4 M it partially I o -4 M Baygon ®, I o - S M aldicarb, or I o-4 M reduced the contractile response (Table I), paraoxon. After developing symptoms of and at Io -3 M it completely abolished the toxicity, the animals were observed to be dead
FIG. 2.--Effect of Io-2M solutions of acetylcholine, succinylcholine, and prostigmine on the neurally evoked contractions of the retractor unguis muscle ofLocusta migratoria. contractions. This effect was not lasting, since normal contractions often recurred in less than I minute after ending exposure to a io -2 M solution. Prostigmine ( X I I I ) is the other ionized anticholinesterase tested on the tissue. As shown in Fig. 2 C, it did not appreciably alter the response of the tissue even though it was tested at a concentration of IO -2 M. The un-ionized anticholinesterase agents ( X V I XX) also did not affect the tissue response at any of the concentrations tested. Test solutions for some of the compounds could not be prepared at concentrations greater than
after 24 hours. This suggests that the insecticidal action of these compounds is not due to any direct effect on the neuromuscular junction, since lethal concentrations for the intact animal had no effect on the isolated nerve-muscle preparation. Since ion barriers would not be a limiting factor for the activity of these compounds, their failure to affect the preparation is strong evidence for noncholinergic transmission. NICOTINE, ATROPINE, AND CURARE
Nicotine ( X X I I ) has cholinomimetic action in vertebrates and insects (Yamamoto,
332
Comp. gen. Pharmac.
McDONALD AND O T H E R S
i965; Cutting, I967; O'Brien, i967). Atropine ( X X I I I ) and D-tubucurarine ( X X I V ) are blocking agents at vertebrate parasympathetic and skeletal neuromuscular junctions respectively (Cutting, I967). As indicated above, in some previous studies these drugs were thought to have an effect at insect
response, similar to the results obtained with 1o-2Natropine. Also likeatropine,3 × i o - 2 M curare abolished the neurally evoked contractions, with recovery on perfusion with control saline. Since these drugs only produced effects at high concentrations, it was considered unlikely that their action reflected
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I L. FIo. 3.--Perfusion with io ~,N" atropine sulphate initially reduced, then potentiated the neurally evoked contractions of the retractor unguis muscle of Locusta migratoria. neuromuscular junctions (Harlow, I958; O ' C o n n o r and others, i965; Larson and others, I966; McCann, I966 ). Nicotine had no effect on the retractor unguis preparation (Table I), while atropine and curare modified the response only at high concentrations. As shown in Fig. 3, solutions of i o -2 N a t r o p i n e initially reduced the neurally evoked contractions of the preparation, but after I-2 minutes the responses were greatly potentiated. When the concentration was increased to 3 × x°-2 dV, the contractions were completely abolished. When control saline was readmitted to the tissue chamber, potentiated contractions appeared abruptly, decreasing gradually in amplitude until stable responses were obtained at about the same contraction force as occurred in control saline before exposure to atropine. Solutions of I o - ~ M curare ( X X I V ) reduced and then potentiated the contractile
a cholinergic mechanism muscular junction.
at
the
neuro-
L-GLUTAMIC ACID
In previous studics with the retractor unguis muscle of S. gregaria, Ushcrwood and Machili ( 1966, 1968) and Usherwood ( 1967 b) obtained evidcncc that L-glutamate m a y be the neuromuscular transmitter. Concentrations greater than Io -~ g. per ml. reduced the amplitude of the neurally evoked contractions of the preparation. In the present experiments the disodium salt of L-glutamate (XXV) was dissolved in Usherwood's saline, and the solutions were tested on several preparations from each of the four orthopteran species of this study. L-Glutamatc was not observed to potentiate the neurally evoked response in any of these experiments. At [ o-4 M it caused the contractions to decrease to about 8o per cent of that
I972, 3
INSECT NEUROMUSCULAR P H A R M A C O L O G Y
w h i c h was o b t a i n e d w i t h perfusion w i t h c o n t r o l saline. D u r i n g perfusion w i t h i o - 3 M L - g l u t a m a t e , the c o n t r a c t i o n s g r a d u a l l y decreased in a m p l i t u d e , a n d after a b o u t i m i n u t e t h e y were c o m p l e t e l y a b o l i s h e d (Fig. 4)- T h i s
FIG.
333
effect was reversible, since perfusion w i t h c o n t r o l saline caused r e c o v e r y o f the c o n t r a c tion force to the s a m e level as t h a t w h i c h was o b t a i n e d before the tissue was exposed to the test solution.
4.--Effect of I o - 3 M L-glutamate on the neurally evoked response of the retractor unguis muscle
ofRomalea microptera. The contractions were completely abolished, but the effect was readily reversible.
FIG. 5.--Effect of I o - 4 M chlorpromazine on the neurally evoked contractions oi the retractor unguis muscle of Romalea microptera.
FIG. 6.---The reversible effect of low p H (4.6) on the neurally evoked contractions of the retractor unguis muscle ofRomalea microptera. The control saline was at p H 6"9 (Usherwood and Machili, 1968 ).
334
McDONALD
MISCELLANEOUS DRUGS N-Ethylmaleimide ( X X V I ) and iodoacetic acid ( X X V I I ) are sulphydryl inhibitors (Webb, 1966). Both iodoacetic acid (i o-2 M)
AND
OTHERS
Comp. gen. Pharmac.
and W-ethylmaleimide (z 0 -4 M) completely abolished the response. However, W-ethylmaleimide was more effective than iodoacetic acid.
FIG. 7.--The irreversible effect of high pH (i I.O) on the neurally evoked contractions of the retractor unguis muscle of Romalea microptera. The control saline was Tris-buffered (see Materials and Methods) at pH 7"3. 100
Phenothiazine derivatives, particularly chlorpromazine ( X X V I I I ) , are extensively used in the treatment of psychosis (Jarvik, 1965). Chloropromazine (Fig. 5) and di.o_ 80 I methylamino-2-methyl-propyl-phenothiazine I ( X X I X ) both reduced the contraction amplitude of the retractor unguis muscle, but much I 0 ~ 60 more slowly than other effective compounds. 1 I1) Fig. 5 shows that the chlorpromazine effect I was reversible. t~ I 3-Nitrosalicyl-4-nitroanilide ( X X X ) also ~ 4O decreased the contraction force at approxiI mately the same rate as the phenothiazine I derivatives, but it was somewhat more effecI ~. 2o tive in depressing the neurally evoked contractions. Some salicylanilides are fungicides I (Esplin, I965) and uncouplers of oxidative phosphorylation (Williamson and Metcalf, w 4 6 8 10 12 1967). Since Io -4 M concentrations of both the phenothiazine derivatives and the salipH cylanilide diminshed the contraction ampliFie,. 8.--The effect of extracellular pH on the tude, these compounds m a y warrant force of the neurally evoked contractions of the retractor unguis muscle of Romalea microptera. The additional studies to determine their site of response was depressed at pH values less than 6 action. and greater than xo. To avoid calcium precipitation, Tris-buffered saline was used at pH values of p H EFFECT 7.0 and higher. At pH values lower than 7.0 the Fig. 6 shows that Usherwood's saline at phosphate buffered saline (Usherwood and p H 4 . 6 reduced completely the neurally Machili, 1968) was used. t-"
"i"
l
1972, 3
INSECT NEUROMUSCULAR P H A R M A C O L O G Y
evoked contractions of the retractor unguis muscle of R. microptera. This low p H effect was reversible, since contractions reappeared when the tissue was again perfused with p H 6. 9 saline. Fig. 7 shows that Tris-buffered saline at p H 11.o also caused the neurally evoked contractions to cease. However, this effect at this p H was irreversible, since the contractions did not reappear when the tissue was perfused with saline at p H 7"3. Tris-buffered saline was used for p H value 7.0 and higher, because Usherwood's saline developed a white precipitate at high p H values. Fig. 8 summarizes the experimental data of the p H experiments of 7 different muscles and shows that muscle responses m a y be obtained during short perfusions (lO minutes) in salines with p H values between 6 and lO. DISCUSSION T h e biogenic amines ( I - I V ) have little or no effect on the retractor unguis preparation, supporting previous reports that they are not the transmitter for insect skeletal muscle (Harlow, 1958; O ' C o n n e r and others, 1965). Although io -2 M 5-hydroxytryptamine (IV) was previously tbund completely to block neuromuscular transmission in the metathoracic leg muscles of S. gregaria (Hill and Usherwood, i96i ; Usherwood and Machili, I968), only a 4 ° per cent reduction in contraction force was obtained in the present experiments. This difference was not considered to be sufficiently great to merit further investigation. I t is very unlikely, from these experiments, that there is a cholinergic mechanism at the neuromuscular junctions of the retractor unguis muscle. Acetylcholine (VI) at IO - 2 M had no effect on the preparation, and cholinergic (VII) and anticholinergic ( V I I I I X ) drugs were ineffective. Since the quaternary a m m o n i u m compounds are ionized at the p H of the perfusion saline (pH 6-9), it was considered possible that they failed to reach the receptor site (O'Brien and Fisher, 1958; O'Brien 1959; Eldefrawi and O'Brien, i967). Un-ionized anticholinesterases ( X V - X X I ) at I o - 4 M had no effect on the preparation, although these drugs at
335
this concentration were lethal to intact locusts. Generally, the ionized cholinesterase inhibitors ( X I I - X I V ) had a slightly greater effect on the preparation than the un-ionized compounds, suggesting that the charge on the molecule was not a limiting factor for these cholinergic compounds. T h e results with atropine ( X X I I , Fig. 3) and curare ( X X I V ) provided further evidence that cholinergic transmission is unlikely. These agents potentiated the neurally evoked muscle response, while they should block transmission if the junction swere cholinergic. T h e high concentration (I o-2 M ) required for potentiation does not correlate with previous reports of the blocking action of these drugs at known cholinergic synapses. T h e y are effective from lO -e to lO -5 M on frog heart and skeletal muscle junctions (Jenkinson, 1957; Orkland, 1968 ). L-Glutamate ( X X V ) was more effective in altering the contraction amplitude than any of the other transmitter candidates ( I - I V , VI), enforcing the idea that the junctions are glutanergic. At I o -3 M it completely abolished the neurally evoked responses, and since only a very slight reduction in contraction force occurred at i o - S M ( 1 . 9 × i o -6 g. per ml.), this was considered to be the threshold concentration. Similar threshold concentrations for Lglutamate were reported for other arthropod nerve-muscle preparations. Crayfish preparations responded to concentrations of Io - S M or more (Robbins, I959; Van Harreveld, 1959; Takeuchi and Takeuchi, 1964). The threshold levels for crab and lobster tissue were between 2 × I O-4 and 5 × Io-5 M (Van Harreveld and Mendelson, I959; Florey and Woodcock, i968 ). For a nerve-muscle preparation from the cockroach, Gromphadorhina portentosa, the threshold was 2"5 x Io -4 M (Faeder and O'Brien, I97O); and for a leg preparation from the American cockroach, it was IO -7 g. per ml. (Kerkut, Leake, Shapira, Cowan, and Walker, 1965 ; Kerkut and others, 1965; K e r k u t and Walker, 1966 ). Usherwood and Machili (1966, 1968) and Usherwood (1967 b) reported that there was a reduction of the neurally evoked contractions at
336
McDONALD AND OTHERS
c o n c e n t r a t i o n s h i g h e r t h a n IO -~ g. p e r ml. using the r e t r a c t o r unguis muscle from S. gregaria. T h e p r e s e n t results confirm their studies, a l t h o u g h the threshold for the p r e s e n t e x p e r i m e n t s was slightly higher. Phenothiazine compounds (XXVIIIXXIX) cause flacid paralysis in i n t a c t insects (Zukel, 1944; Brown, I963) , a n d in the p r e s e n t e x p e r i m e n t s t h e y caused a r e d u c tion in the c o n t r a c t i l e response o f the r e t r a c t o r unguis muscle. I t was n o t d e t e r m i n e d w h e t h e r these c o m p o u n d s interfered w i t h n e u r o n a l c o n d u c t i o n , s y n a p t i c transmission, or the c o n t r a c t i l e process, b u t t h e y c l e a r l y h a v e a n a c t i o n on p e r i p h e r a l tissue. F a e d e r a n d O ' B r i e n (i97o) r e p o r t e d t h a t chlorpromazine (XXVIII) blocked neuronal c o n d u c t i o n in a c o c k r o a c h p r e p a r a t i o n , b e c a u s e a stepwise r e d u c t i o n in the c o n t r a c t i l e force o c c u r r e d d u r i n g n e u r a l stimulation. I n a d d i t i o n to this effect on nerves, however, c h l o r p r o m a z i n e has also b e e n shown to act d i r e c t l y on muscle tissue (Su a n d Lee, I96O; S h i b a t a a n d C a r r i e r , I967). I n the p r e s e n t studies w i t h c h l o r p r o m a z i n e a stepwise r e d u c t i o n in c o n t r a c t i l e force was n o t observed. This m a y h a v e b e e n d u e to the a c t i o n o f the d r u g on a site o t h e r t h a n a x o n a l transmission. T h e n e u r a l l y evoked responses o f the r e t r a c t o r unguis muscle were forceful d u r i n g perfusion w i t h saline at p H 6 - 1 o . The reversible depression w i t h saline b e l o w p H 6 m a y be d u e to the reversible n a t u r e o f molec u l a r p r o t o n a t i o n ( H a f e m a n , x969). I f activation o f the muscle r e q u i r e d a n ionized site w h i c h b e c a m e p r o t o n a t e d a t low p H , the site should r e t u r n to its original state as the p H is increased. T h e irreversible depression a t p H I I m a y b e d u e to hydrolysis or d e n a t u r a tion o f the m e m b r a n e m a c r o m o l e c u l e s . T h i s was suggested b y H a f e m a n (1969) to a c c o u n t for the irreversible effects o f high p H on lobster g i a n t axons.
ACKNOWLEDGEMENTS This study was supported by N I H Training Grant No. ES ooo47 from the National Institute of Environmental Health Sciences (TJM). Grant NB o81 xo-o I from the National Institute of Health (RDF), and research funds from the Agricultural Experiment Station, University of California,
Comp. gen. Pharmac.
Riverside (RDF). The authors thank Dr. T. R. Fukuto for advice and for some of the chemicals used in these experiments.
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Comparative Animal Physiology, pp. 483-486. Philadelphia: W. B. Saunders Co. FLom~% E., and WOODCOCK, B. (1968), ' P r e synaptic excitatory action of glutamate applied crab nerve-muscle preparations ', Comp. Biochem. Physiol., 26, 651-66I. FRIEDMAN, K. J., and CARLSON,A. D. (197o), 'The effects of curare in the cockroach. I. dTC-inducedfailure of leg contraction', 07. exp. Biol., 52, 583-592. FURCHOOTT, R. F., and BURSZTYN, P. (I967) , 'Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathetic receptors ', in Cholinergic Mechanisms (ed. WEYER,E. M., KRAUSS,M., and EHRENPREIS, S.), pp. 882-899. New York: Ann. N.Y. Acad. Sci. FUTAMACHI,K . J . ( x972), ' Acetylcholine: Possible neuromuscular transmitter in Crustacea ', Science, N.Y., 175, I373-I375. HAVEMANN, D. R. (I969), 'Effects of pH and calcium on action potentials of lobster giant axons ', Comp. Biochem. Physiol., 29, I I63-i 173. HARLOW, P. A. (I958), ' T h e action of drugs on the nervous system of the locust (Locusta migratoria) ', Ann. appl. Biol., 46, 55-73. HEATH, D. F. (I961), Organophosphorus Poisons, PP. 259-353. New York: Pergamon Press. HILL, R. B., and USHERWOOD,P. N. R. (196I), 'Action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust, Schistocerca gregaria ', 07. Physiol., Lond.,
I57, 393-4oL HOYLE, G. (I953), 'Potassium ions and insect nerve muscle ', 07. exp. Biol. 30, i 2 i - i 3 5 . IYATOMI, K., and KANESHINA,K. (I958), ' Localization of choline esterase in the American cockroach ', 07ap. 07. appl. Ent. Zool., ~, I-IO. JARVIK, M. E. (I965) , ' Drugs used in the treatment of psychiatric disorders ', in The Pharmacological Basis of Therapeutics, (ed. GOODMAN,L. S., and GILMANA.), pp. I62-I78. New York: The Macmillan Co. JENKINSON, D. H. (I957), ' T h e nature of the antagonism between calcium and magnesium ions at the neuromuscular junction ', aT. Physiol., Lond., I38 , 434-444. KERKUT, G. A., LEAKE, L. D., SHAPIRA, A., COWAN, S., and WALKER,R. J. (1965), ' The presence of glutamate in nerve-muscle perfusates of Helix, Carcinus, and Periplaneta ', Comp. Biochem. Physiol., I5, 485-502. KERKUT, G. A., SI~AVIRA,A., and WALKER, R. J. (i965), ' The effect of aeetyleholine, glutamic acid and GABA on the contractions of the perfnsed cockroach leg ', Comp. Biochem. Physiol., x6, 27-48. KERKUT, G. A., and WALKER, R . J . (i966), ' The effect of L-glutamate, acetylcholine and GABA on the miniature end-plate potentials and contractures of the coxal muscles of the cock-
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LARSEN,J. R., MILLER,D. M., and YAMAMOTO,T, (I966), ' d-Tubocurarine: Effects on insects ',
Science, N.Y., I52, 225-226. McCANN, F. V. (x966), ' C u r a r e as a neuromuscular blocking agent in insects ', Science, N.Y., I54, IO23-Io24. MCCANN, F. V., and REECE, R. W. (I967), ' Neuromuscular transmission in insects: Effect of injected chemical agents ', Comp. Biochem. Physiol., 2x, i i5-i24. McDoNALD, T. J. (I972), ' Free radical mechanism for phenylhydrazine action on the retractor unguis muscle of the grasshopper, Romalea microptera ', Comp. gen. Pharmac. (in press). O'BRIEN, R. D. (I959) , ' Effect of ionization upon penetration of organophosphates to the nerve cord of the cockroach ', 07. econ. Ent. 52, 8x28x6. O'BRIEN, R. D. (I967), Insecticides, Action and Metabolism, pp. 32-IO7, 148-I58. New York: Academic Press. O'BRIEN, R. D., and FISHER, R. W. (I958), ' The relation between ionization and toxicity to insects of some neuropharmacological compounds ', 07. econ. Ent., 5 x, i69-x75. O'CONNOR, A., O'BRIEN, R. D., and SALPETER, M. M. (I965), 'Pharmacology and fine structure of peripheral muscle innervation in the cockroach, Periplaneta americana ', 07. insect Physiol., Ix, I35I-i358. ORKLAND, R. K. (I968), ' Facilitation of heart muscle contraction and its dependence on external calcium and sodium ', 07. Physiol., Lond., x96, 311-325. PITMAN,R. M. (x97I), ' Transmitter substances in insects: a review ', Comp. gen. Pharmac., 2, 347-37 I. O~UISTAD, G. B., FUKUTO, T. R., and METCALF, R. L. ([97o), 'Insecticidal, anticholinesterase, and hydrolytic properties of phosphoramidothiolates ', 07. Agric. F. Chem., I8, 189-I94. ROBEINS,J. (i 959), ' The excitation and inhibition of crustacean muscle by amino acids ', 07. Physiol., Lond., x48, 39-50. ROEDER, K. D., and WEIANT, E. A. (I95O), ' The electrical and mechanical events of neuromuscular transmission in the cockroach, Periplaneta americana ', 07. exp. Biol., ~7, I-I3. SHIBATA, S., and CARRIER, O. (I967), ' A n t a gonizing action of ehlorpromazine, dibenamine and phenoxybenzamine on potassium induced concentrations ', Can. 07. Physiol. Pharmac., 45, 587-596 • Su, C., and LEE, C. Y. (I96O), ' T h e mode of neuromuscular blocking action of chlorpromazinc ', Br. 07. Pharmac., x5, 88~94. TAKEUCHI, A., and TAKEUCHI,N. (I964), ' T h e effect on crayfish muscle of iontophoretically applied glutamate'. 07. Physiol., Lond., x7o, 296-317.
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TREHERNE, J. E. (I966), The Neurochemistry of Arthropods. London : Cambridge
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TRIGGLE, D. J. (i965), Chemical Aspects of the Antonomic Nervous System, pp. 74-I68. New York: Academic Press. TWAROG, B. M., and ROEDI~R, K. D. (1956), ' Properties of the connective tissue sheath of the cockroach nerve cord ', Biol. Bull. mar. biol. Lab., Woods Hole, x H , 278-286. USHERWOOD, P. N. R. (1967a), ' Permeability of insect muscle fibres to potassium and chloride ions ', 07. Physiol., Lond., x9x , 29-3oP. USHERWOOD, P. N. R. (i967b), ' I n s e c t neuromuscular mechanism ', Am. Zool., 7, 553-582. USHERWOOD, P. N. R. (x969), ' G l u t a m a t e sensitivity of denervated insect muscle fibres', Nature, Lond., 223, 411-413. USHERWOOD, P. N. R., COenRANF., D. G., and REES, D. (I968), ' C h a n g e s in the structural, physiological and pharmacological properties of insect excitatory nerve-muscle synapses after motor nerve section ', Nature, Lond., 2x8) 589-591 • USHERWOOD,P. N. R., and MACHILI,P. (x966) , ' C h e m i c a l transmission at the insect neuromuscular synapse ', Nature, Lond., 21o, 634-636. USHERWOOD,P. N. R., and MAGHILI, P. (I968), ' Pharmacological properties of excitatory neuromuscular transmission in the locust ', 07. exp. Biol., 49, 341-36I. USHERWOOD,P. N. R., MACHILI, P., and LEAF, G. (1968), ' L-Glutamate at insect excitatory nerve-muscle synapses', Nature, Lond., 2x9) x I69-I t72.
AND
OTHERS
VANHARREVELD,A. (I959), ' Compounds in brain extracts causing spreading depression of cerebral cortical activity and contraction of crustacean muscle ', 07. Neurochem., 3) 3oo-315 • VAN HARREVELD,A., and MENDELSON,M. (I969) , Glutamate-induced contraction in crustacean muscle ', o7. cell comp. Physiol., 54, 8 5 9 4 . WEBB, J. L. (i966), 'Iodoacetate, Maleate, N-Ethylmaleimide, Alloxan, Quinones, Arsenicals ', in Enzymes and Metabolic Inhibitors, pp. 1-283, 337-365 . New York: Academic Press. WEIDEN, M. H. J., MOOREFIELD,H. H., and PAYNE, L. K. (I965), ' 0-(Methylcatbamoyl) oximes: a new class of carbamate insecticideacaricides ', 07. econ. Ent., 58, x54-I55. WILLIAMSON,R. L., and METCALF,R. L. (I967), ' Salicylanilides: a new group of active uncouplers of oxidative phosphorylation ', Science, N.T., x58, I694-I695. YAMAMOTO, I. (I965), 'Nicotinoids as insecticides ', in Advances in Pest Control Research (ed. METCALF R. L.,), vol. VI, pp. 231-26o. New York: Interscience. ZUKEL, J. W. (I944) , ' Some effects of phenothiazinc, phenothiazone, and thionol on Periplaneta americana ', 07. econ. Ent., 37, 79 T M '
Key Word Index: Acetylcholine, epinephrine, norepinephrine, anticholinesterase agents, atropine, D-tubocurarine, L-glutamic acid, N-ethylmaleimide, iodoacetic acid, chlorpromazine, Romalea microptera, Locusta migratoria, Schistocerca gregaria, Schistocerca vaga, retractor unguis muscle, insect nerve-muscle.
327
PHARMACOLOGICAL PROFILE OF THE E X C I T A T O R Y N E U R O M U S C U L A R SYNAPSES OF THE INSECT R E T R A C T O R U N G U I S MUSCLE T. J. M c D O N A L D , R. D. FARLEY, AND R. B. M A R C H Departments of Entomology and Biology, University of California, Riverside, California 92502
(Received 3° May, I972 ) ABSTRACT i. A series of 3o chemicals was tested for their ability to alter the neurally evoked contractions of the retractor unguis muscle isolated from the grasshopper, Romalea microptera, and the locusts, Locusta migratoria, Schistocercagregaria, and S. vaga. 2. L-Glutamate was one of the most active compounds tested, supporting evidence from previous studies that it may be the insect neuromuscular transmitter at excitatory junctions. Adrenergic and cholinergic drugs and carbamate and organophosphorus esters (anticholinesterase agents) had little or no effect on the responses of the preparation. 3. N-Ethylmaleimide, two phenothiazines, and a dinitrosalicylanilide at io-~M completely abolished the contractile response. 4- Neurally evoked contractions occurred in solutions withpH values between 6 and io. Contractions were reversibly abolished in pH 5 saline but irreversibly abolished in p H I I saline.
THIS study compares L-glutamate with other because acetylcholine, curare, prostigmine, synaptic drugs in their ability to alter the and most anticholinesterase agents were force of the neurally evoked contractions of ineffective. Negative results for cholinergic the insect retractor muscle. As previous transmission were also reported by other studies on this muscle have been limited to the workers (Roeder and Weiant, x95o ; Iyatomi action of L-glutamate and its analogues and Kaneshina, x958 ). The question of cholinergic transmission (Usherwood and Machili, x966, I968; Usherwood, I967 a, b, I969; Usherwood, was reopened when it was found that injected Cochrane, and Rees, x968; Usherwood, curare caused paralysis in insects (Larsen, Machili, and Leaf, 1968), it seemed reasonable Miller, and Yamamoto, 1966). It was further to establish for this preparation whether or reported that injected curare (McCann, not other transmitter substances might be just I966 ) or acetylcholine (McCann and Reece, as effective as L-glutamate. I f the synapses of 1967) resulted, respectively, in blockade or the retractor unguis muscle were glutanergic, depolarization of the neuromuscular juncadrenergic or cholinergic drugs might be tions of the fly, Sarcophaga bullata. Cholinergic transmission further seemed a possibility expected to have little or no action. Previous studies with whole insects and because of recent observations on an organism large complex nerve-muscle preparations related to insects by Futamachi (i972), who have yielded conflicting results on the cholin- suggested that the tonic flexor muscles of a ergic pharmacology of insect neuromuscular crustacean, Procambarus clarkii, were innerjunctions. Harlow (I 958) found that perfused vated by neurons whose transmitter may be solutions of atropine, eserine, nicotine, and acetylcholine. Furthermore, there are difficulties in various choline esters often induced muscular contractions. However, it was considered studying the action of cholinergic compounds unlikely that the synapses were cholinergic, on tissues, because many are ionized and do
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n o t r e a d i l y p e n e t r a t e m e m b r a n e barriers (Hoyle, i 9 5 3 ; T w a r o g a n d R o e d e r , i956; O ' B r i e n a n d Fisher, i958; O ' B r i e n , 1959; Eldefrawi a n d O ' B r i e n , 1967). Consequently, one m i g h t w o n d e r if the n e g a t i v e results of F a e d e r , O ' B r i e n , a n d S a l p e t e r (i97o) a n d F r i e d m a n a n d C a r l s o n (I97O) for a cholinergic m e c h a n i s m at insect n e u r o m u s c u l a r j u n c t i o n s were d u e to tests o f only c h a r g e d molecules on large a n d c o m p l e x n e r v e muscle p r e p a r a t i o n s . T h i s difficulty was r e d u c e d in the p r e s e n t e x p e r i m e n t s , w h e r e a small n e r v e - m u s c l e p r e p a r a t i o n (i 50-350 gin. dia.) was used, a n d w h e r e the threshold c o n c e n t r a t i o n s o f b o t h ionized a n d u n - i o n i z e d cholinergic drugs were c o m p a r e d to t h a t o f L-glutamate. T o give a m o r e c o m p l e t e p h a r m a c o l o g i c a l profile o f the r e t r a c t o r unguis muscle, the p H sensitivity was d e t e r m i n e d . This was because o t h e r e x p e r i m e n t s w i t h isolated nerve a n d m u s c l e tissues have e m p h a s i z e d the i m p o r t a n c e o f e x t r a c e l l u l a r p H on tissue responses. F o r e x a m p l e , Brooks a n d H u t t e r (I962 , i963) f o u n d a n o p t i m a l p H r a n g e o f 5 - 1 o for the electrical a c t i v i t y of frog skeletal muscle. I n a n o t h e r study, a c t i o n potentials of lobster g i a n t fibres were reversibly b l o c k e d b y low p H a n d irreversibly blocked b y high p H ( H a f e m a n n , i969). M A T E R I A L S AND M E T H O D S The retractor unguis muscle was dissected from the metathoracic femurs of the insects and mounted in a Plexiglas chamber (o'25 ml.). A Harvard 975 infusion pump with two ioo-ml. syringes was used to provide a i-o ml. per minute flow of saline or drug'solution through the chamber. The saline (pH 6. 9) was prepared according to the formula of Usherwood and Machili (I968), but the calcium was added just before use to minimize the precipitation of calcium, presumably as calcium phosphate (McDonald, I972). In some instances a Tris-buffered modification of the saline was made to avoid the calcium precipitation problem. The Tris-buffered saline (pH 7'3) contained: x3o m M NaC1, Io m M KC1, 2 m M CaC12, 20 m M glucose, and Io m M tris-(hydroxymethyl)-aminomethane. Since only low concentrations ( i o - s_ i o - ~M) of drugs were used, they were dissolved in saline without compensating for the minute increase in osmotic pressure. The contractile force of the muscle was monitored with a Grass .o 3 FT force displacement transducer. A Grass 5PI D.C. preamplifier
Comp. gen. Pharmac.
excited the strain gauges of the transducer and amplified their output which was displayed on chart paper by a Grass polygraph. Retractor unguis preparations were taken from adult orthopterans obtained from several sources. The grasshopper, Romalea microptera, was collected in Florida (H. Williams, Dunnellon, Florida); Locusta migratoria and Schistocerca gregaria were supplied by the Anti-Locust Research Centre, London, England; and specimens of Schistocerca vaga were reared (Dr. W. Loher, Berkeley, California). The chemicals used in these experiments are listed by number in Table L They were obtained from the sources indicated in the table and were used without further purification. RESULTS BIOOENIC AMINES Solutions o f these amines a n d o t h e r drugs of Table I were a d m i t t e d into the tissue c h a m b e r o n l y after the n e u r a l l y evoked contractions were u n i f o r m in a m p l i t u d e for I o - 2 o minutes in U s h e r w o o d ' s saline. After a u n i f o r m a m p l i t u d e was a t t a i n e d in the test solution, it was d e l i v e r e d for a n o t h e r 5 minutes to allow a d d i t i o n a l time for c o m p l e t e d r u g action. N o r e p i n e p h r i n e (I), e p i n e p h r i n e ( I I ) , 3-hydroxytyramine (III), and 5-hydroxytrypta m i n e (IV) are biogenic a m i n e s t h o u g h t to be n e u r o t r a n s m i t t e r substances in various o r g a n isms ( K e r k u t , S h a p i r a , a n d W a l k e r , i965; A n d e r s o n a n d S h i b u y a , 1966; Elofsson, K a u r i , Nielsen, a n d S t r o m b e r g , I966; Florey, 1966). E x c e p t at high c o n c e n t r a t i o n s , these c o m p o u n d s h a v e very little effect on the n e u r a l l y evoked responses o f the r e t r a c t o r unguis muscle from L. migratoria a n d R. microptera (Table I). T h e y were ineffective at io .3 M ; a n d , as shown in the m y o g r a m s of Fig. I, concentrations o f lO .2 M caused only a slight r e d u c t i o n in c o n t r a c t i o n force. This lack o f sensitivity i n d i c a t e d t h a t these drugs a r e unlikely to be n e u r o h u m o r s at the neurom u s c u l a r j u n c t i o n s of this p r e p a r a t i o n . This conclusion was further s u p p o r t e d b y perfusion tests w i t h D L - a m p h e t a m i n e (V), a d r u g w h i c h is t h o u g h t to cause release o f c a t e c h o l a m i n e s from their storage sites in nerveendings (Carlsson, Fuxe, H a m b e r g e r , a n d Lindqvist, 1966; C a r r a n d M o o r e , I969). A t I o - 4 M it h a d no effect on the c o n t r a c t i l e
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329
response of the retractor unguis preparation, other preparations, that insect neuromuscular although I o - 3 M and l o - 2 M solutions, junctions are insensitive to cholinergic drugs respectively, decreased and totally abolished (Harlow, 1958; O'Conner, O'Brien, and the response. Because relatively high con- Salpeter, I965; Faeder and others, 197o ). centrations of the drug failed to potentiate the response, it was considered unlikely that it ANTICHOLINESTERASE AGENTS Acetylcholine is inactivated at cholinergic caused release of a catecholamine neurojunctions by acetylcholinesterase, an enzyme humor at the junctions.
FIG. I.--Effect of I o-2M solutions of norepinephrine, epinephrine, and 5-hydroxytryptamine on the
neurally evoked contractions of the retractor unguis muscle of Locusta migratoria.
QUATERNARY AMMONIUM COMPOUNDS
Compounds V l - l X act at cholinergic synapses in vertebrate tissue (Triggle, 1965; Cutting, 1967). Acetylcholine (VI) is a common transmitter substance in vertebrates and invertebrates (Triggle, 1965 ; Treherne, 1966); carbamylcholine (VII) is a cholinomimetic (Furchgott and Bursztyn, 1967) , and decamethonium (VIII) and succinylcholine (IX) block cholinergic transmission (Bowman, Hemsworth, and Rand, 1967). These drugs had essentially no effect on the contractile response (Table I and Fig. 2A, B), suggesting that cholinergic receptors are not involved in the synaptic transmission of this tissue. Other quaternary ammonium compounds ( X - X I I ) also had little or no influence on the tissue. These results confirm previous reports for
inhibited by certain carbamate and organophosphorus esters (Heath, 1961 ; Cutting, I967; O'Brien, i967). Since both ionized and un-ionized carbamate and organophosphate esters may be obtained, they are particularly well suited to test whether or not the inactivity of ionized cholinergic drugs is due to their inability to penetrate an ionic barrier. Although results with cholinergic drugs ( V I - X I V , X X I I - X X I V ) indicated that the neuromuscular junctions of the retractor unguis preparation are not likely to be cholinergic, it is possible that the failure of these compounds to affect the tissue was due to their inability to reach the receptor site. As a further test of possible cholinergic activity in this preparation, the tissue was
33 °
Co?tip. gen. Pharmac.
McDONALD AND OTHERS
exposed to b o t h i o n i z e d (XIII-XIV) a n d un-ionized (XVI-XX) anticholinesterase agents. T h e u n - i o n i z e d cholinesterase in-
Table L--CoMPARISON
hibitors,
Baygon ® (XVI) and aldicarb c o m m e r c i a l c a r b a m a t e insecticides (Weiden, Mo o r ef i el d , an d Payne,
(XVII) ar e
OF THE ABILITY OF VARIOUS COMPOUNDS TO CHANGE THE NEURALLY EVOKED CONTRACTIONS OF THE RETRACTOR UNGUIS MUSCLE
The normal response in saline (Usherwood and Machili, i968 ) is designated as ioo per cent. The values shown are typical of those obtained from two or more nerve-muscle preparations taken from the species indicated after each compound: Romalea mieroptera (Rm), Schistocerca gregaria (Sg), S. vaga (Sv), and Locusta migratoria (Lm).
No.
NAME
SPECIES
PERCENTAGE RESPONSE AT MOLAR CONCENTRATIONS i0-5
14 II 4
III 4 IV 4 VIa
Vp ~ VII ix VIII 15 X:6 Xla
XI 4 XII15
XIII 4 XIV19 XV 9 XVI 5 XVI116
XVIII a XIX 9 XX ~ (XI a
KXIP a XXlII* K X I V 15 XXV 1 XXVP a KXVII 7 K X V I I I 1. ~ , . . X I X le KXX a
Norepinephrine Epinephrine 3-Hydroxytyramine 5-Hydroxytryptamine creatinine sulphate DL-Amphetamine hydrochloride Acetylcholine iodide Carbamylcholine chloride Decamethonium bromide Succinylcholine chloride Choline chloride Betaine hydrochloride DL-Carnitine hydroehloride Physostigmine Prostigmine 2-Methyl-acetophenone oximine, W-methyl-carbamate Baygon*, o-isopropoxyphenyl-.N'-methylcarbamate Aldicarb, Temik ®, 2-methyl-2-(methylthio)propionaldehyde-O- (methylcarbamoyl) oxime Paraoxon, diethyl-4-nitrophenyl phosphate Sumioxon *, dimethyl-3-methyl-4-nitrophenyl phosphate Monitor ®, O-methyl-S-methyl phosphoramidothioate Ethyl 4-nitrophenyl-tert-butylphosphonate Nicotine Atropine sulphate D-Tubocurarine chloride L-Glutamate, disodium
Lm Lm Lm Lm, Rm Rm, Sv Sg Lm Lm Lm
10-4
xoo
i0-2
ioo lOO lOO IOO
75 9° 9° 60
IOO
85
O
IOO
lOO
95 lOO IOO IOO
I00
I00
Sg Rm Rm Rm, Sg Lm Rm
i0-3
8o
6o
Ioo
S~
IO0
IO0
IOO
IOO
Rm, Sg
I00
Sg Rm
ioo
ioo
ioo
ioo
Sv
ioo
ioo
I oo lOO
I oo IOO
IOO ioo
ioo 80
lOO
95
9o
o lOO o
IO0
IO
0
3°
o
Rm Rm, Sg Rm, Lm Rm, Sg Lm, Rm, Sg, Sv Rm aV-Ethylmaleimide Iodoacetic acid Rm Chlorpromazine hydrochloride Rm Phenothiazine, lO- (3-dimethylamino-2-methyl- Lm propyl,-, ( + ) - , tartrate 3"Nitr°salicyl'4-nitr°anilide Rm
95 lOO o Ioo
ioo ioo lOO* +~oo* lOO +2oo o o
o o
*Results for normal rather than molar solutions. Sources of Chemicals: 1Aldrich Chemical Co., aAmerican Cyanamid Co., aBen Venue Laboratories, 4Calbiochem., 5Chemagro Corp., eChevron Chemical Corp., ~Eastman Organic Chemicals, SDr. T. R. Fukuto, Dept of Entomology, University of California, Riverside, California, 9Hoffman La-Roche Inc., 1°Merck, Sharp, & Dohme, nNutritional Biochemicals Corp., 1~Sigma Chemical Co., xaSmith, Kline, & French Laboratories, x4Squibb Institute for Medical Research, 15The UpJohn Co., 16Union Carbide Corp.
I972, 3
INSECT NEUROMUSCULARPHARMACOLOGY
331
I965; O'Brien, i967). Paraoxon ( X V I I I ) , Io-4 M, since they have limited water soluSumioxon ® ( X I X ) , and Monitor ® (XX) bility. are un-ionized organophosphorus esters with Although the un-ionized anticholinesterase insecticidal action (Heath, i96x ; Ouistad, agents did not affect the retractor unguis Fukuto, and Metcalf, i97o ). preparation, they were very toxic to intact Physostigmine (XIV) was the only anti- insects at the concentrations tested in Table L cholinesterase which had a significant effect Locusts were dipped for about 3° seconds into on the preparation. At i o -4 M it partially I o -4 M Baygon ®, I o - S M aldicarb, or I o-4 M reduced the contractile response (Table I), paraoxon. After developing symptoms of and at Io -3 M it completely abolished the toxicity, the animals were observed to be dead
FIG. 2.--Effect of Io-2M solutions of acetylcholine, succinylcholine, and prostigmine on the neurally evoked contractions of the retractor unguis muscle ofLocusta migratoria. contractions. This effect was not lasting, since normal contractions often recurred in less than I minute after ending exposure to a io -2 M solution. Prostigmine ( X I I I ) is the other ionized anticholinesterase tested on the tissue. As shown in Fig. 2 C, it did not appreciably alter the response of the tissue even though it was tested at a concentration of IO -2 M. The un-ionized anticholinesterase agents ( X V I XX) also did not affect the tissue response at any of the concentrations tested. Test solutions for some of the compounds could not be prepared at concentrations greater than
after 24 hours. This suggests that the insecticidal action of these compounds is not due to any direct effect on the neuromuscular junction, since lethal concentrations for the intact animal had no effect on the isolated nerve-muscle preparation. Since ion barriers would not be a limiting factor for the activity of these compounds, their failure to affect the preparation is strong evidence for noncholinergic transmission. NICOTINE, ATROPINE, AND CURARE
Nicotine ( X X I I ) has cholinomimetic action in vertebrates and insects (Yamamoto,
332
Comp. gen. Pharmac.
McDONALD AND O T H E R S
i965; Cutting, I967; O'Brien, i967). Atropine ( X X I I I ) and D-tubucurarine ( X X I V ) are blocking agents at vertebrate parasympathetic and skeletal neuromuscular junctions respectively (Cutting, I967). As indicated above, in some previous studies these drugs were thought to have an effect at insect
response, similar to the results obtained with 1o-2Natropine. Also likeatropine,3 × i o - 2 M curare abolished the neurally evoked contractions, with recovery on perfusion with control saline. Since these drugs only produced effects at high concentrations, it was considered unlikely that their action reflected
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I
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.
.,,
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:
:
.
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.
.
.
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I L. FIo. 3.--Perfusion with io ~,N" atropine sulphate initially reduced, then potentiated the neurally evoked contractions of the retractor unguis muscle of Locusta migratoria. neuromuscular junctions (Harlow, I958; O ' C o n n o r and others, i965; Larson and others, I966; McCann, I966 ). Nicotine had no effect on the retractor unguis preparation (Table I), while atropine and curare modified the response only at high concentrations. As shown in Fig. 3, solutions of i o -2 N a t r o p i n e initially reduced the neurally evoked contractions of the preparation, but after I-2 minutes the responses were greatly potentiated. When the concentration was increased to 3 × x°-2 dV, the contractions were completely abolished. When control saline was readmitted to the tissue chamber, potentiated contractions appeared abruptly, decreasing gradually in amplitude until stable responses were obtained at about the same contraction force as occurred in control saline before exposure to atropine. Solutions of I o - ~ M curare ( X X I V ) reduced and then potentiated the contractile
a cholinergic mechanism muscular junction.
at
the
neuro-
L-GLUTAMIC ACID
In previous studics with the retractor unguis muscle of S. gregaria, Ushcrwood and Machili ( 1966, 1968) and Usherwood ( 1967 b) obtained evidcncc that L-glutamate m a y be the neuromuscular transmitter. Concentrations greater than Io -~ g. per ml. reduced the amplitude of the neurally evoked contractions of the preparation. In the present experiments the disodium salt of L-glutamate (XXV) was dissolved in Usherwood's saline, and the solutions were tested on several preparations from each of the four orthopteran species of this study. L-Glutamatc was not observed to potentiate the neurally evoked response in any of these experiments. At [ o-4 M it caused the contractions to decrease to about 8o per cent of that
I972, 3
INSECT NEUROMUSCULAR P H A R M A C O L O G Y
w h i c h was o b t a i n e d w i t h perfusion w i t h c o n t r o l saline. D u r i n g perfusion w i t h i o - 3 M L - g l u t a m a t e , the c o n t r a c t i o n s g r a d u a l l y decreased in a m p l i t u d e , a n d after a b o u t i m i n u t e t h e y were c o m p l e t e l y a b o l i s h e d (Fig. 4)- T h i s
FIG.
333
effect was reversible, since perfusion w i t h c o n t r o l saline caused r e c o v e r y o f the c o n t r a c tion force to the s a m e level as t h a t w h i c h was o b t a i n e d before the tissue was exposed to the test solution.
4.--Effect of I o - 3 M L-glutamate on the neurally evoked response of the retractor unguis muscle
ofRomalea microptera. The contractions were completely abolished, but the effect was readily reversible.
FIG. 5.--Effect of I o - 4 M chlorpromazine on the neurally evoked contractions oi the retractor unguis muscle of Romalea microptera.
FIG. 6.---The reversible effect of low p H (4.6) on the neurally evoked contractions of the retractor unguis muscle ofRomalea microptera. The control saline was at p H 6"9 (Usherwood and Machili, 1968 ).
334
McDONALD
MISCELLANEOUS DRUGS N-Ethylmaleimide ( X X V I ) and iodoacetic acid ( X X V I I ) are sulphydryl inhibitors (Webb, 1966). Both iodoacetic acid (i o-2 M)
AND
OTHERS
Comp. gen. Pharmac.
and W-ethylmaleimide (z 0 -4 M) completely abolished the response. However, W-ethylmaleimide was more effective than iodoacetic acid.
FIG. 7.--The irreversible effect of high pH (i I.O) on the neurally evoked contractions of the retractor unguis muscle of Romalea microptera. The control saline was Tris-buffered (see Materials and Methods) at pH 7"3. 100
Phenothiazine derivatives, particularly chlorpromazine ( X X V I I I ) , are extensively used in the treatment of psychosis (Jarvik, 1965). Chloropromazine (Fig. 5) and di.o_ 80 I methylamino-2-methyl-propyl-phenothiazine I ( X X I X ) both reduced the contraction amplitude of the retractor unguis muscle, but much I 0 ~ 60 more slowly than other effective compounds. 1 I1) Fig. 5 shows that the chlorpromazine effect I was reversible. t~ I 3-Nitrosalicyl-4-nitroanilide ( X X X ) also ~ 4O decreased the contraction force at approxiI mately the same rate as the phenothiazine I derivatives, but it was somewhat more effecI ~. 2o tive in depressing the neurally evoked contractions. Some salicylanilides are fungicides I (Esplin, I965) and uncouplers of oxidative phosphorylation (Williamson and Metcalf, w 4 6 8 10 12 1967). Since Io -4 M concentrations of both the phenothiazine derivatives and the salipH cylanilide diminshed the contraction ampliFie,. 8.--The effect of extracellular pH on the tude, these compounds m a y warrant force of the neurally evoked contractions of the retractor unguis muscle of Romalea microptera. The additional studies to determine their site of response was depressed at pH values less than 6 action. and greater than xo. To avoid calcium precipitation, Tris-buffered saline was used at pH values of p H EFFECT 7.0 and higher. At pH values lower than 7.0 the Fig. 6 shows that Usherwood's saline at phosphate buffered saline (Usherwood and p H 4 . 6 reduced completely the neurally Machili, 1968) was used. t-"
"i"
l
1972, 3
INSECT NEUROMUSCULAR P H A R M A C O L O G Y
evoked contractions of the retractor unguis muscle of R. microptera. This low p H effect was reversible, since contractions reappeared when the tissue was again perfused with p H 6. 9 saline. Fig. 7 shows that Tris-buffered saline at p H 11.o also caused the neurally evoked contractions to cease. However, this effect at this p H was irreversible, since the contractions did not reappear when the tissue was perfused with saline at p H 7"3. Tris-buffered saline was used for p H value 7.0 and higher, because Usherwood's saline developed a white precipitate at high p H values. Fig. 8 summarizes the experimental data of the p H experiments of 7 different muscles and shows that muscle responses m a y be obtained during short perfusions (lO minutes) in salines with p H values between 6 and lO. DISCUSSION T h e biogenic amines ( I - I V ) have little or no effect on the retractor unguis preparation, supporting previous reports that they are not the transmitter for insect skeletal muscle (Harlow, 1958; O ' C o n n e r and others, 1965). Although io -2 M 5-hydroxytryptamine (IV) was previously tbund completely to block neuromuscular transmission in the metathoracic leg muscles of S. gregaria (Hill and Usherwood, i96i ; Usherwood and Machili, I968), only a 4 ° per cent reduction in contraction force was obtained in the present experiments. This difference was not considered to be sufficiently great to merit further investigation. I t is very unlikely, from these experiments, that there is a cholinergic mechanism at the neuromuscular junctions of the retractor unguis muscle. Acetylcholine (VI) at IO - 2 M had no effect on the preparation, and cholinergic (VII) and anticholinergic ( V I I I I X ) drugs were ineffective. Since the quaternary a m m o n i u m compounds are ionized at the p H of the perfusion saline (pH 6-9), it was considered possible that they failed to reach the receptor site (O'Brien and Fisher, 1958; O'Brien 1959; Eldefrawi and O'Brien, i967). Un-ionized anticholinesterases ( X V - X X I ) at I o - 4 M had no effect on the preparation, although these drugs at
335
this concentration were lethal to intact locusts. Generally, the ionized cholinesterase inhibitors ( X I I - X I V ) had a slightly greater effect on the preparation than the un-ionized compounds, suggesting that the charge on the molecule was not a limiting factor for these cholinergic compounds. T h e results with atropine ( X X I I , Fig. 3) and curare ( X X I V ) provided further evidence that cholinergic transmission is unlikely. These agents potentiated the neurally evoked muscle response, while they should block transmission if the junction swere cholinergic. T h e high concentration (I o-2 M ) required for potentiation does not correlate with previous reports of the blocking action of these drugs at known cholinergic synapses. T h e y are effective from lO -e to lO -5 M on frog heart and skeletal muscle junctions (Jenkinson, 1957; Orkland, 1968 ). L-Glutamate ( X X V ) was more effective in altering the contraction amplitude than any of the other transmitter candidates ( I - I V , VI), enforcing the idea that the junctions are glutanergic. At I o -3 M it completely abolished the neurally evoked responses, and since only a very slight reduction in contraction force occurred at i o - S M ( 1 . 9 × i o -6 g. per ml.), this was considered to be the threshold concentration. Similar threshold concentrations for Lglutamate were reported for other arthropod nerve-muscle preparations. Crayfish preparations responded to concentrations of Io - S M or more (Robbins, I959; Van Harreveld, 1959; Takeuchi and Takeuchi, 1964). The threshold levels for crab and lobster tissue were between 2 × I O-4 and 5 × Io-5 M (Van Harreveld and Mendelson, I959; Florey and Woodcock, i968 ). For a nerve-muscle preparation from the cockroach, Gromphadorhina portentosa, the threshold was 2"5 x Io -4 M (Faeder and O'Brien, I97O); and for a leg preparation from the American cockroach, it was IO -7 g. per ml. (Kerkut, Leake, Shapira, Cowan, and Walker, 1965 ; Kerkut and others, 1965; K e r k u t and Walker, 1966 ). Usherwood and Machili (1966, 1968) and Usherwood (1967 b) reported that there was a reduction of the neurally evoked contractions at
336
McDONALD AND OTHERS
c o n c e n t r a t i o n s h i g h e r t h a n IO -~ g. p e r ml. using the r e t r a c t o r unguis muscle from S. gregaria. T h e p r e s e n t results confirm their studies, a l t h o u g h the threshold for the p r e s e n t e x p e r i m e n t s was slightly higher. Phenothiazine compounds (XXVIIIXXIX) cause flacid paralysis in i n t a c t insects (Zukel, 1944; Brown, I963) , a n d in the p r e s e n t e x p e r i m e n t s t h e y caused a r e d u c tion in the c o n t r a c t i l e response o f the r e t r a c t o r unguis muscle. I t was n o t d e t e r m i n e d w h e t h e r these c o m p o u n d s interfered w i t h n e u r o n a l c o n d u c t i o n , s y n a p t i c transmission, or the c o n t r a c t i l e process, b u t t h e y c l e a r l y h a v e a n a c t i o n on p e r i p h e r a l tissue. F a e d e r a n d O ' B r i e n (i97o) r e p o r t e d t h a t chlorpromazine (XXVIII) blocked neuronal c o n d u c t i o n in a c o c k r o a c h p r e p a r a t i o n , b e c a u s e a stepwise r e d u c t i o n in the c o n t r a c t i l e force o c c u r r e d d u r i n g n e u r a l stimulation. I n a d d i t i o n to this effect on nerves, however, c h l o r p r o m a z i n e has also b e e n shown to act d i r e c t l y on muscle tissue (Su a n d Lee, I96O; S h i b a t a a n d C a r r i e r , I967). I n the p r e s e n t studies w i t h c h l o r p r o m a z i n e a stepwise r e d u c t i o n in c o n t r a c t i l e force was n o t observed. This m a y h a v e b e e n d u e to the a c t i o n o f the d r u g on a site o t h e r t h a n a x o n a l transmission. T h e n e u r a l l y evoked responses o f the r e t r a c t o r unguis muscle were forceful d u r i n g perfusion w i t h saline at p H 6 - 1 o . The reversible depression w i t h saline b e l o w p H 6 m a y be d u e to the reversible n a t u r e o f molec u l a r p r o t o n a t i o n ( H a f e m a n , x969). I f activation o f the muscle r e q u i r e d a n ionized site w h i c h b e c a m e p r o t o n a t e d a t low p H , the site should r e t u r n to its original state as the p H is increased. T h e irreversible depression a t p H I I m a y b e d u e to hydrolysis or d e n a t u r a tion o f the m e m b r a n e m a c r o m o l e c u l e s . T h i s was suggested b y H a f e m a n (1969) to a c c o u n t for the irreversible effects o f high p H on lobster g i a n t axons.
ACKNOWLEDGEMENTS This study was supported by N I H Training Grant No. ES ooo47 from the National Institute of Environmental Health Sciences (TJM). Grant NB o81 xo-o I from the National Institute of Health (RDF), and research funds from the Agricultural Experiment Station, University of California,
Comp. gen. Pharmac.
Riverside (RDF). The authors thank Dr. T. R. Fukuto for advice and for some of the chemicals used in these experiments.
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Key Word Index: Acetylcholine, epinephrine, norepinephrine, anticholinesterase agents, atropine, D-tubocurarine, L-glutamic acid, N-ethylmaleimide, iodoacetic acid, chlorpromazine, Romalea microptera, Locusta migratoria, Schistocerca gregaria, Schistocerca vaga, retractor unguis muscle, insect nerve-muscle.