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Proctolin: A peptide transmitter candidate in insects
Life Sciences Vol . 17, pp . Printed in the U.S .A .
PROCTOLIN:
Perqamon Press
1241-1252
A PEPTIDE TRANSMITTER CANDIDATE IN INSECTS Brian E . Brown
Research Institute, Agriculture Canada, University Sub P.O ., London, Canada N6A 5B7 (Received in final form September 15, 1975) Summary The slow, striated muscles of the proctodeum (hindgut) of the cockroach, Periplaneta americana (L .), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from Periplaneta recently isolated and identified . The graded contractions evoked by repetitive nerve stimulation were simulated b~r9 5-HT and proctolin at threshold concentrations -$f about 10 and 10 M respectively ; responses to glutamate (10 M) were not similarly graded . The 5-HT receptors are distinct from other receptors, including the postsynaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide . Proctolin was fully active on TTXtreated or surgically denervated muscle indicating that the proctolin receptors are located on ~he muscle fibre menbrane . Tyramine, at threshold levels of 5x10 M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the S-I-fr and glutamate responses . Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate . Pharmacologically, the proctolin and postsynaptic receptors appear to be identical and distinct from the glutamate and S-1-fr receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosecretory granules in addition to electron-lucent, synaptic vesicles . Interest in peptides as transmitter substances or as modulators of neuronal activity dates fran the discovery of substance P bY von E~ler and Gaddum (1) . Substance P has now been sequenced and synthesized (2,3) and recent evidence suggests that it and other peptides may indeed function as neurotransmitters (4) . Substance P may be the excitatory transmitter of primary afferent neurones in the spinal cord (5-7) and studies by Nicoll and Barker indicate that recurrent inhibition of neurones of the supraoptic nucleus may be mediated by the synaptic action of vasopressin (B) . At the ultrastructural level, additional evidence is provided by the "peptidergen Synapsen" described by Bargmann et al (9) in the cat hypothesis . The present report extends the peptide trânsm~tter hypothesis to invertebrates. Etitidence is presented which suggests that insects possess a distinctive class of motor nerve cell in which synaptic transmission is mediated by a small peptide . 1241
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I suggested earlier that a myotropic substance associated with an efferent pathway of the proctodeal (hindgut) innervation of the cockroach, Perip l aneta americana (L .), might function as an excitatory transmitter substance in insect visceral muscle (10) . The active principle was associated with a small particulate fraction (synaptic vesicles :) and caused graded contractions of the proctodeal muscles similar to those evoked by repetitive nerve stimulation (10) . Subsequent studies described the innervation of these fibres and the bioelectrics underlying their mechanical behaviour (11-16) . Proctolin, the active compound of the initial study (10), was recently isolated in pure form (17) . The following coR¢nunication (18) describes the sequential analysis and synthesis of proctolin, a pentapeptide . The current report presents pharmacological data consistent with proctolin~s proposed transmitter function . The concept of peptidergic motor neurones in insects is discussed in respect to the ultrastructural canponents of the present motor nerve terminals which are similar to others in insects (19) but quite distinct from those terminating in the fast, skeletal fibres of insects, wherein glutamate is the putative transmitter (e .q . 20) . Methods and Materials Anatomical details of the proctodeum and its bilateral innervation in P. americana have been described previously (12) . All experiments except that in Fiq. 6 were performed with the whole proctodeum and intact nerve supply isolated from adult, male cockroaches . The sixth abdominal ganglion, in which the motor nerve cell bodies are located, was crushed or removed . The left and right cercal nerve (N XI), from which the fine proctodeal nerves branch, were tied together to ensure the introduction of both proctodeal nerves into the stimulating suction electrode. The results i.n Fig. 6 were obtained with the isolated rectum only (10,12) . The isolated hindgut or rectum was suspended by silk threads in a 4-ml organ bath and perfused with oxygenated insect saline (21) . 5-200 ul quantities of drug solutions were added rapidly to the organ bath via Hamilton syringes and r~noved by fast perfusion. Nerve stimulation was achieved by a suction electrode fixed in the side wall of the perfusion chamber. Stimulation was supramaximal with single stimuli of 0.5 meet duration . Contractions were recorded isotonically under 250 mg tension. Unless otherwise indicated, preparations were rested for 3-4 min between responses to drugs or nerve stimulation except for glutamate when the rest period was 10 min. Approximately 200 preparations were examined using both a highly purified aliquot of natural proctolin (17) and the synthetic peptide (18) . The records presented herein were obtained with synthetic proctolin but the results are identical to those obtained with natural proctoli.n . Experiments were performed at room temperature (22-25°C) . Results Normal responses to agonists and nerve stimulation : Typical examples of responses of the isolated proctodeum to 5-ffP, nerve stimulation, proctoli.n and glutamate are shown in Fiq . lA and 2A . The contractions evoked by 5-HT, proctolin and repetitive nerve stimulation are graded responses to drug concentration or to frequency of nerve stimulation . Maximum neurally evoked responses occur at about 50 Hz and their magnitude can be attained by increased concentrations of both 5-HT and proctolin. In contrast, the glutamate responses are not similarly graded nor do they ever approach the magnitude of the maximal neural response . Continuous nerve stimulation at moderate frequencies of 10 to 20 Hz evokes contractions which are sustained for at least several minutes and
Proctolin :
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A Peptide Transmitter
often for much longer . Sustained contractions are also characteristic of the S-I-fP and proctolin responses but, in agreement with Holman and Cook (22), they are not characteristic of the glutamate responses (see also Fig. 5) . The small glutamate contractions are not due to slight contraction of all muscles . Significantly (see Discussion), glutamate contractions are restricted mainly to muscles comprising the rectal valve, a short region of the proc todeum just anterior to the rectum (12) . By contrast, contractions evoked by 5-HT, proctolin, and nerve stimulation involve all or most fibres of the entire proctodeum . Branolysergic acid diethylamide: The effect of bravo LSD, a well known antagonist of indolealkylamines, is shown in Fig. 1B . The responses to 5-HT are blocked whereas the other responses are unaffected . Clearly, the 5-HT receptors are distinct from the other receptors including those of the postsynaptic meJnbrane. Tetrodotoxin and muscle denervation; At other junctions, TTX blockade is limited to the nerve action potential ; it neither interferes with the release of transmitter nor does it block postsynaptic receptors (23) . TTX has already shown to rapidly block the excitatory postsynaptic potential (EPSP) in fibres of the rectum of Periplaneta (15) . I have further examined the TTX blockade in these fibres to ensure that TTX has no effect on the postsynaptic receptors . TTX indeed causes a rapid failure of the EPSP but,
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FIG . 1 The effect of bromolysergic acid diethylamide (bravo LSD) on proctodeal muscle responses to 5-HT, nerve stimulation, proctolin and glutamate. Drugs and stimulation applied for 20 sec . A - Normal responses . B - Responses of same preparation after a 10-min exposure to bravo LSD at 100 ng per ml .
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A Peptide Transmitter
A Normal
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TTX -Treated I min
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0~5 I :10 -4 M
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FIG. 2 The effect of tetroxotoxin (TTX) on proctodeal muscle responses to 5-HP, nerve stimulation, proctolin, and glutamate. Drugs and stimulation applied for 10 sec. A - Normal responses. B - Responses of same preparation in the presence of 4x10 -7 M TTX. even after prolonged treatment, miniature EPSP~s (11) were unaffected . Presumably theSi,the action of TTX at this junction, as elsewhere, is limited to neural blockade . As shown in Fig. 2B, TTX completely abolishes the neurally evoked response but is without effect on the proctolin responses or on the responses evoked by 5-H'i' and glutamate. Therefore, neither proctolin nor the other agoniats are myotropic by reason of initiating nerve action potentials . Proctolin~s lack of effect at the neural or presynaptic level was confirmed by studies with a limited number of surgically denervated preparations . (10,12) . Following nerve section, EPSP~s in rectal fibres can be recorded intracellularly up to approximately 72 hours, following which junctional transmission fails completely (Nagai an3 Brown, unpublished) . The present study reports on the mechanical responses of three preparations from insects that survived 5 days of bilateral neurotomy (mortality was about 80 percent) . As éxpected, the denervated muscle failed to respond mechanically to repetitive nerve stimulation. However, in all cases, the denervated proctodeum displayed normal contractions and sensitivity to proctolin, as well as to 5-HT and glutamate. Therefore, proctolin~s activity cannot be attributed to neuroexcitation or to initiating the release of presynaptic stores of the natural transmitter . Judging from the EPSP failure 3 days after nerve section, presynaptic transmitter stores must be radically depleted 5 days after neurotomy . Parenthetically, it should be noted that proctolin is 50 percent de-
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pleted from the rectum 4 days after nerve section and completely lost sometime between the fourth and eighth day (10) . amine: In an examination of the pharmacological effects of catecholamines and analogues on the hindgut, it was found that two compounds were Especially effective were tyramine (ß(4 useful proctolin antagonists . hydroxyphenyl)ethylamine) and its ß-hydroxy analogue, octopamine . Other analogues were inactive or less effective as antagonists, especially the diphenols (e .g ., dopamine) and tyrosine . Of significance to the transmitter candidacy of proctolin, tyramine also suppresses neurally evoked contractions . In most preparations, the threshold inhibitory concentration of tyramine is similar for both the proctolin and neurally evoked responses and both responses are similarly suppressed at higher levels of tyramine (Fig . 3) . There has been some deviation from this observation but not in any consistent manner . The deviation may be attributed to the apparently competitive nature of the tyramine blockade (see Discussion) .
Ss10~
3e10~
I :10" 8 Y
Tyremine
FIG. 3 The effects of tyramine on proctodeal muscle responses to nerve stimulation at 10 Hz for 10 sec (~) and to 1x10-8 M proctolin for 10 sec . (~) . Threshold inhibition of both responses at 5x10 B M tyramine . Both responses equally suppressed at higher concentrations of tyramine . The specificity of the tyramine antagonism is demonstrated in another preparation (Fig . 4) . In this case, t~e proctolin response was unaffected -fit 3x10 M tyramine, diminished at 3x10 M, and strongly suppressed at 3x10 M tyramine (Fig . 4A) . The same levels of tyramine had no effect on the contractions evoked by 5-HT (Fig . 4C) or by glutamate (Fig . 4D) . In a few preparations, some inhibition of 5-HT r_~sponses has been observed at relatively high concentrations of tyramine ( 10 M and higher) . Thus, the tyramine effect appears to be highly selective with threshold inh_jrbition gf the proctolin and neurally evoked contractions occurring in the 10 to lÔ M range. As an example of the structual requirement of the antagonist molecule referred to earlier, the same concentrations of the ortho diphenol analogue, dopamine, have no effect on the proctolin responses (Fig . 4B) . Effects of subthreshold levels of proctolin and glutamate on neurally evoked contractions : The graded responses of proctodeal muscles to repetitive nerve stimulation (10) are coupled mainly to EPSP summation with some
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Psroctolin :
A
A Peptide Transmitter
Proctolin Response :
ô
Tyramine
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Proctolin Responses
Dopamine 6lutam ats Responses
Tyramlne
Tyromlne FIG. 4
Specificity of tyramine antagonism . In each quadrant (A,B,C,D), the first and last of the five responses are normal contractions to 2x10 -9 M proctolin (A,B), 6x10-7 M 5-HT (C), and 1 .5x10 -4 M glutamate (D), each applied for 20 sec. The three remaining contractions in each quadrant were similarly evoked lut in the presence of (from left to right) 3x10 -9 , 3x10 -8 , and 3x10 -7 M tyramine (A,C,D) or dopamine (B) . All records from one preparation . contribution from slow action potentials which may be superimposed on the summated EPSP (13) . Routinely, neurally evoked contractions of the proctodeum can reflect a difference of one Impulse per sec in the range of 6-15 Hz (e .g ., Fig. 7B) . Thus, the postsynaptic receptors are particularly sensitive to small changes in the concentration of natural transmitter within the synaptic cleft . Given these circwnstances, it is reasonable to expect that subthreshold concentrations of the transmitter substance in the organ bath should be additive to amounts released by nerve stimulation . As shown in Fiq. 5, this is indeed true of proctolin but is not tnae of glutamate. Neurally evoked contractions are increasingly potentiated at 2, 4, and 8x10 -lOM proctolin ; at 8x10 -10 M proctolin, there is a slight rise in the base line indicating the threshold nature of this concentration. In contrast, subthreshold levels of glutamate do not potentiate the neurally evoked response . In many instances, as shown in Fig . 5, the first observed effect of glutamate is one of inhibition . At 10 -4 M glutamate there is a typical brief contraction (Fig . 5, arrow) following which the evoked contractions are further suppressed . Higher concentrations of glutamate (2 to 5x10 -~i) will almost or co¢npletely abolish the neurally evoked response . As indicated earlier, the glutamate contractions are restricted mainly to a relatively small group of longitudinal muscles in the region of the rectal valve and therefore the glutamate responses never approach the maximal neurally evoked contraction of the whole proctodeum . However, the antagonistic effect of glutamate (Fig . 5) extends to most or all proctodeal muscle fibres . This observation has been confirmed in current studies with three separate nPrve-
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Proctolin:
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FIG. 5 The effect of subthreshold concentrations of proctolin and glutamate on neurally evoked contractions of proctodeal muscle . Neurally evoked responses initiated every 30 sec by a 3-sec train at 8 Hz . Note brief contraction at 10 -4 M glutamate (arrow) . muscle preparations consisting of the rectum, rectal valve, and anterior proctodeum . Thus, the longitudinal muscle straps of the rectum, i .e ., those fibres which have been the subject of most of our studies (11-16), do not contract in response to applied glutamate but their neurally evoked contractions are almost -4 M glutamate (Fig . 6) . With or completely abolished at levels of about 10 this important exception, the pharmacology of the rectal straps is similar to that of the whole proctodeum . Release of proctolin during nerve stimulation : Subsequent to the isolation, characterization and synthesis of proctolin (17,18), comparative bioassay of an extract of 500 hindguts determined that one hindgut contained only about 1600 pg proctolin. Therefore, it seemed unlikely that sufficient quantities of proctolin would accumulate in the perfusate of a stimulated preparation to allow chromatographic analysis (17) . This was indeed found to be the case and, in fact, experiments had to be designed to employ the most sensitive bioassay available, i .e ., the potentiation of neurally evoked responses which occurs with as little as 50-70 pg proctolin per ml . The most successful of these experiments employed a classic, double nerve-muscle preparation in which one
II~IIIIIIIIIIIIIIIIIIIIIIIVII~~ I
2
4t10- s
I
2~t10-4 Y
61uta :not~ FIG. 6 The effect of glutamate on neurally evoked contractions of the rectal longitudinal muscles. Isolated rectum only . Contractions evoked every 30 sec by a 2-sec train at 15 Hz . Note inhibition without a glutamate contraction.
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preparation served as the donor (Fig . 7A) while the other provided a continual monitoring system (Fig . 7B) . In the experiment shown (Fig . 7), 340 pq proctolin added to the 6-ml bath caused a marked potentiation of the neurally evoked responses of the assay gut. Subsequently, stimulation of the donor preparation at 50 Hz for 6 min released a substance which caused an approximately equal potentiation . A 6-min control
B- ASSAY
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IOlit
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CONTROL
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FIG . 7 Release of an active substance with similar pharmacological properties as proctolin by repetitive nerve stimulation of donor preparation (A) . Both donor (A) and assay preparation (B) in same organ bath (vol . 6 ml) . First nine responses in B were evoked by 5-sec trains at 8,9, and 10 Hz ; remaining contractions at 8 Hz . Addition of 340 pg of proctolin potentiates neurally evoked responses in B. Stopping perfusion (control) has no effect on B. With perfusion stopped, nerve stimulation of donor preparation at 50 Hz for 6 min leads to potentiation of responses in assay muscle . period (perfusion stopped, no stimulation of donor) had no effect on the assay gut. It is evident that the active substance released by nerve stimulation is not glutamate, since glutamate does not potentiate (Fig . 5) . However, the results do not deny the possibility that glutamate is also released . It can only be claimed that nerve stimulation released a substance which, as far as could be tested, had similar pharmacological activity as proctolin. Assuming that the active substance is proctolin, then approximately 300 pg accumulated in the organ bath during donor stimulation (Fig . 7), representing about 20 percent of the average amount present in one unstimulated proctodeum . It has not yet been possible to obtain quantitative data relating the amount of proctolin released to the frequency of stimulation .
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Discussion Extensive studies pertaining to the chemistry of neuromuscular transmission in the fast, skeletal fibres of insects appear to have reached a consensus that glutamate is the excitatory mediator (24) . In the slow, visceral muscles of insects the identity of the excitatory transmitter s) is still a matter of conjecture . I originally suggested that an unidentified substance present in efferent axons of the proctodeal nerve of Periplaneta might function as the excitatory mediator (10) . Holman and Cook (25) demonstrated that the active substance was a small peptide but the authors maintained that glutamate was the transmitter (22,26) and that the peptide functioned as a neutohormone involved in regulating visceral muscle activity (25) . The isolation, identification, and synthesis of the peptide, proctolin, has now been completed (17,18) . In the interim, the nature of the innervation and the bioelectrics underlying the mechanical activity of rectal muscle fibres in Periplaneta (11-13) were elucidated to an extent which allows a more intelligent appraisal of pharmacological data . Single fibres receive a distributed, polyaxonal innervation consisting of similar axons originating from the terminal ganglion of the central nervous system (CNS) . Slow action potentials can be initiated by pacemaker potentials or by EPSP~s and elicit small, ~unit~ contractions . Larger, graded contractions in response to nerve stimulation at 5 to 50 Hz are coupled mainly to summated EPSP~s, with or without superimposed action potentials (11-13) . The present study has examined pharmacologically the mechanical responses of proctodeal muscle to three agonists and to nerve stimulation . The results confirm the original statsnent (10) that only the proctolin responses are pharmacologically similar to the neurally-evoked contractions . Both 5-HT and proctolin initiate graded contractions of all or most of the proctodeal muscles similar to those evoked neurally . It is evident from the use of bromo LSD (Fig . 1) that the 5-HT receptors are distinct from the other receptors including those of the postsynaptic membrane . It follows that 5-Hf cannot be an excitatory transmitter in these muscles or, if so, then serotonergic neurones comprise a small fraction of the total excitatory innervation. In agreement with this conclusion, Colhoun could not detect S-I-if, or the appropriate decarboxylase for its synthesis in the viscera of Periplaneta (27) . In contrast to the 5-HT and proctolin responses, the glutamate contractions were not similarly graded, were often not sustained, and never approached the magnitude of the maximum neurally evoked contraction . Glutamate con tractions were localized to muscles in the region of the rectal valve . Recently, microelectrode studies were extended to fibres in this region and, contrary to our previous experience with rectal longitudinal fibres (11-13), spontaneous single and multiple EPSP~s were recorded in the absence of connection to thk central nervous systan (Brown and Nagai, unpublished) . Thus, fibres of the rectal valve appear to receive input from hitherto unsuspected peripheral nerve cells and it seems possible that this condition is related to their glutamate sensitivity . In another cockroach, Leucophaea maderae, Cook and Holman (26) have recently described spontaneous EPSP~s which they interpreted to be miniature EPSP~s . This interpretation is not consistent with their large size (cf 11) or with their sensitivity to TTX . As reported herein, and at other arthropod junctions (28), miniature potentials are not affected by TTX . It seems likely that the potentials recorded in Leuco haea (26) represent the spontaneous activity of peripheral nerve cells as ust described in Peri lp aneta . Apparently then, the proctodeal innervation of both species consists of a central component and a peripheral component, although morphological confirmation of the latter is lacking (12,26) . Given the existence of two excitatory pathways it is entirely possible
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that junctional transmission in each is mediated by different transmitters . The transmitter involvement of glutamate in the presumed peripheral neural component is a possibility that requires further study. Perhaps species variation in the distribution of the peripheral neural élanents could account for the difference between the rather localized glutamate contractions reported herein and the apparently more extensive response reported in Leucophaea (22) . In any event, it is evident that glutamate is not the excitatory transmitter in those motor neurones which originate from the CNS. The latter nerve cells constitute the sole or main excitatory innervation of the longitudinal muscle straps on the rectum of Periplaneta (11-13), but these muscles are canpletely refractory to excitation by glutamate (Fig . 6) . Furthernwre, there is no indication that glutamate potentiates the neurally evoked response. The sole action of glutamate consists of inhibition of the neurally evoked response (Fig . 6) . Since there is no indication of prior excitation, the latter effect cannot be described as desensitization of postsynaptic receptors, as attributed to glutamate in insect twitch fibres (20,24) . Indeed, since the inhibitory and excitatory activities of glutamate are clearly separable, the effects could involve entirely different mechanisms . The claim that glutamate mimics the neurally evoked contraction of the hindgut of Leucophaea (22) is only true if nerve stimulation is brief . With continuous stimulation at moderate frequencies of 10 to 20 Hz, the proctodeal muscles of Periplaneta (and presumably of Leucophaea ) respond with substantial contractions which are sustained for at least several mirnites . The sustained response suggests that the postsynaptic receptors are not readily susceptible to transmitter desensitization . A similar situation is evident in the slow muscles of frogs (29) and birds (30) in which ACh evoxes long lasting contractions . Thus, a reasonable prerequisite for transmitter status in the present slow muscles is that the candidate mimics the sustained neurally evoked contraction. In agreement with the results of Holmen and Cook (22,25,26) this criterion is satisfied by proctolin but mt by glutamate. Additionally, the present study dsnonstrates that both the proctolin and neurally evoked responses are antagonized by tyramine (Fig . 3) . In kinetic studies now in progress, we have found that the percent inhibition bY tyramine of the proctolin and neurally evoked responses is greatly reduced at higher proctolin doses or higher stimulation frequencies respectively (Brown and ICrupka, unpublished) . Thus, tyramine behaves as a competitive antagonist . Since dopamine is relatively inactive (Fig . 4), it is also apparent that the monophenolic structure is required for full antagonistic activity . Hence it is probably significant that proctolin contains a tyrosyl residue (18) and it may be speculated that the phenolic group of tyramine has considerable affinity for the same receptor site as the phenolic group of proctolin. The specificity of the tyramine antagonism (Fig . 4) indicates that the blockade does not occur at the contractile level nor would interfer~ce with excitation-contraction coupling be anticipated . On the other hand, the results with TTX and denervated muscle dsnonstrate convincingly that proctolin does not evoke contraction by acting at a neural or presynaptic site . Thus, the combined results indicate that either (1) the proctolin and postsynaptic receptors are distinct but, coincidentally, are both canpetitively blocked by tyramine, or (2) the postsynaptic receptor is the site of action common to proctolin, the natural transmitter, and tyramine . Obviously the latter interpretation is the simpler and is consistent with proctolin~s proposed role as excitatory transmitter . With the availability of synthetic proctolin (1B), this tentative conclusion can now be examined by iontophoretic techniques . The well known association of peptide neurohotmones and elsnentary neurosecretory granules led Holmen and Cook to assume a similar relationship between
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their peptide and the neurosecretory axons (1300-3900 ~ granules) which are located peripherally in the proctodeal nerve (25) . In my view, it is highly significant that the proctodeal motor axons originating in the CNS and terminating on rectal muscle fibres (12) also possess ~neurosecretory~ characteristics (10, Brown and Graham, unpublished) . Similar terminals elsewhere in insects have been referred to as neurosecretory or motosecretory (19) and are quite distinct from the ~glutamate~ terminals on insect fast fibres (20) . On this basis alone, the transmitter involvement of glutamate in the present fibres is suspect . In the rectal fibres of Periplaneta , the motor nerve endings contain numbers of electron-dense granules (1000-2000 ~ diam ., no halo) which appear to fragment to form electron-lucent vesicles as in the A fibres described by Knowles (30) . The vesicles migrate into typical presynaptic clusters and thus the terminals are strikingly similar to the peptidergic terminals described by Barcgnann and associates in the cat hypophysis (9) . The latter work has been cited by Nicoll (31) as supporting evidence for the neurotransmitter activity of vasopressin (8) . Thus, while it may be premature to define such terminals in insects as peptidergic, it is evident that peptidergic transmission at the present junctions is not without some basis at the ultrastructural level . Acknowledgments I am grateful for the technical assistance of Miss D . N . Mindenhall and Mr . N . Jerry . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .
U . S . VON EULER and J . H . GADDUM, J . Physiol . 72, 74-87 (1931) . M . M . CHANG, S . E . LEEf7AN, and H . D . NTALL, Nature, New Biol . 232, 86-87 (1971) . G . W . TREGFAR, H . D . MALL, J . T . POTTS, S . E . T .RFMAN , and M . M . CHANG, Nature, New Biol . 232, 87-89 (1971) . L . L . IVERSEN, Nature , 252, 630 (1974) . M . OTSUKA, S . KONISHI, and T . TAKAHASHI, Proc . Jap . Acad . _48, 747-752 (1972) . T . TAKAHASHI, S . KONISHI, D . POWELL, S . E . T .RFMAN , and M . OTSUKA, Brain Res . 73, 59-69 (1974) . S . KONISH2 and M . OTSUKA, Nature _252, 734-735 (1974) . R . A . NICOLL and J . L . BARKER, Brain Res . 35, 501-511 (1971) . W . BARCi+lAta1, E . LIImNER, and K . H . ANDRES, Z . Zellforsch . 77, 282-298 (1967) . B . E . BROWN, Science , 155, 595-597 (1967) . P . BELTON and B . E . BR(7G7N, Camp . Biochor . Physiol . 28, 853-863 (1969) . B . E . BROWN and T . NAGAI, J . Insect Physiol . 15, 1767-1783 (1969) . T . NAGAI and B . E . BROWN, J . Insect Physiol . 15, 2151-2167 (1969) . T . NAGAI, J . Insect Physiol . 16, 437-448 (1970) . T . NAGEAI, J . Insect Physiol . 18, 2299-2318 (1972) . T . NAGAI, J . Insect Physiol . _19, 1753-1764 (1973) . B . E . BROWN and A . N. STARRATT, J . Insect Ph siol ., in press . A . N . STARRATT and B. E . BROWN, L1fe Sci . , 3-1256 (1975) . T . M ILLER and D . REES, Amer . Zool . _13, 299-313 (1973) . P . N . R . USHERWOOD and P . MACHILI, J . E Biol . 49, 341-361 (1968) . B . E . BROWN, Gen. Carp . Endocrin . 5, 38 - " 1 1965) . G . M . HOLMAN and B . J . COOK, J . Insect Physiol . _16, 1891-1907 (1970) . C . Y . CAO, Phaxmacol . Rev . 18, 997-1049 (1966) . T . J . McDONALD, Ann . Rev . Entomol . 20, 151-166 (1975) . G . M . HOLMAN and B . J . COOK, Biol . Bull . 142, 446-460 (1972) . B . J . COOK and G . M . HOLMAN, Canes . Biochor .Physiol . 50C, 137-146 (1975) .
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E. H. COLI-~UN, Experientia 19, 9-10 (1963) . M. OZEKI, A. R. FREEMAN, andH. GRUNDFEST, J. Gen. Physiol . _49, 1319-1331 (1966) . S . W. KOFFLER and E . M. VAUGHN WILLIAMS, J. Physiol . _121, 318-340 (1953) . A . P. SAMLYO and A. V . SAMLYO, Fed. Proc . 28, 1634-1642 (1969) . F. G. W. KNOWLES, Phil . Trans. Ray . 3oc . Ldnd . B249 , 435-455 (1965) . R. A. NICOLL, Neurosci . Res . Prog . Bull . 10, 202-206 (1972) .
PROCTOLIN:
Perqamon Press
1241-1252
A PEPTIDE TRANSMITTER CANDIDATE IN INSECTS Brian E . Brown
Research Institute, Agriculture Canada, University Sub P.O ., London, Canada N6A 5B7 (Received in final form September 15, 1975) Summary The slow, striated muscles of the proctodeum (hindgut) of the cockroach, Periplaneta americana (L .), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from Periplaneta recently isolated and identified . The graded contractions evoked by repetitive nerve stimulation were simulated b~r9 5-HT and proctolin at threshold concentrations -$f about 10 and 10 M respectively ; responses to glutamate (10 M) were not similarly graded . The 5-HT receptors are distinct from other receptors, including the postsynaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide . Proctolin was fully active on TTXtreated or surgically denervated muscle indicating that the proctolin receptors are located on ~he muscle fibre menbrane . Tyramine, at threshold levels of 5x10 M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the S-I-fr and glutamate responses . Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate . Pharmacologically, the proctolin and postsynaptic receptors appear to be identical and distinct from the glutamate and S-1-fr receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosecretory granules in addition to electron-lucent, synaptic vesicles . Interest in peptides as transmitter substances or as modulators of neuronal activity dates fran the discovery of substance P bY von E~ler and Gaddum (1) . Substance P has now been sequenced and synthesized (2,3) and recent evidence suggests that it and other peptides may indeed function as neurotransmitters (4) . Substance P may be the excitatory transmitter of primary afferent neurones in the spinal cord (5-7) and studies by Nicoll and Barker indicate that recurrent inhibition of neurones of the supraoptic nucleus may be mediated by the synaptic action of vasopressin (B) . At the ultrastructural level, additional evidence is provided by the "peptidergen Synapsen" described by Bargmann et al (9) in the cat hypothesis . The present report extends the peptide trânsm~tter hypothesis to invertebrates. Etitidence is presented which suggests that insects possess a distinctive class of motor nerve cell in which synaptic transmission is mediated by a small peptide . 1241
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I suggested earlier that a myotropic substance associated with an efferent pathway of the proctodeal (hindgut) innervation of the cockroach, Perip l aneta americana (L .), might function as an excitatory transmitter substance in insect visceral muscle (10) . The active principle was associated with a small particulate fraction (synaptic vesicles :) and caused graded contractions of the proctodeal muscles similar to those evoked by repetitive nerve stimulation (10) . Subsequent studies described the innervation of these fibres and the bioelectrics underlying their mechanical behaviour (11-16) . Proctolin, the active compound of the initial study (10), was recently isolated in pure form (17) . The following coR¢nunication (18) describes the sequential analysis and synthesis of proctolin, a pentapeptide . The current report presents pharmacological data consistent with proctolin~s proposed transmitter function . The concept of peptidergic motor neurones in insects is discussed in respect to the ultrastructural canponents of the present motor nerve terminals which are similar to others in insects (19) but quite distinct from those terminating in the fast, skeletal fibres of insects, wherein glutamate is the putative transmitter (e .q . 20) . Methods and Materials Anatomical details of the proctodeum and its bilateral innervation in P. americana have been described previously (12) . All experiments except that in Fiq. 6 were performed with the whole proctodeum and intact nerve supply isolated from adult, male cockroaches . The sixth abdominal ganglion, in which the motor nerve cell bodies are located, was crushed or removed . The left and right cercal nerve (N XI), from which the fine proctodeal nerves branch, were tied together to ensure the introduction of both proctodeal nerves into the stimulating suction electrode. The results i.n Fig. 6 were obtained with the isolated rectum only (10,12) . The isolated hindgut or rectum was suspended by silk threads in a 4-ml organ bath and perfused with oxygenated insect saline (21) . 5-200 ul quantities of drug solutions were added rapidly to the organ bath via Hamilton syringes and r~noved by fast perfusion. Nerve stimulation was achieved by a suction electrode fixed in the side wall of the perfusion chamber. Stimulation was supramaximal with single stimuli of 0.5 meet duration . Contractions were recorded isotonically under 250 mg tension. Unless otherwise indicated, preparations were rested for 3-4 min between responses to drugs or nerve stimulation except for glutamate when the rest period was 10 min. Approximately 200 preparations were examined using both a highly purified aliquot of natural proctolin (17) and the synthetic peptide (18) . The records presented herein were obtained with synthetic proctolin but the results are identical to those obtained with natural proctoli.n . Experiments were performed at room temperature (22-25°C) . Results Normal responses to agonists and nerve stimulation : Typical examples of responses of the isolated proctodeum to 5-ffP, nerve stimulation, proctoli.n and glutamate are shown in Fiq . lA and 2A . The contractions evoked by 5-HT, proctolin and repetitive nerve stimulation are graded responses to drug concentration or to frequency of nerve stimulation . Maximum neurally evoked responses occur at about 50 Hz and their magnitude can be attained by increased concentrations of both 5-HT and proctolin. In contrast, the glutamate responses are not similarly graded nor do they ever approach the magnitude of the maximal neural response . Continuous nerve stimulation at moderate frequencies of 10 to 20 Hz evokes contractions which are sustained for at least several minutes and
Proctolin :
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A Peptide Transmitter
often for much longer . Sustained contractions are also characteristic of the S-I-fP and proctolin responses but, in agreement with Holman and Cook (22), they are not characteristic of the glutamate responses (see also Fig. 5) . The small glutamate contractions are not due to slight contraction of all muscles . Significantly (see Discussion), glutamate contractions are restricted mainly to muscles comprising the rectal valve, a short region of the proc todeum just anterior to the rectum (12) . By contrast, contractions evoked by 5-HT, proctolin, and nerve stimulation involve all or most fibres of the entire proctodeum . Branolysergic acid diethylamide: The effect of bravo LSD, a well known antagonist of indolealkylamines, is shown in Fig. 1B . The responses to 5-HT are blocked whereas the other responses are unaffected . Clearly, the 5-HT receptors are distinct from the other receptors including those of the postsynaptic meJnbrane. Tetrodotoxin and muscle denervation; At other junctions, TTX blockade is limited to the nerve action potential ; it neither interferes with the release of transmitter nor does it block postsynaptic receptors (23) . TTX has already shown to rapidly block the excitatory postsynaptic potential (EPSP) in fibres of the rectum of Periplaneta (15) . I have further examined the TTX blockade in these fibres to ensure that TTX has no effect on the postsynaptic receptors . TTX indeed causes a rapid failure of the EPSP but,
A
B
0 "2
Normal
Bromo LSD-Treefed
0-S 5-HT
FOtIOaM
4
6
18 Ne
NEURAL
I
2
4eH~11
PROCTOLIN
2
4
" >
GLUTAMATE
FIG . 1 The effect of bromolysergic acid diethylamide (bravo LSD) on proctodeal muscle responses to 5-HT, nerve stimulation, proctolin and glutamate. Drugs and stimulation applied for 20 sec . A - Normal responses . B - Responses of same preparation after a 10-min exposure to bravo LSD at 100 ng per ml .
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A Normal
B
TTX -Treated I min
t 0~5 Ix10 -6 M
8 I6Hz
0~6 1~2x10 -9 M
0~5 I :10 -4 M
5-H T
NEURA L
PROGTOLIN
GLUTAMATE
FIG. 2 The effect of tetroxotoxin (TTX) on proctodeal muscle responses to 5-HP, nerve stimulation, proctolin, and glutamate. Drugs and stimulation applied for 10 sec. A - Normal responses. B - Responses of same preparation in the presence of 4x10 -7 M TTX. even after prolonged treatment, miniature EPSP~s (11) were unaffected . Presumably theSi,the action of TTX at this junction, as elsewhere, is limited to neural blockade . As shown in Fig. 2B, TTX completely abolishes the neurally evoked response but is without effect on the proctolin responses or on the responses evoked by 5-H'i' and glutamate. Therefore, neither proctolin nor the other agoniats are myotropic by reason of initiating nerve action potentials . Proctolin~s lack of effect at the neural or presynaptic level was confirmed by studies with a limited number of surgically denervated preparations . (10,12) . Following nerve section, EPSP~s in rectal fibres can be recorded intracellularly up to approximately 72 hours, following which junctional transmission fails completely (Nagai an3 Brown, unpublished) . The present study reports on the mechanical responses of three preparations from insects that survived 5 days of bilateral neurotomy (mortality was about 80 percent) . As éxpected, the denervated muscle failed to respond mechanically to repetitive nerve stimulation. However, in all cases, the denervated proctodeum displayed normal contractions and sensitivity to proctolin, as well as to 5-HT and glutamate. Therefore, proctolin~s activity cannot be attributed to neuroexcitation or to initiating the release of presynaptic stores of the natural transmitter . Judging from the EPSP failure 3 days after nerve section, presynaptic transmitter stores must be radically depleted 5 days after neurotomy . Parenthetically, it should be noted that proctolin is 50 percent de-
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pleted from the rectum 4 days after nerve section and completely lost sometime between the fourth and eighth day (10) . amine: In an examination of the pharmacological effects of catecholamines and analogues on the hindgut, it was found that two compounds were Especially effective were tyramine (ß(4 useful proctolin antagonists . hydroxyphenyl)ethylamine) and its ß-hydroxy analogue, octopamine . Other analogues were inactive or less effective as antagonists, especially the diphenols (e .g ., dopamine) and tyrosine . Of significance to the transmitter candidacy of proctolin, tyramine also suppresses neurally evoked contractions . In most preparations, the threshold inhibitory concentration of tyramine is similar for both the proctolin and neurally evoked responses and both responses are similarly suppressed at higher levels of tyramine (Fig . 3) . There has been some deviation from this observation but not in any consistent manner . The deviation may be attributed to the apparently competitive nature of the tyramine blockade (see Discussion) .
Ss10~
3e10~
I :10" 8 Y
Tyremine
FIG. 3 The effects of tyramine on proctodeal muscle responses to nerve stimulation at 10 Hz for 10 sec (~) and to 1x10-8 M proctolin for 10 sec . (~) . Threshold inhibition of both responses at 5x10 B M tyramine . Both responses equally suppressed at higher concentrations of tyramine . The specificity of the tyramine antagonism is demonstrated in another preparation (Fig . 4) . In this case, t~e proctolin response was unaffected -fit 3x10 M tyramine, diminished at 3x10 M, and strongly suppressed at 3x10 M tyramine (Fig . 4A) . The same levels of tyramine had no effect on the contractions evoked by 5-HT (Fig . 4C) or by glutamate (Fig . 4D) . In a few preparations, some inhibition of 5-HT r_~sponses has been observed at relatively high concentrations of tyramine ( 10 M and higher) . Thus, the tyramine effect appears to be highly selective with threshold inh_jrbition gf the proctolin and neurally evoked contractions occurring in the 10 to lÔ M range. As an example of the structual requirement of the antagonist molecule referred to earlier, the same concentrations of the ortho diphenol analogue, dopamine, have no effect on the proctolin responses (Fig . 4B) . Effects of subthreshold levels of proctolin and glutamate on neurally evoked contractions : The graded responses of proctodeal muscles to repetitive nerve stimulation (10) are coupled mainly to EPSP summation with some
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Psroctolin :
A
A Peptide Transmitter
Proctolin Response :
ô
Tyramine
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Proctolin Responses
Dopamine 6lutam ats Responses
Tyramlne
Tyromlne FIG. 4
Specificity of tyramine antagonism . In each quadrant (A,B,C,D), the first and last of the five responses are normal contractions to 2x10 -9 M proctolin (A,B), 6x10-7 M 5-HT (C), and 1 .5x10 -4 M glutamate (D), each applied for 20 sec. The three remaining contractions in each quadrant were similarly evoked lut in the presence of (from left to right) 3x10 -9 , 3x10 -8 , and 3x10 -7 M tyramine (A,C,D) or dopamine (B) . All records from one preparation . contribution from slow action potentials which may be superimposed on the summated EPSP (13) . Routinely, neurally evoked contractions of the proctodeum can reflect a difference of one Impulse per sec in the range of 6-15 Hz (e .g ., Fig. 7B) . Thus, the postsynaptic receptors are particularly sensitive to small changes in the concentration of natural transmitter within the synaptic cleft . Given these circwnstances, it is reasonable to expect that subthreshold concentrations of the transmitter substance in the organ bath should be additive to amounts released by nerve stimulation . As shown in Fiq. 5, this is indeed true of proctolin but is not tnae of glutamate. Neurally evoked contractions are increasingly potentiated at 2, 4, and 8x10 -lOM proctolin ; at 8x10 -10 M proctolin, there is a slight rise in the base line indicating the threshold nature of this concentration. In contrast, subthreshold levels of glutamate do not potentiate the neurally evoked response . In many instances, as shown in Fig . 5, the first observed effect of glutamate is one of inhibition . At 10 -4 M glutamate there is a typical brief contraction (Fig . 5, arrow) following which the evoked contractions are further suppressed . Higher concentrations of glutamate (2 to 5x10 -~i) will almost or co¢npletely abolish the neurally evoked response . As indicated earlier, the glutamate contractions are restricted mainly to a relatively small group of longitudinal muscles in the region of the rectal valve and therefore the glutamate responses never approach the maximal neurally evoked contraction of the whole proctodeum . However, the antagonistic effect of glutamate (Fig . 5) extends to most or all proctodeal muscle fibres . This observation has been confirmed in current studies with three separate nPrve-
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FIG. 5 The effect of subthreshold concentrations of proctolin and glutamate on neurally evoked contractions of proctodeal muscle . Neurally evoked responses initiated every 30 sec by a 3-sec train at 8 Hz . Note brief contraction at 10 -4 M glutamate (arrow) . muscle preparations consisting of the rectum, rectal valve, and anterior proctodeum . Thus, the longitudinal muscle straps of the rectum, i .e ., those fibres which have been the subject of most of our studies (11-16), do not contract in response to applied glutamate but their neurally evoked contractions are almost -4 M glutamate (Fig . 6) . With or completely abolished at levels of about 10 this important exception, the pharmacology of the rectal straps is similar to that of the whole proctodeum . Release of proctolin during nerve stimulation : Subsequent to the isolation, characterization and synthesis of proctolin (17,18), comparative bioassay of an extract of 500 hindguts determined that one hindgut contained only about 1600 pg proctolin. Therefore, it seemed unlikely that sufficient quantities of proctolin would accumulate in the perfusate of a stimulated preparation to allow chromatographic analysis (17) . This was indeed found to be the case and, in fact, experiments had to be designed to employ the most sensitive bioassay available, i .e ., the potentiation of neurally evoked responses which occurs with as little as 50-70 pg proctolin per ml . The most successful of these experiments employed a classic, double nerve-muscle preparation in which one
II~IIIIIIIIIIIIIIIIIIIIIIIVII~~ I
2
4t10- s
I
2~t10-4 Y
61uta :not~ FIG. 6 The effect of glutamate on neurally evoked contractions of the rectal longitudinal muscles. Isolated rectum only . Contractions evoked every 30 sec by a 2-sec train at 15 Hz . Note inhibition without a glutamate contraction.
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preparation served as the donor (Fig . 7A) while the other provided a continual monitoring system (Fig . 7B) . In the experiment shown (Fig . 7), 340 pq proctolin added to the 6-ml bath caused a marked potentiation of the neurally evoked responses of the assay gut. Subsequently, stimulation of the donor preparation at 50 Hz for 6 min released a substance which caused an approximately equal potentiation . A 6-min control
B- ASSAY
8
9
IOlit
30 Hi
PROCTOLIN
(340 pq)
CONTROL
(no pwfl
FIG . 7 Release of an active substance with similar pharmacological properties as proctolin by repetitive nerve stimulation of donor preparation (A) . Both donor (A) and assay preparation (B) in same organ bath (vol . 6 ml) . First nine responses in B were evoked by 5-sec trains at 8,9, and 10 Hz ; remaining contractions at 8 Hz . Addition of 340 pg of proctolin potentiates neurally evoked responses in B. Stopping perfusion (control) has no effect on B. With perfusion stopped, nerve stimulation of donor preparation at 50 Hz for 6 min leads to potentiation of responses in assay muscle . period (perfusion stopped, no stimulation of donor) had no effect on the assay gut. It is evident that the active substance released by nerve stimulation is not glutamate, since glutamate does not potentiate (Fig . 5) . However, the results do not deny the possibility that glutamate is also released . It can only be claimed that nerve stimulation released a substance which, as far as could be tested, had similar pharmacological activity as proctolin. Assuming that the active substance is proctolin, then approximately 300 pg accumulated in the organ bath during donor stimulation (Fig . 7), representing about 20 percent of the average amount present in one unstimulated proctodeum . It has not yet been possible to obtain quantitative data relating the amount of proctolin released to the frequency of stimulation .
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Discussion Extensive studies pertaining to the chemistry of neuromuscular transmission in the fast, skeletal fibres of insects appear to have reached a consensus that glutamate is the excitatory mediator (24) . In the slow, visceral muscles of insects the identity of the excitatory transmitter s) is still a matter of conjecture . I originally suggested that an unidentified substance present in efferent axons of the proctodeal nerve of Periplaneta might function as the excitatory mediator (10) . Holman and Cook (25) demonstrated that the active substance was a small peptide but the authors maintained that glutamate was the transmitter (22,26) and that the peptide functioned as a neutohormone involved in regulating visceral muscle activity (25) . The isolation, identification, and synthesis of the peptide, proctolin, has now been completed (17,18) . In the interim, the nature of the innervation and the bioelectrics underlying the mechanical activity of rectal muscle fibres in Periplaneta (11-13) were elucidated to an extent which allows a more intelligent appraisal of pharmacological data . Single fibres receive a distributed, polyaxonal innervation consisting of similar axons originating from the terminal ganglion of the central nervous system (CNS) . Slow action potentials can be initiated by pacemaker potentials or by EPSP~s and elicit small, ~unit~ contractions . Larger, graded contractions in response to nerve stimulation at 5 to 50 Hz are coupled mainly to summated EPSP~s, with or without superimposed action potentials (11-13) . The present study has examined pharmacologically the mechanical responses of proctodeal muscle to three agonists and to nerve stimulation . The results confirm the original statsnent (10) that only the proctolin responses are pharmacologically similar to the neurally-evoked contractions . Both 5-HT and proctolin initiate graded contractions of all or most of the proctodeal muscles similar to those evoked neurally . It is evident from the use of bromo LSD (Fig . 1) that the 5-HT receptors are distinct from the other receptors including those of the postsynaptic membrane . It follows that 5-Hf cannot be an excitatory transmitter in these muscles or, if so, then serotonergic neurones comprise a small fraction of the total excitatory innervation. In agreement with this conclusion, Colhoun could not detect S-I-if, or the appropriate decarboxylase for its synthesis in the viscera of Periplaneta (27) . In contrast to the 5-HT and proctolin responses, the glutamate contractions were not similarly graded, were often not sustained, and never approached the magnitude of the maximum neurally evoked contraction . Glutamate con tractions were localized to muscles in the region of the rectal valve . Recently, microelectrode studies were extended to fibres in this region and, contrary to our previous experience with rectal longitudinal fibres (11-13), spontaneous single and multiple EPSP~s were recorded in the absence of connection to thk central nervous systan (Brown and Nagai, unpublished) . Thus, fibres of the rectal valve appear to receive input from hitherto unsuspected peripheral nerve cells and it seems possible that this condition is related to their glutamate sensitivity . In another cockroach, Leucophaea maderae, Cook and Holman (26) have recently described spontaneous EPSP~s which they interpreted to be miniature EPSP~s . This interpretation is not consistent with their large size (cf 11) or with their sensitivity to TTX . As reported herein, and at other arthropod junctions (28), miniature potentials are not affected by TTX . It seems likely that the potentials recorded in Leuco haea (26) represent the spontaneous activity of peripheral nerve cells as ust described in Peri lp aneta . Apparently then, the proctodeal innervation of both species consists of a central component and a peripheral component, although morphological confirmation of the latter is lacking (12,26) . Given the existence of two excitatory pathways it is entirely possible
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that junctional transmission in each is mediated by different transmitters . The transmitter involvement of glutamate in the presumed peripheral neural component is a possibility that requires further study. Perhaps species variation in the distribution of the peripheral neural élanents could account for the difference between the rather localized glutamate contractions reported herein and the apparently more extensive response reported in Leucophaea (22) . In any event, it is evident that glutamate is not the excitatory transmitter in those motor neurones which originate from the CNS. The latter nerve cells constitute the sole or main excitatory innervation of the longitudinal muscle straps on the rectum of Periplaneta (11-13), but these muscles are canpletely refractory to excitation by glutamate (Fig . 6) . Furthernwre, there is no indication that glutamate potentiates the neurally evoked response. The sole action of glutamate consists of inhibition of the neurally evoked response (Fig . 6) . Since there is no indication of prior excitation, the latter effect cannot be described as desensitization of postsynaptic receptors, as attributed to glutamate in insect twitch fibres (20,24) . Indeed, since the inhibitory and excitatory activities of glutamate are clearly separable, the effects could involve entirely different mechanisms . The claim that glutamate mimics the neurally evoked contraction of the hindgut of Leucophaea (22) is only true if nerve stimulation is brief . With continuous stimulation at moderate frequencies of 10 to 20 Hz, the proctodeal muscles of Periplaneta (and presumably of Leucophaea ) respond with substantial contractions which are sustained for at least several mirnites . The sustained response suggests that the postsynaptic receptors are not readily susceptible to transmitter desensitization . A similar situation is evident in the slow muscles of frogs (29) and birds (30) in which ACh evoxes long lasting contractions . Thus, a reasonable prerequisite for transmitter status in the present slow muscles is that the candidate mimics the sustained neurally evoked contraction. In agreement with the results of Holmen and Cook (22,25,26) this criterion is satisfied by proctolin but mt by glutamate. Additionally, the present study dsnonstrates that both the proctolin and neurally evoked responses are antagonized by tyramine (Fig . 3) . In kinetic studies now in progress, we have found that the percent inhibition bY tyramine of the proctolin and neurally evoked responses is greatly reduced at higher proctolin doses or higher stimulation frequencies respectively (Brown and ICrupka, unpublished) . Thus, tyramine behaves as a competitive antagonist . Since dopamine is relatively inactive (Fig . 4), it is also apparent that the monophenolic structure is required for full antagonistic activity . Hence it is probably significant that proctolin contains a tyrosyl residue (18) and it may be speculated that the phenolic group of tyramine has considerable affinity for the same receptor site as the phenolic group of proctolin. The specificity of the tyramine antagonism (Fig . 4) indicates that the blockade does not occur at the contractile level nor would interfer~ce with excitation-contraction coupling be anticipated . On the other hand, the results with TTX and denervated muscle dsnonstrate convincingly that proctolin does not evoke contraction by acting at a neural or presynaptic site . Thus, the combined results indicate that either (1) the proctolin and postsynaptic receptors are distinct but, coincidentally, are both canpetitively blocked by tyramine, or (2) the postsynaptic receptor is the site of action common to proctolin, the natural transmitter, and tyramine . Obviously the latter interpretation is the simpler and is consistent with proctolin~s proposed role as excitatory transmitter . With the availability of synthetic proctolin (1B), this tentative conclusion can now be examined by iontophoretic techniques . The well known association of peptide neurohotmones and elsnentary neurosecretory granules led Holmen and Cook to assume a similar relationship between
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their peptide and the neurosecretory axons (1300-3900 ~ granules) which are located peripherally in the proctodeal nerve (25) . In my view, it is highly significant that the proctodeal motor axons originating in the CNS and terminating on rectal muscle fibres (12) also possess ~neurosecretory~ characteristics (10, Brown and Graham, unpublished) . Similar terminals elsewhere in insects have been referred to as neurosecretory or motosecretory (19) and are quite distinct from the ~glutamate~ terminals on insect fast fibres (20) . On this basis alone, the transmitter involvement of glutamate in the present fibres is suspect . In the rectal fibres of Periplaneta , the motor nerve endings contain numbers of electron-dense granules (1000-2000 ~ diam ., no halo) which appear to fragment to form electron-lucent vesicles as in the A fibres described by Knowles (30) . The vesicles migrate into typical presynaptic clusters and thus the terminals are strikingly similar to the peptidergic terminals described by Barcgnann and associates in the cat hypophysis (9) . The latter work has been cited by Nicoll (31) as supporting evidence for the neurotransmitter activity of vasopressin (8) . Thus, while it may be premature to define such terminals in insects as peptidergic, it is evident that peptidergic transmission at the present junctions is not without some basis at the ultrastructural level . Acknowledgments I am grateful for the technical assistance of Miss D . N . Mindenhall and Mr . N . Jerry . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .
U . S . VON EULER and J . H . GADDUM, J . Physiol . 72, 74-87 (1931) . M . M . CHANG, S . E . LEEf7AN, and H . D . NTALL, Nature, New Biol . 232, 86-87 (1971) . G . W . TREGFAR, H . D . MALL, J . T . POTTS, S . E . T .RFMAN , and M . M . CHANG, Nature, New Biol . 232, 87-89 (1971) . L . L . IVERSEN, Nature , 252, 630 (1974) . M . OTSUKA, S . KONISHI, and T . TAKAHASHI, Proc . Jap . Acad . _48, 747-752 (1972) . T . TAKAHASHI, S . KONISHI, D . POWELL, S . E . T .RFMAN , and M . OTSUKA, Brain Res . 73, 59-69 (1974) . S . KONISH2 and M . OTSUKA, Nature _252, 734-735 (1974) . R . A . NICOLL and J . L . BARKER, Brain Res . 35, 501-511 (1971) . W . BARCi+lAta1, E . LIImNER, and K . H . ANDRES, Z . Zellforsch . 77, 282-298 (1967) . B . E . BROWN, Science , 155, 595-597 (1967) . P . BELTON and B . E . BR(7G7N, Camp . Biochor . Physiol . 28, 853-863 (1969) . B . E . BROWN and T . NAGAI, J . Insect Physiol . 15, 1767-1783 (1969) . T . NAGAI and B . E . BROWN, J . Insect Physiol . 15, 2151-2167 (1969) . T . NAGAI, J . Insect Physiol . 16, 437-448 (1970) . T . NAGEAI, J . Insect Physiol . 18, 2299-2318 (1972) . T . NAGAI, J . Insect Physiol . _19, 1753-1764 (1973) . B . E . BROWN and A . N. STARRATT, J . Insect Ph siol ., in press . A . N . STARRATT and B. E . BROWN, L1fe Sci . , 3-1256 (1975) . T . M ILLER and D . REES, Amer . Zool . _13, 299-313 (1973) . P . N . R . USHERWOOD and P . MACHILI, J . E Biol . 49, 341-361 (1968) . B . E . BROWN, Gen. Carp . Endocrin . 5, 38 - " 1 1965) . G . M . HOLMAN and B . J . COOK, J . Insect Physiol . _16, 1891-1907 (1970) . C . Y . CAO, Phaxmacol . Rev . 18, 997-1049 (1966) . T . J . McDONALD, Ann . Rev . Entomol . 20, 151-166 (1975) . G . M . HOLMAN and B . J . COOK, Biol . Bull . 142, 446-460 (1972) . B . J . COOK and G . M . HOLMAN, Canes . Biochor .Physiol . 50C, 137-146 (1975) .
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E. H. COLI-~UN, Experientia 19, 9-10 (1963) . M. OZEKI, A. R. FREEMAN, andH. GRUNDFEST, J. Gen. Physiol . _49, 1319-1331 (1966) . S . W. KOFFLER and E . M. VAUGHN WILLIAMS, J. Physiol . _121, 318-340 (1953) . A . P. SAMLYO and A. V . SAMLYO, Fed. Proc . 28, 1634-1642 (1969) . F. G. W. KNOWLES, Phil . Trans. Ray . 3oc . Ldnd . B249 , 435-455 (1965) . R. A. NICOLL, Neurosci . Res . Prog . Bull . 10, 202-206 (1972) .