Cardioacceleratory action of tachykinin-related neuropeptides and proctolin in two coleopteran insect species1☆,☆☆

Peptides 22 (2001) 209 –217

Cardioacceleratory action of tachykinin-related neuropeptides and proctolin in two coleopteran insect species夞,夞夞 Joanna Sliwowskaa,b, Grzegorz Rosinskib, Dick R. Na¨ssela* a

Department of Zoology, Stockholm University, S-10691 Stockholm, Sweden Department of Animal Physiology, A. Mickiewicz University, 61-701 Poznan, Poland

b

Received 10 March 2000; received in revised form accepted 7 August 2000

Abstract Several cardioactive peptides have been identified in insects and most of them are likely to act on the heart as neurohormones. Here we have investigated the cardioactive properties of members of a family of insect tachykinin-related peptides (TRPs) in heterologous bioassays with two coleopteran insects, Tenebrio molitor and Zophobas atratus. Their effects were compared with the action of the pentapeptide proctolin. We tested the cardiotropic activity of LemTRP-4 isolated from the midgut of the cockroach Leucophaea maderae, CavTK-I and CavTK-II isolated from the blowfly Calliphora vomitoria. The semi-isolated hearts of the two coleopteran species were strongly stimulated by proctolin. We observed a dose dependent increase in heartbeat frequency (a positive chronotropic effect) and a decrease in amplitude of contractions (a negative inotropic effect). In both beetles the TRPs are less potent cardiostimulators and exert lower maximal frequency responses than proctolin. LemTRP-4 applied at 10⫺9–10⫺6 M was cardiostimulatory in both species inducing an increase of heart beat frequency. The amplitude of contractions was stimulated only in Z. atratus. CavTK-I and CavTK-II also exerted cardiostimulatory effects in Z. atratus at 10⫺9–10⫺6 M. Both peptides stimulated the frequency, but only CavTK-II increased the amplitude of the heart beat. In T. molitor, however, the CavTKs induced no significant effect on the heart. Immunocytochemistry with antisera to the locust TRPs LomTK-I and LomTK-II was employed to identify the source of TRPs acting on the heart. No innervation of the heart by TRP immunoreactive axons could detected, instead it is possible that TRPs reach the heart by route of the circulation. The likely sources of circulating TRPs in these insects are TRP-immunoreactive neurosecretory cells of the median neurosecretory cell group in the brain with terminations in the corpora cardiaca and endocrine cells in the midgut. In conclusion, LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and also exert stimulatory rather than inhibitory action on amplitude of contractions. The differences in the responses to proctolin and TRPs suggest that the peptides regulate heart activity by different mechanisms. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Myotropic neuropeptides; Tachykinin; Proctolin; Neurohormone; Insect nervous system; Heart muscle; Circulation

1. Introduction A large number of insect neuropeptides display cardioacceleratory actions [see 9,10,39,41]. It is well known that Abbreviations used: CavTK, callitachykinin; CCAP, crustacean cardioactive peptide; LemTRP, Leucophaea tachykinin-relaetd peptide; LomTK, locustatachykinin; LTK-LI, LomTK-like immunoreactive. 夞 Supported by a grant from the Swedish Natural Science Research Council (D.R.N.), a scholarship from the Swedish Institute (J.S.) and a staff award from A. Mickiewicz University (G.R.). 夞夞 Taken from a paper presented at the Winter Neuropeptide Conference 2000, Invertebrate Division, Hua Hin, Thailand, January 10 –15, 2000. * Corresponding author. Tel.: ⫹46-8-164077; fax: ⫹46-8-167715. E-mail address: [email protected] (D. R. Na¨ssel).

many of the cardioactive peptides as well as other myotropic peptides may play additional functional roles. For example, crustacean cardioactive peptide (CCAP; previously designated CAP2A) of the moth Manduca sexta which is cardioacceleratory [3,21], also plays an important role in generation of motor activity associated with ecdysis behaviour [12,46]. Another example is corazonin, which was first isolated as a cardioacceleratory peptide from the American cockroach [42] and additionally has a function as a melanization hormone in locusts [40]. Thus it is possible that a range of hormonal peptides in insects have myostimulatory actions simply to increase circulation of hemolymph to promote their own spread through the hemocoel, as has been suggested for peptides evoking oviduct movements and writhing of Malpighian tubules [4,19]. In a screen for fur-

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ther biological activities of insect tachykinin-related peptides (TRPs) we have in the present paper investigated the cardioactive properties of a few TRPs. A number of TRPs have been isolated from locust, cockroach, blowfly and mosquito [see 28,39]. The insect TRPs are characterized by a carboxy terminus pentapeptide FX1GX2Ramide, where X1 and X2 are variable amino acid residues. The first bioactivity demonstrated for the insect TRPs was stimulation of contractions in locust oviduct and cockroach hindgut [39]. Later a variety of actions have been shown in vitro for various members of the TRP family: myostimulatory action on cockroach foregut [29], locust midgut [20] and Malpighian tubules [4], myomodulatory action on extensor tibia muscle of locust hindleg [7], neurostimulatory action on identified neurons in locust thoracic ganglia [22] and stimulation of release of adipokinetic hormone in locust corpora cardiaca [30,31]. The TRPs are distributed widely in interneurons of the central nervous system and in endocrine cells of the midgut of insects studied [see 28]. In some insects there are also efferent TRP-containing neurons innervating different parts of the intestine [26,29] suggesting a direct peptidergic regulation of certain target muscles (close range signalling). A hormonal control of some targets by TRPs also seems possible. For instance the muscle of cockroach and locust oviducts and Malpighian tubules respond to TRPs, but are not innervated by TRP-immunoreactive axons [4,19,28]. The source of hormonal TRPs is not known. In most insects, studied so far, no TRP-immunoreactive neurosecretory cells have been found in the typical neurosecretory systems. Instead it has been assumed that TRPs could be released from the endocrine cells of the midgut, but evidence is lacking. An exception is the moth Spodoptera litura, however, where cells of the median neurosecretory cell group with axon terminals in the corpora cardiaca and anterior aorta were found TRPimmunoreactive [14]. Here we have investigated the actions of three insect TRPs on the heart of two coleopteran insects Tenebrio molitor and Zophobas atratus. As a comparison we tested the action of proctolin (RYLPT) known to be a strong cardioaccelerator in insects [6,17,35]. These beetles have been used extensively in studies of actions of different insect neuropeptides [11,15–18,35,36]. We tested the cardiotropic activity of LemTRP-4 (APSGFMGMRamide) isolated from the midgut of the cockroach Leucophaea maderae [25] as well as CavTK-I (APTAFYGVRamide) and CavTK-II (GLGNNAFVGVRamide) isolated from the blowfly Calliphora vomitoria [24]. To determine the possible source of TRPs acting on the heart we performed immunocytochemistry on several tissues with antisera to locust TRPs (LomTK-I and II), known to label neurons in the brain of T. molitor [44]. Since the hearts of the two beetle species appear not to be directly innervated by TRP immunoreactive axons it is likely that TRP action is mediated by circulating peptide. Two possible sources of circulating TRPs have been identified in the

beetles, neurosecretory cells with axons to the corpora cardiaca storage lobe and endocrine cells in the midgut.

2. Method 2.1. Insects Studies were carried out on adults of two beetle species, Tenebrio molitor L. and Zophobas atratus Fabr. which were maintained in laboratory cultures (Department of Animal Physiology, A. Mickiewicz University, Poznan, Poland). T. molitor was reared as described previously [37] and Z. atratus was reared according to the procedure described by Quennedey et al. [33]. 2.2. Peptides Proctolin was purchased from Peninsula Laboratories (Belmont, CA, USA). LemTRP-4, [see 25] was synthesized with Fmoc chemistry and purified at the Department of Medical Biochemistry and Microbiology at the Biomedical Center of Uppsala University, Sweden by Dr. Å. Engstro¨m. CavTK-I and CavTK-II [see 24] were customs synthesized by Ferring-Euro-Diagnostica, Malmo¨, Sweden). 2.3. Heart bioassay Peptides were bioassayed in vitro in a semi-isolated heart preparation according to Rosinski and Ga¨de [36]. In brief, insects were decapitated and the abdomen was removed as close to the metathorax as possible. The ventral body wall of the abdomen was trimmed away so that lateral spiracular structures remained attached to the dorsal sclerites. The fat body, digestive organs, and Malpighian tubules were removed from the abdominal dorsum. The final preparation consisted of the dorsal vessel (i.e. the heart), alary muscles, internal body muscles, the tracheae, and the dorsal cuticle. The heart preparations were selected on the basis of frequency and regularity of beating and then they were superfused in Tenebrio saline (274 mM NaCl, 19 mM KCl, 9 mM CaCl2, 5 mM glucose, and 5 mM HEPES, pH 7.0) for stabilization (during 20 min.). An open perfusion system was used, with an injection port (for peptides) 70 mm above the superfusion chamber. The heart was subjected to a constant perfusion with fresh saline at the rate of about 140 ␮l/min. Thus the injected peptide would reach the heart after about 15–20 seconds (this was tested with injections of methylene blue). During bioassay, saline flowed directly from the open point to the perfusion chamber onto the caudal portion of the heart. The saline flowed through the length of the heart and was removed by suction with a Whatman paper at the cephalic end of the preparation. The activity of the heart could be observed through the transparent cuticle and was recorded automatically using a Microdensitometer MD-100 (Carl Zeiss, Germany). All tested

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samples were applied at the injection port with a Hamilton syringe. Many pulse applications of samples could be sequentially assayed in a single preparation. The open system was designed to enable samples to be added without causing a change in pressure. All assays were performed at room temperature. Typically the control frequency and amplitude in saline was monitored for one minute, followed by peptide application and after 20 sec measurement of heart frequency and amplitude for one minute. The activities of tested peptides are presented as percentage changes in the control frequency and amplitude of the heart contractions. The Wilcoxon single rank test was used for statistical evaluation of experimental data. Dose response graphs were made in Harvard Graphics 2.1 (Software Publishing Corp.).

shown in Figs. 1A and B. The recordings show that the frequency and amplitude effects interact. The increase in T. molitor and Z. atratus heartbeat frequencies were observed in similar concentration ranges of peptide (concentrations of 10⫺11–10⫺7 M were tested), although the T. molitor heart appeared slightly more sensitive to proctolin: the concentration yielding the half-maximal response (EC50) was about 3 ⫻ 10⫺10 M in T. molitor and about 10⫺9 M in Z. atratus. In both insects it could be seen that when the amplitude is reduced after peptide application, the heart action is shifted to the systolic phase in the cycle (Figs. 1A, B).

2.4. Immunocytochemistry

Dose response tests on the hearts of the two insects were made with the TRPs LemTRP-4, Cav-TK-I and CavTK-II (applied at 10⫺11 to 10⫺6 M). These TRPs were chosen in order to test peptides with some differences in their sequences (see introduction) since we do not know the structure of the endogeneous beetle TRPs. All three peptides evoked purely cardioexcitatory responses in T. molitor and in Z. atratus. In the two insects the TRPs are less potent cardiostimulators than proctolin and evoke smaller maximal chronotropic responses. Typical responses to LemTRP-4 in the heart of the two insects are shown in Figs. 1C and D. In T. molitor LemTRP-4 induced an increase in the frequency of the heart beat (Fig. 1C) with a threshold for observable effect at about 5 ⫻ 10⫺10 M. The maximal increase in the response was about 20% above the control frequency and observed at about 5 ⫻ 10⫺8 M (Fig. 2A). The EC50 value is about 10⫺9 M. LemTRP-4 does not have any effect on the amplitude of the contractions in the concentration range 10⫺11 to 10⫺6 M (P ⬎ 0.05; Fig. 3A). The response to LemTRP-4 was more complex in Z. atratus. In this beetle LemTRP-4 evoked a cardioacceleratory effect at lower concentrations (up to 5 ⫻ 10⫺8 M), but at higher concentrations exhibited less prominent stimulatory effects (Fig. 2B). In the low concentration range (10⫺9– 10⫺8 M) cardioexcitation was manifested primarily as an increase in frequency of the heart beat (Fig. 1D, 2B), but also in a significant increase of the amplitude of contractions (P ⬍ 0.05; Fig. 3B). At higher concentrations LemTRP-4 application gave an immediate decrease in the frequency and amplitude of the contractions and the actions of the heart became diastolic. This response was followed by a period of increased frequency lasting as long as the peptide remained in the superfusate (not shown). The degree of cardioinhibition was stronger when increasing the LemTRP-4 concentration to 10⫺6 M. The blowfly peptides Cav-TK-I and CavTK-II exerted significant cardiostimulatory effects only in Z. atratus. In T. molitor both CavTKs were weakly cardiostimulatory (chronotropic action) in the concentration range tested (10⫺11– 10⫺6 M). The peptide-induced responses were, however, not found significantly higher than the saline controls (P ⬎

Standard immunocytochemistry technique was employed to localize TRP-like material in tissues of the two beetles. We used brains, chains of ventral nerve cords, hearts and intestines from adults of both species. Tissues were fixed for 4 h in 4% paraformaldehyde in 0.1 M sodium phosphate buffer. Tissues were either sectioned on a cryostat or used for wholemounts. The antisera used were: antiLomTK-I [23,27] (diluted 1:1000) and anti-LomTK-II (kind gift of Dr. H.J. Agricola, Jena, Germany; [see 1,43]) (diluted 1:4000); both were raised in rabbit. The peroxidaseantiperoxidase technique was utilized for detection of the primary antisera as described by Muren et al. [26] and Na¨ssel et al. [29]. The LomTK-II antiserum produced the most prominent immunolabeling and was used for all immunocytochemistry described in this paper. This antiserum is known to cross react with the other LomTKs and several other insect TRPs [28a,43,44]. As a control the LomTK-II antiserum was preabsorbed with 50 nmol LomTK-I to 1 ml diluted antiserum (over night at 4° C). This preabsorbed antiserum was applied to brain sections and whole ganglion chains (as described above). The immunocytochemical preparations were analyzed on a Zeiss Axioplan II microscope equipped with interference contrast optics. Images were captured with an integrated chilled color CCD camera (Hamamatsu, Hamamatsu City, Japan). The images were edited in Adobe Photoshop 4.0.

3. Results 3.1. Effects of proctolin on heart beat The semi-isolated heart preparations from T. molitor and Z. atratus were stimulated strongly by the pentapeptide proctolin. The peptide caused an increase in heartbeat frequency (a positive chronotropic effect; Figs. 1A, B, 2) and decrease in amplitude of contractions (a negative inotropic effect; Figs. 1A, B, 3); frequency and amplitude are monitored simultaneously. Examples of such responses are

3.2. Effects of tachykinin-related peptides on heart beat

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Fig. 1. Myograms displaying typical responses on the spontaneous activity of heart muscle to proctolin and LemTRP-4. Peptide application is indicated by arrow. A. Response of the heart of T. molitor to 0.1 nM proctolin. Note that both frequency and amplitude of contractions are affected (positive chronotropic and negative inotropic effects). B. The effect of 10⫺9 M proctolin on the heart of Z. atratus. Also evident are strong chronotropic and inotropic effects. C. The response of the T. molitor heart to 10⫺9 M LemTRP-4 is mainly chronotropic. D. Similarily the Z. atratus heart responds to 10⫺9 M LemTRP-4 by an increase in heart beat frequency, but also by an increased amplitude of contractions.

0.05) even at concentrations up to 10⫺6 M (Fig. 4A). The CavTKs also had no inotropic effect in T. molitor (Fig. 5A). In Z. atratus both CavTKs are cardiostimulatory producing significant chronotropic effects at concentrations of about 10⫺8–10⫺6 M (Fig. 4B). CavTK-II was found slightly more potent as a cardiostimulator than CavTK-I, but induced a slightly smaller maximal response (Fig. 4B). Of the CavTKs only CavTK-II increased the amplitude of the spontaneous heart contractions in Z. atratus (P ⬍ 0.05; Fig. 5B). 3.3. Immunocytochemical localization of TRPs We used well characterized antisera raised against two of the TRPs of the locust Locusta migratoria (LomTK-I and II) for immunocytochemical detection of TRPs in the two

beetle species. The most intense and distinct immunolabeling was obtained with the antiserum to LomTK-II (also used in an earlier study of T. molitor; [44]) and the results shown here were obtained with this antiserum. As a control this antiserum was preabsorbed with LomTK-I which has a C-terminus heptapeptide that is identical to LomTK-II. No immunolabeling was obtained with the preabsorbed antiserum on cryostat sections of the brain or on whole ganglion chains or intestines. To screen for possible sources of TRPs acting on the heart we applied the LomTK antisera to cryostat sections of brains and whole chains of ventral nerve cords, abdominal hearts and intestines of the two species. We found no innervation of the abdominal heart by LomTK-like immunoreactive (LTK-LI) neuron processes in

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Fig. 2. Chronotropic effects of peptides on the heart. Dose response curves for proctolin (䡩) and LemTRP-4 (●) applied to the hearts of T. molitor and Z. atratus. The percent increase in heart beat frequency was measured. Each point is the mean ⫾ SEM of 4 –17 measurements. Significant differences (*P ⬍ 0.05; **P ⬍ 0.01) from the controls are indicated by asterisks (Wilcoxon single rank test). In both insects proctolin is far more potent than LemTRP-4. A. T. molitor. B. Z. atratus. Note a decrease in frequency at a LemTRP-4 concentration of 10⫺7 M. For the higher concentrations only the cardiostimulatory response is shown.

Fig. 3. Inotropic effects of peptides on the heart. Dose response curves for proctolin (䡩) and LemTRP-4 (●) applied to the hearts of T. molitor (A) and Z. atratus (B). The percent increase (or decrease) in heart beat amplitude was measured. Each point is the mean ⫾ SEM of 4 –12 measurements. Significant differences (*P ⬍ 0.05; **P ⬍ 0.01) from the controls are indicated by asterisk (Wilcoxon single rank test). Proctolin has a strong negative inotropic effect in both species, whereas LemTRP-4 either has no significant effect on heart beat amplitude (T. molitor) or has a significant positive inotropic effect (Z. atratus).

the two species studied. In the thoracic and abdominal ganglia there were no immunoreactive neurosecretory cells or processes in neurohemal structures. Only in the brain we found LTK-LI neurosecretory cells. About eight cell bodies of the median neurosecretory cell group were labeled with the LomTK antisera (Figs. 6A, B). These neurons supply axons to the storage lobe of corpora cardiaca (Figs. 6C, D) suggesting that they are neurosecretory in nature and may release TRPs into the circulation. Another possible source of circulating TRPs is the midgut where we found LTK-LI endocrine cells scattered in the epithelium of both species (Figs. 6E, F) similar to all insects investigated previously [1,20,23,26,43]. No LTK-LI nerve fibers were seen associated with the intestine.

the three TRPs tested the most robust responses were obtained with the cockroach peptide LemTRP-4, but also the blowfly peptides CavTK-I and II gave reproducible results. The heart of Z. atratus was more sensitive to the TRPs, whereas the effect was less significant on that of T. molitor. In both beetles LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and the TRPs primarily evoke positive chronotropic effects. However, in Z. atratus LemTRP-4 and CavTK-II also induce a simultaneous increase in amplitude of contractions (positive inotropic effect). This is in contrast to proctolin, which induces a negative inotropic effect. These differences in the nature of the heart response to the tachykinins and proctolin suggest that the peptides regulate the myocardium by different mechanisms. Chronotropic and inotropic properties of the tachykinins tested are similar to the action of the neuropeptide CCAP, the so far only native cardioacceleratory peptide isolated and sequenced from T. molitor [8]. In T. molitor also other cardioactive factors were isolated from the corpora cardiacum-corpus allatum complex peptides but their structures have not yet been elucidated [36]. These factors

4. Discussion We have provided the first evidence that tachykininrelated peptides (TRPs) stimulate contractions in the insect heart by investigating two species of coleopteran insects. Of

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Fig. 4. Dose response curves for chronotropic effects of CavTK-I (●), CavTK-II (Œ) and proctolin (䡩) on the heart of the two beetles. Each point is the mean ⫾ SEM of 4 – 8 measurements. Significant differences (P ⬍ 0.05) from the controls are indicated by asterisk (Wilcoxon single rank test). A. In T. molitor there is no significant effect of CavTK-I and II at the concentrations tested here (in the range of 10⫺11–10⫺6 M). B. In Z. atratus, however, both CavTKs induce significant chronotropic responses in concentrations above 10⫺9 M.

increased heart beat frequency and decreased the amplitude of contractions. The T. molitor heart is excited by LemTRP-4 at concentrations of 10⫺9 ⫺ 5 ⫻ 10⫺6 M, whereas when applying LemTRP-4 to the heart of Z. atratus it is excited at lower concentrations and display a biphasic response at higher concentrations. Thus there seems to be a species-specific action of the TRPs which might depend on the sequence of the TRPs used. Possibly LemTRP-4 is structurally more related to a native TRP of Z. atratus (and thus a better agonist) than to peptides of T. molitor. It is also possible that the inhibitory action of LemTRP-4 seen in Z. atratus at higher concentrations is due to a desensitization of the TRP receptors. At present there is no information on the chemical structure of the native TRPs of the two beetles studied here or any other coleopteran insect. So far the only evidence for presence of TRPs in the nervous system and intestine of these insects is provided by immunocytochemistry using antisera to the locust TRPs LomTK-I and II [44; this study]. In the insects analyzed so far the TRPs have a fairly well

Fig. 5. Inotropic effects of CavTK-I (●), CavTK-II (Œ) and proctolin (䡩) on the heart of the two beetles. Each point is the mean ⫾ SEM of 4 – 6 measurements. Significant differences (P ⬍ 0.05) from the controls are indicated by asterisk (Wilcoxon single rank test). A. T. molitor. B. Z. Atratus. No significant changes in amplitude were detected in T. molitor and in Z. atratus only CavTK-II changed the amplitude. The effect was a stimulation (positive inotropic effect) in contrast to the negative effect of proctolin.

preserved structure, especially the carboxy terminus which appears to be the active core [28,39,45]. Even the crustacean TRPs isolated from a crab and a shrimp have a very similar sequence [2,32] and both insect and crustacean TRPs are bioactive in heterologous bioassays [see 28]. Thus it is likely that the native beetle TRPs are structurally related to the ones used here in the assays. Still slight differences in sequence may account for the differences in action of LemTRP-4 in the two species studied here, and may also explain why the CavTKs are less bioactive. Also other neuropeptides existing in multiple related isoforms are known to modulate contractile activity of hearts of insects in a differential way. The semi-isolated hearts of the locust Schistocerca gregaria [5,34] and the blowfly Calliphora vomitoria [6] respond to some isoforms of FMRFamiderelated peptides (FaRPs) and not others, or the nature of the response varies with isoform applied. The actions of FaRP isoforms on the S. gregaria heart can be excitatory, inhibitory or biphasic and in C. vomitoria some isoforms (encoded on the same gene) are inactive even at high concentrations [6,34]. The site of action of the TRPs on the beetle hearts is not

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Fig. 6. Localization of LomTK-like immunoreactive (LTK-LI) material in tissues of Z. atratus (anti-LomTK-II; peroxidase anti-peroxidase detection system). A and B. In frontal cryostat sections of the brain LTK-LI material is located in cell bodies (arrow) in the median neurosecretory cell (MNC) group of the protocerebrum. Two adjacent sections are shown. Also immunoreactive neuron processes in the pars intercerebralis can be seen below the cell bodies. C and D. Cryostat section of the corpora cardiaca with LTK-LI processes derived from the MNC neurons of the brain. In C the immunoreactive axons in one of the corpora cardiaca nerves (NCC 1) are seen at arrow entering the storage lobe (SL). E. Wholemount of the midgut seen from the outer surface. Immunoreactivity is seen in cells (arrows) with morphologies resembling endocrine cells. F. One of the LTK-LI endocrine-like cells seen in side view. Note the apical portion (arrow) with the nucleus) and a basal elongation projecting towards the gut lumen (to the left). Scale bars: A–E ⫽ 50 ␮m; F ⫽ 25 ␮m.

know, but it could be either on muscle fibers or pacemaker regions (if they exist). It is likely that the TRP action is hormonal since no TRP-immunoreactive neuronal processes were found in the segmental or lateral heart nerves or in the myocardium. The likely sources of hormonal TRPs are the endocrine cells of the midgut and/or the neurosecretory cells of the median neurosecretory cell group of the brain with terminals in corpus cardiacum, both of which display TRP immunoreactivity. It is yet to be demonstrated that TRPs are indeed released from these tissues in coleopteran insects. In L. migratoria release of LomTKs has been shown in vitro

from the midgut (Winther and Na¨ssel, in preparation), which is rewarding since also in this species there is evidence of LomTK actions on target muscle that is not innervated by LomTK-containing nerve fibers [4,7,19]. It has also been shown that the heart of pharate adults of the moth Manduca sexta respond to a few insect TRPs by cardioacceleration, although the heart is not innervated (Skaer NJV, Tublitz NJ, MacGraw HF, Na¨ssel DR, Maddrell SHP, unpublished data). A question is whether the action of TRPs on the heart of the two insects studied here is physiologically relevant. As

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mentioned earlier many hormonal peptides in insects display myotropic activity although the primary action may be different. Thus the TRPs may be released into the circulation and by increasing the heart beat frequency the further spread of the peptide is promoted. Alternatively the action on the heart may be linked to some other action requiring a higher rate of circulation. The insect TRPs display a variety of actions in assays in vitro [28]. Many of these appear to be functionally unrelated. For example in the locust L. migratoria LomTKs stimulate contractions in the oviduct and midgut, modulate contraction in extensor tibia muscle of the metathoracic leg, induce release of adipokinetic hormone from corpora cardiaca and activate neurons in thoracic and abdominal ganglia [7,19,20,22,30]. It is not clear whether all these actions are induced by LomTKs in vivo and if so under which conditions. It is likely that some of the above actions are not synchronized in vivo. Rather it is likely that independent neuronal systems release TRPs and thus serve distinct functions. Several of the neurostimulatory and myostimulatory actions of TRPs may be modulatory in nature and serve in fine tuning of responses to other neurotransmitters or neuropeptides. This would be supported by the rather weak and transient action of insect TRPs. Evidence for a neuromodulatory role of TRPs in combination with GABA is available from the crayfish visual system [13]. On the other hand some TRP actions may be coordinated: e. g. action on the different parts of the intestine and heart may serve to increase gut motility, release of digestive enzymes and circulation and thus play roles in food transport and digestion. The myostimulatory action on the locust oviducts has in fact been suggested to be another means to increase circulation in the abdominal body cavity [19]. So far we do not have data on TRP action on the locust heart and conversely we do not know whether in beetles the TRPs stimulate the additional targets identified in locust. In the cockroach L. maderae, however, we have shown cardioacceleratory action of LemTRPs additional to action on hindgut and oviduct [28; Rudwall, Sliwowska and Na¨ssel, in prep.]. Thus we can propose that TRPs may have a general role in increasing circulation by increasing the rate of spontaneous contractions in different muscle in the body cavity.

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Acknowledgment We thank Anne Karlsson for technical assistance, Aurelia Rudwall for help with immunocytochemistry and Dr. J. E. Muren for comments on the paper. The study was supported by the Swedish Natural Science Research Council (NFR) to D. R. Na¨ssel and an A. Mickiewicz University staff award to G. Rosinski. J. S. was the recipient of a travel grant from the Swedish Institute.

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