Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca

Regu tory ides ELSEVIER

Regulatory Pcptides 57 (1995)297-310

Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca Dick R. Nassel"~ *, Paul C.C.M. Passier b, Karoly Elekes c, Heinrich Dircksen d, Henk G.B. Vullings b, Rafael Cantera ~ ~DepartmeJTt q[' Zooh~go'. Stockhohn Unive~MO', Svante A rrhenius Wig 16, S- 1069 l Stockholm, Sweden b Department f?/'Eaperimetmd Zoology, Uzrecht UniversiO', Padualaan 8, NL-3584 CH Utrecht, The Netherlands "Department of E.xperimenta/ Zooh~g3 , Balaton Limnologk'al Research Institute o! the Hungarian Academy of Sciences, P.O. Box 35, H-8237 IThany, Hungao" d Departmem o[' Zooph3wioh)gv, Universio' Bomt. Emlenk'her A llee 11-13, D-53115 Bonn. Germany

Received 10 December 1994: revised version received 9 February 1995, accepted 10 February 1995

Abstract

The glandular cells oF the corpus cardiacum of the locust Locusta migratoria, known to synthesize and release adipokinetic hormones (AKH), are contacted by axons immunoreactive to an antiserum raised against the locust neuropeptide locustatachykinin I (LomTK I). Electron-microscopical immunocytochemistry reveals L o m T K immunoreactive axon terminals, containing granular vesicles, in close contact with the glandular cells cells. Release of A K H I from isolated corpora cardiaca of the locust has been monitored in an in vitro system where the amount of A K H I released into the incubation saline is determined by reversed phase high performance liquid chromatography with fluorometric detection. We could show that L o m T K I induces release of A K H from corpora cardiaca in a dose-dependent manner when tested in a range of 10-200/~M. This is thus the first clear demonstration of a substance inducing release of A K H , correlated with the presence of the substance in fibers innervating the AKH-synthesizing glandular cells, in the insect corpora cardiaca. K e y w o r d s . Locusta migratoria: Neuropeptide; Locustatachykinin; Adipokinetic hormone; Hormone release; Corpus car-

diacum

* Corresponding author. E-mail: [email protected]. Fax: + 46 8 167715. 0167-0115,'95,'$9.50 © 1995 Elsevier Science B.V. All rights reserved S S D I 0 1 6 7 - 0 1 1 5 ( 9 5 ) 0 0 0 4 3 -7

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1. Introduction

Three isoforms of adipokinetic hormone (AKH), designated AKH I - I l l , have been isolated from the locust Locusta migratoria [1,2]. The presence of AKH I and II in the glandular cells of the locust corpus cardiacum has been demonstrated with antisera raised against the two peptides [3-6] and by analysis of AKH precursor transcripts [6,7]. AKH is released into the hemolymph from the glandular cells during extended flight and induces lipid mobilization from the fat body, thus fueling flight metabolism [8-10]. The release of AKH from the glandular cells is affected by lipid levels of the hemolymph and is inhibited by injection of trehalose during flight, suggesting the existence of feedback mechanisms in regulation of circulating AKH levels [ 1,10-12 ]. Numerous studies have been undertaken to unravel the pathways by which AKH release is controlled. It has been demonstrated that the glandular lobes of the corpus cardiacum are supplied by fibers derived from neurons in the brain [5,13-18]. Several neuroactive compounds have been indicated in the corpus cardiacum of the locust: octopamine, dopamine and serotonin [15,19-21] and it has been suggested that the release of AKH is controlled by octopamine and cyclic AMP [22,23]. Octopamine immunoreactive fibers could, however, not be demonstrated in the glandular lobe nor in the storage lobe of the corpus cardiacum [24] and more recent studies failed to support the finding that octopamine induces AKH release [25,26]. Among other candidate factors for control of AKH release, as suggested from immunocytochemistry, are serotonin [21], peptides of the FMRFamide family [27,28] and the locustatachykinins [29]. The effects of serotonin and dopamine on AKH release have been described elsewhere [26] and the FMRFamide-related peptides, which appear to inhibit A K H release [28], will be dealt with in a separate communication. Here we focus on the possible role of locustatachykinins. Five isoforms oflocustatachykinins (LomTK l-V)

were isolated from brains and corpora cardiaca of L. migratoria [30-32]. The locustatachykinins were isolated as myotropic peptides, as determined in a cockroach hindgut contraction bioassay, but have also been shown to stimulate contractions of the locust foregut and oviduct in vitro [30-32]. Immunocytochemical mapping of LomTKs in the locust and the blowfly Calliphora vomitoria has furthermore suggested that these peptides may function as neurotransmitters or neuromodulators in interneurons of the central nervous system and as regulators in the midgut [29,33,34]. In the locust, L. rnigratoria, LomTK immunoreactive fibers were also detected in the glandular lobes of the corpus cardiacum [29] suggesting that it would be worthwhile to further investigate a possible role of LomTKs in regulation of AKH release. In the present paper we show by electronmicroscopical immunocytochemistry that LomTK immunoreactive fibers contact the AKH-producing glandular cells and that LomTK I induces release of AKH I in a dose-dependent manner. 2. Material and methods

2.1. Animals The African migratory locust Locusta migratoria was used in all experiments. For the immunocytochemistry we used adult male and female locusts from a colony kept at the Balaton Limnological Research Institute, Tihany, Hungary. For the release experiments locusts from a colony at the University of Utrecht were used (see details below). For conventional electron microscopy, locusts were obtained from a local breeder at Bergisch-Gladbach, Germany'.

2.2. Immunoo,tochemisto' (Lore TK )

of

locustatachykinin

For light-microscopical immunocytochemistry the insects were decapitated and the opened heads were

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Re~ulat~rv Peptides 57:1995) 297-310

immersed overnight at 4 ~C in a fixative consisting of 4°;, paraformaldehyde in 0.1 M sodium phosphate buffer. After fixation the corpora cardiaca-corpora allata complexes were dissected in buffer and processed for immunocytochemistry either as wholemounts or after cryostat sectioning. The rabbit antiserum to L o m T K I (code 9206-8) employed here, has previously been extensively tested for its specificity in enzyme linked immunosorbent assay (ELISA), immuno-dot blots on nitrocellulose membrane, and preabsorption tests on locust tissue [29,34,35 ]. This antiserum was used at a dilution of 1:2000 in a dilution buffer (DiB) consisting of 0.01 M phosphate-buffered saline (PBS), 0.5°Jo bovine serum albumin and 0.25°~, Triton X-100. The peroxidase anti-peroxidase (PAP) method was employed on cryostat sections and whole tissues as described previously, using unlabeled swine anti-rabbit immunoglobulins (DAKO, Copenhagen), rabbit PAP complex (DAKO) and diaminobenzidine (Sigma) as chromogen [29]. For electron-microscopical immunocytochemistry we followed a pre-embedding PAP protocol devised by Nassel and Elekes [36]. In short, the tissue was briefly fixed in 4°0 paraformaldehyde in 0.1 M sodium phosphate buffer and then transferred to a mixture of 4? o paraformaldehyde and 0.050 o glutaraldehyde in the same buffer for 12 h at 4°C. The immunocytochemistry was performed as described above on whole dissected corpora cardiaca, except that Triton X-100 was omitted from the washing steps. After the peroxidase reaction, the tissue was post-fixed for 1 h in l~,i, OsO 4 in 0.15 M sodium cacodylate buffer (pH 7.2), dehydrated and embedded in resin (Durcupan, Fluka). Serial sections were cut at 25 /xm on a sliding microtome. After microphotography, selected sections with immunolabeled fibers in the glandular lobe were embedded in Durcupan for subsequent ultrathin sectioning. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and then analyzed in a Tesla BS 500 electron microscope.

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2.3. Double labeling imrnunocytochemistrv To obtain details of the relations between L o m T K immunoreactive fibers and the glandular cells in the corpora cardiaca, we performed double labeling immunocytochemistry on cryostat sections with antisera to L o m T K I and A K H I. To detect A K H I we employed a rabbit antiserum raised against the N-terminus of locust A K H I (code 433) at a dilution of 1:1000 in DiB. This antiserum was generously donated by Prof. H. Schooneveld (Wageningen, The Netherlands). Also the specificity of this antiserum has been well characterized [4]. First, the L o m T K antiserum was employed for 48 h and detected as described above, using the PAP method with diaminobenzidine as a chromogen. After blockage with normal rabbit serum, we applied the A K H I antiserum for 48 h. As a second layer fluorescein isothiocyanate (FITC) conjugated swine anti-rabbit immunoglobulins (DAKO), was applied at 1:30 for 1 h. The distribution of the two immunoreactive peptides in the tissue sections was analyzed by alternating between FITC epifluorescence and regular light microscopy or by a combination of the two techniques using a Zeiss Axiophot microscope.

2.4. Con ventional electron microscopy The locusts used for conventional electron microscopy were kept in the laboratory at Bonn University at 3 0 o c under a 12:12 h light/dark regime and fed fresh grass or rolled oats and water. The brain-retrocerebra[ complexes were dissected adhering in toto to the dorsal aorta in ice-chilled saline [37] and immediately fixed according to a fixation procedure slightly modified after Dircksen et al. [ 38]. It consisted of a primary fixation in 2 °/jo paraformaldehyde, 2°o glutaraldehyde, 0.1 picric acid in 0.1 M sodium cacodylate buffer, pH 7.4, containing 5 mM CaC12, for 2-3 h at room temperature followed by extensive washes in the same buffer supplemented with 0.1 M sucrose. Postfixation was per-

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Fig. 1. LomTK-[I fibers in the glandular lobes of the locust corpus cardiacum. (a) Tracing of immunoreactive fibers in the glandular lobe from consecutive 25/lm sections (Durcopan embedded). The fibers enter via the NCC I1 nerves (arrow indicates the left nerve) from protocerebrum, Five axons were seen in each nerve. A. aorta. (b) Micrograph of one of the sections used for the tracing. Scale for a and b, 50/~m.

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w a s h e d in t h e c a c o d y l a t e - b u f f e r , s p e c i m e n s w e r e d e h y d r a t e d in a g r a d e d e t h a n o l s e r i e s a n d e m b e d d e d

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Fig. 2. Double labeling with antisera to LomTK I (PAP method) and AKH I (FITC fluorescence). (a) LomTK-LI fibers in the glandular lobes (GL). No immunoreactivity is seen in the storage lobe (SL). (b) The same section viewed in epifluorescence, AKH immunoreactive glandular cells are located in the same areas as the LomTK-LI fibers. This micrograph is from a slightly different focal plane than Fig. 3a and some other LomTK-LI fibers can be seen adjacent to the fluorescent cells (arrow). (c and d) A pair of micrographs of the same section showing the relationship between LomTK-LI fibers and AKH immunoreactive glandular cells. The arrow in (a) indicates LomTK-LI fibers in the NCC I1, Scale for a-c, 50 #m.

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grey colour ultrathin cross-sections of the c o r p o r a c a r d i a c a / a o r t a area were counterstained for 10 min with a uranylacetate/ethanol (1:1) mixture and for 3 min with lead citrate. They were viewed and phot o g r a p h e d in a Zeiss EM 109 electron m i c r o s c o p e operating at 50 kV. 2.5. Release o f A K H I in vitro F o r the release experiments adult male specimens o f L. migraloria were used, 14 days after their final moult (at this age the animals are able to fly). These animals had been reared under controlled conditions at a temperature o f 30 ° C, 40
The a m o u n t s o f A K H I released into the m e d i a of the first and second incubation were measured by reversed phase high performance liquid c h r o m a t o graphy ( R P - H P L C ) on a C18 column (Spherisorb 250, 4 mm, 5 um). The column was eluted with a rising gradient of acetonitrile at a flow rate of 0.90 ml/min. F l u o r e s c e n c e was detected with a spect r o p h o t o m e t r i c detector ( S h i m a d z u R F 1 0 A , excitation 276 nm, emission 340 nm). Peaks of A K H I were integrated with the use of P h a r m a c i a P e a k m a s ter software ( P h a r m a c i a L K B Biochrom Ltd.). The mean ratio between the a m o u n t s o f A K H I released during the second and the first incubation period was used as a p a r a m e t e r for the A K H release inducing capacity, of L o m T K I. The following final concentrations of L o m T K I were tested: 0, 10, 25, 50, 75, 100 and 200 # M . As a control the spontaneous release of A K H I was m e a s u r e d in the first and second incubation period by' incubation the tissues only with the ISB during both incubation periods. C o n c o m i t a n t with each test incubation, a control incubation was carried out. The experimental d a t a are given as mean values + S.E.M. of n experiments. The significance o f the observed differences between control and test values per concentration was established using an unpaired t-test.

3. Results 3.1. Immunoo'tochemistry and electron microscopy L i g h t - m i c r o s c o p i c a l i m m u n o c y t o c h e m i s t r y revealed locustatachykinin immunoreactive ( L o m T K LI) fibers invading the glandular lobes of the corpus

Fig. 3. Electron micrographs of LomTK-LI axon terminals in the glandular Iobc. (a) Two LomTK-LI axon profiles recognized by containing black peroxidase reaction product and granular vesicles. At the arrow the close association of one of the LomTK-LI axons and a adipokinetic cell (GC) can be seen. The adipokinetic cells contain larger electron-dense vesicles. Scale: 1.1 /lm. (b) Slightly higher magnification of another pair of LomTK-L1 axon profilcs. The granular vcsiclcs of one of them are indicated by small arrows. Onc of the LomTK-LI axons is closely apposed (large arrow) to a adipokinetic cell (GC). Two non-immunoreactive axon profiles, indicated by asterisks, contain granular vesicles similar to the ones in the LomTK-LI profiles. Scale: 0.6 btm.

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c a r d i a c u m via the N C C II nerves from the protocerebrum (Fig. 1). The varicose L o m T K - L I fibers were found to arborize extensively among the glandular cells of the glandular lobe. The immunoreactive fibers do not seem to invade the storage lobe of the corpus c a r d i a c u m (Fig. 2a). In 25 gin-thick sections of o s m i u m post-fixed tissue the varicose L o m T K - L I fibers could be seen contacting the glandular cells. To be sure of the identity of the cells in the glandular lobe that are c o n t a c t e d by the L o m T K - L I fibers, we performed double labeling i m m u n o c y tochemistry with antisera to L o c u s t a A K H I and L o m T K 1. It could be clearly shown that the glandular cells that are supplied by L o m T K - L I fibers are A K H i m m u n o r e a c t i v e (Fig. 2 a - d ) . In o r d e r to investigate the relationships between the L o m T K - L I fibers and the glandular cells in more detail we performed electron-microscopical immunocytochemistry. Analysis of r e - e m b e d d e d sections with L o m T K - L I fibers revealed that these fibers contain granular vesicles ( 1 5 0 - 2 0 0 nm) and it can be seen that they c o n t a c t the glandular cells (Figs. 3 and 4a). The glandular cells are characterized by their contents of large granular vesicles (around 350 nm) (see Refs. [5,17]). Due to the rather p o o r tissue preservation caused by the p r e e m b e d d i n g immunocytochemical technique, we could not resolve synaptic or synaptoid m e m b r a n e specializations. A m o n g the L o m T K - L I axon profiles and the glandular cells there are also some n o n - i m m u n o r e a c t i v e neuronal terminals which contain granular vesicles (Fig. 3b), similar to those in the L o m T K - L I terminals. Also these terminals can be seen contacting thc glandular cells.

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Electron m i c r o s c o p y of conventionally fixed tissue was performed in o r d e r to obtain a better resolution o f m e m b r a n e contacts between fibers o f the typc reacting with L o m T K antiserum a n d the glandular cells (Fig. 4b,c). It was possible to reveal synaptoid c o n t a c t s between terminals with granular vesicles (150-200 nm) and the glandular cells (Fig. 4c). These specialized a x o - s o m a t i c contacts are characterized by close m e m b r a n e contacts and often pre- and p o s t s y n a p t i c densities. O c c a s i o n a l l y clusters of small clear vesicles (Fig. 4c) and exocytosis figures (not shown) can be seen on the presynaptic side. 3.2. Release experiments

Release of A K H I from the c o r p o r a c a r d i a c a in vitro, as determined by H P L C with fluorometric detection, could be induced by L o m T K I applied in a c o n c e n t r a t i o n range o f 5 0 - 2 0 0 # M . A typical H P L C c h r o m a t o g r a m with identification of released A K H I is shown in Fig. 5. The absolute amounts of A K H I released s p o n t a n e o u s l y and during stimulation with L o m T K I were in this particular experiment approx. 6 pmol (first incubation) and 14 pmol (second incubation) per pool o f 5 c o r p o r a cardiaca. The a m o u n t released in the second incubation (about 3 pmol per corpus c a r d i a c u m ) matches very well the physiological level which induces lipid mobilization [40]. It a p p e a r e d that the amounts o f A K H I released by different pools o f c o r p o r a c a r d i a c a varied somewhat. The ratio between the amounts o f A K H I released during the two consecutive incubation periods, however, remained fairly constant. This ratio, therefore,

Fig. 4. (a) Electron microscopical immunocytochemistry reveals that a LomTK-LI terminal with granular vesicles contact an adipokinetic cell (asterisk), characterized by large vesicles. Due to the poor tissue preservation small clear vesicles are hard to resolve. Scale: 0.8 ,urn. (b and c) By conventional electron microscopy it is possible to identify axon terminals with granular vesicles similar to the LomTK-LI terminals. These terminals contact the adipokinetic cells characterized by large dense vesicles. In (b) a terminal with granular and clear vesicles contact an adipokinetic cell at small arrows. The larger arrow indicates a synaptoid contact with an aggregation of small clear vesicles at the presynaptic side. In (c) a synaptoid contact is seen between a putative peptidergic axon terminal (asterisk) and an adipokinetic cell. Synaptoid contacts are seen at arrows. Note aggregation of clear vesicles in the area around the asterisk. Scales: b = 0.5 /~m: c - 0.25 /xm.

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Fig. 5. H P L C chromatogram showing the response of a pool of 5 corpora cardiaca to 75 ktM locustatachykinin I (LomTK I). The response is release of A K H l (with known retention time in the system used) as measured by spcctrophotometry (excitation 276 nm, emission 340 nm). Control: first incubation of 30 rain in insect saline buffer (ISB). Test: same corpora cardiaca are incubated in ISB containing 75 # M L o m T K I. Gradient: percentage of solvent B (90"o acetonitrile) during chromatography. AKH: adipokinetic hormone I.

was used as a parameter for the potential effect of L o m T K I on A K H release. L o m T K I applied in concentrations below 50 ~M gave no significant release as compared with controls in which only insect saline buffer was used for incubation. As shown in the dose response curve of Fig. 6 the amount of A K H I released was dependent on the dose of L o m T K I applied in vitro. 4. Discussion

4.1. The search,~r a releasing.~lctor ~f AKH A long quest for the factor(s) responsible for release of the metabolically important A K H s from the

Fig. 6. Dose response curve for A K H l release in vitro for L o m T K I. Each point represents the means_+ S.E.M. for the number of experiments in parenthesis. Since the number of control experiments is much higher (n = 27) their S.E.M. value is much smaller.

corpora cardiaca of insects has until now been unsuccessful in providing an unambiguous candidate substance. Octopamine (OA) and cyclic A M P have been shown to induce release of A K H from isolated corpora cardiaca of Locusta migratoria [22,23]. Moreover, OA has been demonstrated biochemically in the glandular lobe lobes of the corpus cardiacum of L. Migratoria [19,20]. More recent experiments, however, have failed to support the action of OA in A K H release [25,26]. Also the lack of OA immunoreactive fibers in the locust corpora cardiaca [24] is an argument against OA being a releasing factor. The absence of OA in the corpora cardiaca has been confirmed by chromatographical separation of homogenized corpora cardiaca with RPH P L C followed by electrochemical detection [26]. The latter authors thus conclude that OA is not the neuroactive compound that initiates the release of A K H . Other substances that may be implicated in the regulation of A K H are serotonin and dopamine. Both substances have been demonstrated immunocytochemically in the storage lobe of the corpus

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cardiacum and not in the glandular lobes [21,41] and this was confirmed by R P - H P L C followed by electrochemical detection [26]. It appears that these amines do not initiate A K H release, but only have a modulatory effect. In the present investigation, however, we have shown that locustatachykinins, or at least L o m T K I, may be involved in the regulation of A K H release. Two lines of evidence have been presented here. The first is the immunocytochemical demonstration of L o m T K - L I fibers directly contacting the glandular cells in the corpora cardiaca. Secondly, it was possible to induce release of A K H I from isolated corpora cardiaca in a dose-dependent manner by addition of L o m T K I to the incubation medium.

4.2. Immunoo, tochemistrv indicates control of AKH release by peptidergic neurons Locustatachykinins I - V were isolated from dissected brains and corpora cardiaca-corpora allata complexes of Locusta migratoria, the species used in the present investigation, and have been shown to have stimulatory actions on locust visceral and skeletal muscle in vitro [30-32]. The antiserum raised to L o m T K I has been shown to cross react with the carboxy terminus of L o m T K I - I I I and to a lesser extent with that of L o m T K IV, but not with neuropeptides unrelated to L o m T K s [29,34]. The cross-reactivity with L o m T K V has not yet been tested, but this peptide which has a primary structure very similar to L o m T K I [32], will probably also be recognized by the antiserum. Consequently, the antiserum employed here is capable of labeling L o m T K I - V in tissue sections, but not any of the known unrelated peptides of the locust. In the locust brain it has been shown that the L o m T K antiserum labels a large number of interneurons and some cells in the lateral neurosecretory cell group and fibers were detected in the glandular lobe of corpora cardiaca [29]. When discovering that the L o m T K - L I fibers surround the glandular cells in the corpora cardiaca, the possibility surfaced that the

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L o m T K s may be involved in control of release or synthesis of A K H s . Electron microscopy confirmed the close association between L o m T K - L I fiber terminals and the glandular cells. The characteristic large granular vesicles of the latter cells have been shown by electron-microscopical immunocytochemistry to contain colocalized A K H I and A K H II [5]. There are also axon terminals with granular vesicles that do not display L o m T K imnmnoreactivity contacting the glandular cells, indicating the presence of a second substance involved in control of release. In fact an antiserum to F M R F a m i d e was shown to label such processes and FMRFamide-related peptides applied to corpora cardiaca in vitro inhibited cyclic A M P induced release of A K H I [28]. The cell bodies that give rise to the L o m T K - L I fibers innervating the glandular cells have not been identified, but the immunoreactive axons reach the corpora cardiaca by route of the second set of corpora cardiaca nerves termed N C C II. Small L o m T K - L I cell bodies can be seen in the region of the lateral neurosecretory cell group (LNC) in the protocerebrum, but due to the very extensive immunolabeling of neuronal processes it is not possible to identify the specific neurons sending axons to the N C C II [29]. From tracing experiments [14,18,42] the conclusion can be drawn that in Locusta axons from the brain reach the glandular cells only via the N C C II. The N C C II contains fibers from three separate cell clusters [24], but only one of these contains the cell bodies of the approx. 15 neurons which invade the glandular lobe of the corpus cardiacum [14]. In another study the presence of FMRFamide-immunoreactive neurons in the L N C was reported [28]. It is possible that the fibers of the latter neurons represent the non-immunoreactive fibers seen in the glandular lobe in the present study. The identity of the LomTK- and F M R F a m i d e - L I cell bodies supplying the fibers to the corpus cardiacum will be revealed by backfilling the NCC II and then applying antisera against the two peptides for immunocytochemistry.

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4.3. LomTK I induces release of A K H in vitro

L o m T K I induces release of A K H I in vitro from the locust corpora cardiaca in a dose-dependent manner when applied in the concentration range of 50-200 gM. The high concentrations of L o m T K I needed to obtain release of A K H may be explained by the use of intact corpora cardiaca causing a low accessibility of the receptors for this peptide. Additionally, it is known that under in vivo conditions the concentration of a neurotransmitter in the synaptic cleft after presynaptic activity builds up very rapidly to concentrations in the millimolar range [43 ]. Since A K H I and II are colocalized in glandular cells in a ratio of about 6:1 [44], the amount of A K H II released is about one sixth of the A K H I release measured here. Preliminary experiments have shown that A K H III is not released by the same mechanism as A K H I and I! (Passier and Vullings, unpublished data). The mechanisms by which L o m T K induces release of A K H are not known at present. It has been shown that cyclic A M P can induce A K H release [22,23]. Preliminary studies of the action of L o m T K related peptides callitachykinins I and II isolated from the blowfly Calliphora vomitoria [35], have indicated that these peptides activate adenylate cyclase in blowfly brain homogenates when applied in concentrations in the range of 10 ~-10 11 M [45]. It would thus be important to investigate the action of L o m T K s on the production of c A M P in the locust corpus cardiacum. The functional implications of the presence of several isoforms of locustatachykinins are not understood. In the stimulation on visceral muscle contraction all isoforms are active [30-32], but there are no data from other target cells to reveal whether the different isoforms have differential actions. In the present investigation we only tested the action of L o m T K I on A K H release. It would thus be of great interest to test the other four isopeptides to determine whether there are differences in their potency to induce A K H release. Finally, the possible antago-

nistic actions of FMRFamide-like peptides and L o m T K s in the regulation of A K H release are important to investigate further and so is the question as to whether any of the monoamines serotonin, dopamine and octopamine have roles to play in this system.

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