Vertebrate Tachykinins Research Articles

Functional homology of tachykinin signalling: The influence of human substance P on the immune system of the mealworm beetle, Tenebrio molitor L.

Tachykinin-related peptides (TRPs) are one of the most prominent families of neuropeptides in the animal kingdom. Insect TRPs display strong structural and functional homology to vertebrate tachykinins (TKs). To study functional homologies between these two neuropeptide families, the influence of human substance P (SP, one of the essential vertebrate TKs) on the immune system of the mealworm beetle, Tenebrio molitor L., was analysed.Human SP influences the phagocytic abilities of T. molitor haemocytes. Peptide injection leads to an increase in the number of haemocytes participating in the phagocytosis of latex beads. In contrast, incubation of haemocytes from non-injected beetles in a solution of physiological saline and SP causes a decrease in phagocytic activity. Treatment with human SP also led to increased adhesion of haemocytes, but no changes in the arrangement of the F-actin cytoskeleton were observed. Interestingly, 6 h after human SP injection, increased DNA integrity in T. molitor haemocytes was reported. The opposite effects were observed 24 h after SP injection.Human SP caused the upregulation of humoral immune responses, such as phenoloxidase (PO) activity in the T. molitor haemolymph, and the downregulation of immune-related genes encoding coleoptericin A, tenecin 3 and Toll receptor. However, genes encoding attacin 2 and cecropin were upregulated. Despite these differences, the antimicrobial activity of T. molitor haemolymph was significantly lower in beetles injected with SP than in control beetles. Moreover, an analysis of the direct influence of SP on lysozyme activity was performed. Our results suggest that SP at a concentration of 10−6 M can directly inhibit lysozyme activity. However, an opposite effect was reported after the application of SP at a concentration of 10−4 M.The presented results suggest structural and functional homology between TK signalling in vertebrates and insects. Primarily, this was visible in the context of the humoral response and general antimicrobial activity of T. molitor haemolymph. However, some of the results related to haemocyte function may also indicate the importance of the TK and TRP sequences for evoking immunological effects.

Intrinsic sources of tachykinin-related peptide in the thoracic ganglion mass of the crab, Cancer borealis

Neuropeptides comprise the largest class of neural and neuroendocrine signaling molecules. Vertebrate tachykinins (TKs) and the structurally-related invertebrate tachykinin-related peptides (TRPs) together form the largest neuropeptide superfamily, with a number of conserved neural and neuroendocrine functions across species. Arthropods, including crustaceans, have provided many insights into neuropeptide signaling and function. Crustacean tachykinin-related peptide occurs in endocrine organs and cells and in two of the major crustacean CNS components, the supraoesophageal ganglion (“brain”) and the stomatogastric nervous system. However, little is known about TRP sources in the remaining major CNS component, the thoracic ganglion mass (TGM). To gain further insight into the function of this peptide, we aimed to identify intrinsic TRP sources in the TGM of the Jonah crab, Cancer borealis. We first adapted a clearing protocol to improve TRP immunoreactivity specifically in the TGM, which is a dense, fused mass of multiple ganglia in short-bodied crustaceans such as Cancer species of crabs. We verified that the clearing protocol avoided distortion of cell body morphology yet increased visibility of TRP immunoreactivity. Using confocal microscopy, we found TRP-immunoreactive (TRP-IR) axon tracts running the length of the TGM, TRP-IR neuropil in all ganglia, and approximately 110 TRP-IR somata distributed throughout the TGM, within and between ganglia. These somata likely represent both neural and neuroendocrine sources of TRP. Thus, there are many potential intrinsic sources of TRP in the TGM that are positioned to regulate behaviors such as food intake, locomotion, respiration, and reproduction.

Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms

It has been previously shown that octopus venoms contain novel tachykinin peptides that despite being isolated from an invertebrate, contain the motifs characteristic of vertebrate tachykinin peptides rather than being more like conventional invertebrate tachykinin peptides. Therefore, in this study we examined the effect of three variants of octopus venom tachykinin peptides on invertebrate and vertebrate tissues. While there were differential potencies between the three peptides, their relative effects were uniquely consistent between invertebrate and vertebrae tissue assays. The most potent form (OCT-TK-III) was not only the most anionically charged but also was the most structurally stable. These results not only reveal that the interaction of tachykinin peptides is more complex than previous structure–function theories envisioned, but also reinforce the fundamental premise that animal venoms are rich resources of novel bioactive molecules, which are useful investigational ligands and some of which may be useful as lead compounds for drug design and development.

The Leucokinin Pathway and Its Neurons Regulate Meal Size in Drosophila

Total food intake is a function of meal size and meal frequency, and adjustments to these parameters allow animals to maintain a stable energy balance in changing environmental conditions. The physiological mechanisms that regulate meal size have been studied in blowflies but have not been previously examined in Drosophila. Here we show that mutations in the leucokinin neuropeptide (leuc) and leucokinin receptor (lkr) genes cause phenotypes in which Drosophila adults have an increase in meal size and a compensatory reduction in meal frequency. Because mutant flies take larger but fewer meals, their caloric intake is the same as that of wild-type flies. The expression patterns of the leuc and lkr genes identify small groups of brain neurons that regulate this behavior. Leuc-containing presynaptic terminals are found close to Lkr neurons in the brain and ventral ganglia, suggesting that they deliver Leuc peptide to these neurons. Lkr neurons innervate the foregut. Flies in which Leuc or Lkr neurons are ablated have defects identical to those of leucokinin pathway mutants. Our data suggest that the increase in meal size in leuc and lkr mutants is due to a meal termination defect, perhaps arising from impaired communication of gut distension signals to the brain. Leucokinin and the leucokinin receptor are homologous to vertebrate tachykinin and its receptor, and injection of tachykinins reduces food consumption. Our results suggest that the roles of the tachykinin system in regulating food intake might be evolutionarily conserved between insects and vertebrates.

Differential mRNA splicing and precursor processing of neurokinin B in neuroendocrine tissues

The tachykinin neurokinin B which is encoded on the tachykinin 3 precursor, has prominent roles in both neuronal and endocrine systems, yet little is known about its evolution, potential splice variants and the manner in which it is processed. Here, we deduce the diversity within the vertebrate tachykinin 3 precursors, and identify novel tachykinin 3 splice variants and precursors. A total of 35 different tachykinin 3 precursors were identified in mammals, birds and reptiles. Nine additional alternatively spliced tachykinin 3 mRNA transcripts were also discovered in humans leading to the formation of three tachykinin 3 precursors (named α, β and γ tachykinin 3), but no novel tachykinin. γ tachykinin 3, albeit rarer, was not found to encode neurokinin B. Differential processing of the tachykinin 3 precursor in the human placenta leads to the formation of potential NH 2-terminally extended forms of neurokinin B. Moreover, we found increased proteolytic cleavage of the tachykinin 3 precursor during the pregnancy syndrome of pre-eclampsia. We have established neurokinin B to be an evolutionarily conserved peptide, nonetheless the significance of the three different tachykinin 3 precursors is not clear, but could represent an evolutionarily redundant splicing mechanism once employed by an ancestral gene that encoded two tachykinins. Our results indicate that differential mRNA splicing and precursor processing is likely to play an important role in differentiating the actions of the tachykinin 3 gene products in both neuronal and endocrine tissues.

Characterization and distribution of NKD, a receptor for Drosophila tachykinin-related peptide 6

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila and are referred to as drosotachykinins, or DTKs. Two Drosophila G protein-coupled receptors, designated NKD (neurokinin receptor from Drosophila; CG6515) and DTKR ( Drosophila tachykinin receptor; CG7887), display sequence similarities to mammalian tachykinin receptors. Whereas DTKR was shown to be activated by DTKs [Birse RT, Johnson EC, Taghert PH, Nässel DR. Widely distributed Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptides. J Neurobiol 2006;66:33–46; Poels J, Verlinden H, Fichna J, Van Loy T, Franssens V, Studzian K, et al. Functional comparison of two evolutionary conserved insect neurokinin-like receptors. Peptides 2007;28:103–8] and was localized by immunocytochemistry in Drosophila central nervous system (CNS), agonist-dependent activation and distribution of NKD have not yet been investigated in depth. In the present study, we have challenged NKD-expressing mammalian and insect cells with a library of Drosophila neuropeptides and discovered DTK-6 as a specific agonist that can induce a calcium response in these cells. In addition, we have produced antisera to sequences from NKD protein to analyze receptor distribution. We found that NKD is less abundantly distributed in the central nervous system than DTKR, and only NKD was found in the intestine. In fact, the two receptors are distributed in mutually exclusive patterns in the CNS. The combined distribution of the receptors in brain neuropils corresponds well with the distribution of DTKs. Most interestingly, NKD appears to be activated only by DTK-6, known to possess an Ala-substitution in an otherwise conserved C-terminal core motif. Our findings suggest that NKD and DTKR provide substrates for two functionally and spatially separated peptide signaling systems.

A Novel Biological Role of Tachykinins as an Up-Regulator of Oocyte Growth: Identification of an Evolutionary Origin of Tachykininergic Functions in the Ovary of the Ascidian, Ciona intestinalis

Tachykinins (TKs) and their receptors have been shown to be expressed in the mammalian ovary. However, the biological roles of ovarian TKs have yet to be verified. Ci-TK-I and Ci-TK-R, characterized from the protochordate (ascidian), Ciona intestinalis, are prototypes of vertebrate TKs and their receptors. In the present study, we show a novel biological function of TKs as an inducible factor for oocyte growth using C. intestinalis as a model organism. Immunostaining demonstrated the specific expression of Ci-TK-R in test cells residing in oocytes at the vitellogenic stage. DNA microarray and real-time PCR revealed that Ci-TK-I induced gene expression of several proteases, including cathepsin D, chymotrypsin, and carboxy-peptidase B1, in the ovary. The enzymatic activities of these proteases in the ovary were also shown to be enhanced by Ci-TK-I. Of particular significance is that the treatment of Ciona oocytes with Ci-TK-I resulted in progression of growth from the vitellogenic stage to the post-vitellogenic stage. The Ci-TK-I-induced oocyte growth was blocked by a TK antagonist or by protease inhibitors. These results led to the conclusion that Ci-TK-I enhances growth of the vitellogenic oocytes via up-regulation of gene expression and enzymatic activities of the proteases. This is the first clarification of the biological roles of TKs in the ovary and the underlying essential molecular mechanism. Furthermore, considering the phylogenetic position of ascidians as basal chordates, we suggest that the novel TK-regulated oocyte growth is an "evolutionary origin" of the tachykininergic functions in the ovary.

A novel biological role of tachykinins as an upregulator of oocyte growth: evolutionary origin of tachykinergic functions in the ovary

Tachykinins (TKs) and their receptors have been shown to be expressed in the mammalian ovary. However, the biological roles of ovarian TKs have yet to be verified. Ci‐TK‐I and Ci‐TK‐R, characterized from the protochordate (ascidian), Ciona intestinalis, are prototypes of vertebrate TKs and their receptors. We show a novel biological function of TKs as an inducible factor for oocyte growth using C. intestinalis as a model organism. Immunostaining demonstrated the specific expression of Ci‐TK‐R in test cells residing in oocytes at the vitellogenic stage. DNA microarray and real‐time PCR revealed that Ci‐TK‐I induced gene expression of several proteases including cathepsin D, chymotrypsin, and carboxypeptidase B1 in the ovary. The enzymatic activities of these proteases in the ovary were also shown to be enhanced by Ci‐TK‐I. Of particular significance is that the treatment of Ciona oocytes with Ci‐TK‐I resulted in progression of growth from the vitellogenic stage to the postvitellogenic stage. The Ci‐TK‐I‐induced oocyte growth was blocked by a TK antagonist or by protease inhibitors. These results led to the conclusion that Ci‐TK‐I enhances growth of the vitellogenic oocytes via upregulation of gene expression and enzymatic activities of the proteases. This is the first clarification of the biological roles of TKs in the ovary and the underlying essential molecular mechanism.

Identification and characterization of a tachykinin-containing neuroendocrine organ in the commissural ganglion of the crabCancer productus

A club-shaped, tachykinin-immunopositive structure first described nearly two decades ago in the commissural ganglion (CoG) of three species of decapod crustaceans has remained enigmatic, as its function is unknown. Here, we use a combination of anatomical, mass spectrometric and electrophysiological techniques to address this issue in the crab Cancer productus. Immunohistochemistry using an antibody to the vertebrate tachykinin substance P shows that a homologous site exists in each CoG of this crab. Confocal microscopy reveals that its structure and organization are similar to those of known neuroendocrine organs. Based on its location in the anterior medial quadrant of the CoG, we have named this structure the anterior commissural organ (ACO). Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry shows that the ACO contains the peptide APSGFLGMRamide, commonly known as Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). Using the same technique, we show that CabTRP Ia is also released into the hemolymph. As no tachykinin-like labeling is seen in any of the other known neuroendocrine sites of this species (i.e. the sinus gland, the pericardial organ and the anterior cardiac plexus), the ACO is a prime candidate to be the source of CabTRP Ia present in the circulatory system. Our electrophysiological studies indicate that one target of hemolymph-borne CabTRP Ia is the foregut musculature. Here, no direct CabTRP Ia innervation is present, yet several gastric mill and pyloric muscles are nonetheless modulated by hormonally relevant concentrations of the peptide. Collectively, our findings show that the C. productus ACO is a neuroendocrine organ providing hormonal CabTRP Ia modulation to the foregut musculature. Homologous structures in other decapods are hypothesized to function similarly.

Widely distributedDrosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptides

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G-protein-coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. We determined that HEK-293 cells transfected with DTKR displayed dose-dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR-green fluorescent protein (GFP) fusion constructs in HEK-293 cells. We generated specific antireceptor antisera and showed that DTKR is widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. Our findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand-receptor system plays multiple functional roles.

The distribution and activity of tachykinin-related peptides in the blood-feeding bug, Rhodnius prolixus

The invertebrate tachykinin-related peptides (TRPs) with the conserved C-terminal sequence FX 1GX 2Ramide shows sequence similarity to the vertebrate tachykinins after which they are named, and are hypothesized to be ancestrally related. In this study a polyclonal antiserum generated against a locust tachykinin (LomTK I), was used to demonstrate the presence and describe the distribution of LomTK-like immnoreactivity in the CNS and gut of Rhodnius prolixus. Reverse phase high performance liquid chromatography (RP-HPLC) was used in combination with a sensitive radioimmunoassay (RIA) to demonstrate picomolar amounts of immunoreactive material in the CNS, and femptomolar amounts associated with the hindgut. Furthermore, the results from CNS extracts separated by RP-HPLC, suggest that at least two tachykinin isoforms exist in R. prolixus. A hindgut contraction assay was developed to quantify the myotropic effects of selected LomTKs on R. prolixus hindgut contraction. Both LomTK I and II caused an increase in the frequency of hindgut contractions with EC 50 values of 3.6 × 10 −8 M and 3.8 × 10 −8 M for LomTK I and II, respectively.

Tachykinin and Tachykinin Receptor of an Ascidian, Ciona intestinalis: EVOLUTIONARY ORIGIN OF THE VERTEBRATE TACHYKININ FAMILY

Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian gamma-preprotachykinin A (gamma-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH(2) vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.

Identification of multiple urechistachykinin peptides, gene expression, pharmacological activity, and detection using mass spectrometric analyses

Urechistachykinin I and II (Uru-TK I and II) are invertebrate tachykinin-related peptides (TRPs), which have been isolated from echiuroid worms. The cDNA sequence encoding the Uru-TK I and II revealed that the precursor also encoded five TRP-like peptides. Here, we report the characterization of these Uru-TK-like peptides named as Uru-TK III-VII. Northern and Southern blot analyses demonstrated that Uru-TK mRNA is localized in nerve tissue. In addition, the presence of the Uru-TK-like peptides as matured forms in the nerve tissue was detected by mass spectrometric analysis, and identified these peptides were shown to exhibit a contractile activity on cockroach hindgut that was as potent as that of Uru-TK II. Furthermore, synthetic Uru-TK-like peptide analogs which contained Met-NH 2 instead of Arg-NH 2 at their C-termini were shown to possess a potential to bind to a mammalian tachykinin receptor, indicating that Uru-TK-like peptides are likely to correspond to vertebrate tachykinins, except for the difference at the C-terminal residue. These findings show that Uru-TK-like peptides are essentially equivalent to Uru-TK I and II, leading to the proposal that Uru-TK-like peptides play an essential role as invertebrate tachykinin neuropeptides.

Characterization of a receptor for insect tachykinin-like peptide agonists by functional expression in a stable Drosophila Schneider 2 cell line.

STKR is an insect G protein-coupled receptor, cloned from the stable fly Stomoxys calcitrans. It displays sequence similarity to vertebrate tachykinin [or neurokinin (NK)] receptors. Functional expression of the cloned STKR cDNA was obtained in cultured Drosophila melanogaster Schneider 2 (S2) cells. Insect tachykinin-like peptides or "insectatachykinins," such as Locusta tachykinin (Lom-TK) III, produced dose-dependent calcium responses in stably transfected S2-STKR cells. Vertebrate tachykinins (or neurokinins) did not evoke any effect at concentrations up to 10(-5) M, but an antagonist of mammalian neurokinin receptors, spantide II, inhibited the Lom-TK III-induced calcium response. Further analysis showed that the agonist-induced intracellular release of calcium ions was not affected by pretreatment of the cells with pertussis toxin. The calcium rise was blocked by the phospholipase C inhibitor U73122. In addition, Lom-TK III was shown to have a stimulatory effect on the accumulation of both inositol 1,4,5-trisphosphate and cyclic AMP. These are the same second messengers that are induced in mammalian neurokinin-dependent signaling processes.

Tachykinin-like peptides and their receptors. A review.

Tachykinin-like peptides have been identified in many vertebrate and invertebrate species. On the basis of the data reviewed in this paper, these peptides can be classified into two distinct subfamilies, which are recognized by their respective sequence characteristics. All known vertebrate tachykinins and a few invertebrate ones share a common C-terminal sequence motif, -FXGLMa. The insect tachykinins, which have a common -GFX1GX2Ra C-terminus, display about 30% of sequence homology with the first group. Tachykinins are multifunctional brain/gut peptides. In mammals and insects, various isoforms play an important neuromodulatory role in the central nervous system. They are involved in the processing of sensory information and in the control of motor activities. In addition, members of both subfamilies elicit stimulatory responses on a variety of visceral muscles. The receptors for mammalian and insect tachykinins show a high degree of sequence conservation and their functional characteristics are very similar. In both mammals and insects, angiotensin-converting enzyme (ACE) plays a prominent role in tachykinin peptide metabolism.

Distribution of tachykinin-related neuropeptide in the developing central nervous system of the moth spodoptera litura

Neuropeptides with similarities to vertebrate tachykinins, designated tachykinin-related peptides (TRPs), have been identified in several insect species. In this investigation we have utilized an antiserum raised to one of the locust TRPs, locustatachykinin-I (LomTK-I), to determine the distribution pattern of LomTK-like immunoreactive (LTKLI) neurons in the developing nervous system of the moth Spodoptera litura. A number of LTKLI neurons could be followed from the larval to the adult nervous system: a set of median neurosecretory cells (MNCs) in the brain, a pair of brain descending neurons and a few sets on neurons in the ventral nerve cord. The distribution of LTKLI neurons in the adult brain is very similar to that seen in other insect species with prominent arborizations in the central body, antennal lobes, mushroom body calyces, optic lobe neuropils and other distinct neuropil areas in the protocerebrum and tritocerebrum. A new finding is the presence of LTKLI neurosecretory cells with axon terminals in the anterior aorta and corpora cardiaca, suggesting for the first time a neurohormonal role of tachykinin-related peptide(s) in insects. During postembryonic development the number of LTKLI neurons in the ventral nerve cord decreases somewhat, whereas the number increases in the brain. Thus the functional roles of TRPs may change to some extent during development.

Peptidergic neurons in the snail Helix pomatia: distribution of neurons in the central and peripheral nervous systems that react with an antibody raised to the insect neuropeptide, leucokinin I.

In this study, an antiserum raised against an insect myotropic peptide, leucokinin I (DPAFNSWGamide), was used for mapping leucokinin-like immunoreactive (LK-LI) neurons in the gastropod mollusc, Helix pomatia. Immunocytochemistry performed on both whole-mounts and cryostat sections demonstrated LK-LI neurons in all ganglia of the central nervous system (CNS), except the visceral ganglion. Altogether about 700 immunolabelled neurons have been found, with nearly one-half (46%) in the cerebral ganglia. A large proportion of the LK-LI neurons have small cell bodies and are likely to be interneurons. The most prominent LK-LI cell group is represented by the entire neuron population of the mesocerebri, which is the major source of a thick fiber bundle system, encircling and innervating the whole CNS. One single LK-LI giant neuron was found, which is located in the left pedal ganglion and is termed GLPdLKC (giant left pedal leucokinin immunoreactive cell). This cell has not been identified previously. The ganglion neuropils are heavily innervated by varicose LK-LI fiber arborizations. Some integrative centers, such as the medullary neuropil of the procerebri, reveal an extreme density of LK-LI innervation. All major peripheral nerves contain a large number of LK-LI axons, and LK-LI innervation is found in the musculature of different peripheral organs (buccal mass, lip, tentacles, oviduct, intestine). Among the peripheral organs investigated, the intestine contains a rich varicose LK-LI network, composed of both intrinsic and extrinsic elements. Radioimmunoassay (RIA) demonstrates a very high content of LK-LI material in Helix ganglion extracts (about 50 pmol/CNS). This is the first report on the occurrence of a substance resembling the myotropic neuropeptide leucokinin I in a phylum outside arthropods. Based on our immunocytochemical observations, a role for leucokinin-like peptides in both central and peripheral regulatory processes in Helix is suggested. According to double-labelling experiments, only a small number of the LK-LI neurons are labelled with an antibody to the vertebrate tachykinin substance P.

Locustatachykinin immunoreactivity in the blowfly central nervous system and intestine.

An antiserum raised against locustatachykinin I, one of four myotropic peptides that have been isolated from the locust brain and corpora cardiaca, was characterized by enzyme-linked immunosorbent assay (ELISA) and used for immunocytochemical detection of neurons and endocrine cells in the nervous system and intestine of the blowfly Calliphora vomitoria. The ELISA characterization indicated that the antiserum recognizes the common C-terminus sequence of the locustatachykinins I-III. Hence, the cross reaction with locustatachykinin IV is less, and in competitive ELISAs no cross reaction was detected with a series of vertebrate tachykinins tested. It was also shown that the antiserum recognized material in extracts of blowfly heads, as measured in ELISA. In high-performance liquid chromatography the extracted locustatachykinin-like immunoreactive (LomTK-LI) material eluted in two different ranges. A fairly large number of LomTK-LI neurons was detected in the blowfly brain and thoracicoabdominal ganglion. A total of about 160 LomTK-LI neurons was seen in the proto-, deuto-, and tritocerebrum and subesophageal ganglion. Immunoreactive processes from these neurons could be traced in many neuropil regions of the brain: superior and dorsomedian protocerebrum, optic tubercle, fan-shaped body and ventral bodies of the central complex, all the glomeruli of the antennal lobes, and tritocerebral and subesophageal neuropil. No immunoreactivity was seen in the mushroom bodies or the optic lobes. In the fused thoracicoabdominal ganglion, 46 LomTK-LI neurons could be resolved. The less evolved larval nervous system was also investigated to obtain additional information on the morphology and projections of immunoreactive neurons. In neither the larval nor the adult nervous systems could we identify any efferent or afferent immunoreactive axons or neurosecretory cells. The widespread distribution of LomTK-LI material in interneurons suggests an important role of the native peptide(s) as a neurotransmitter or neuromodulator within the central nervous system. Additionally a regulatory function in the intestine is indicated by the presence of immunoreactivity in endocrine cells of the midgut.

Callitachykinin I and II, two novel myotropic peptides isolated from the blowfly, Calliphora vomitoria, that have resemblances to tachykinins

Two peptides, related to the locust myotropic peptides locustatachykinin I–IV, were isolated from the blowfly Calliphora vomitoria. Whole, frozen flies were used for extraction with acidified methanol. A cockroach hindgut muscle contraction bioassay was used for monitoring fractions during subsequent purification steps. A series of eight different high performance liquid chromatography column systems was required to obtain optically pure peptides. Two peptides were isolated and their sequences determined by Edman degradation and confirmed by mass spectrometry and chemical synthesis as APTAFYGVR-NH 2 and GLGNNAFVGVR-NH 2. They were named callitachykinin I and II. The peptides have sequence similarities to the locustatachykinins and vertebrate tachykinins. Both callitachykinins were recognized by an antiserum to locustatachykinin I in enzyme-linked immunosorbent assay (ELISA) tests and callitachykinin II was additionally recognized by an antiserum to the vertebrate tachykinin kassinin, suggesting that immunolabeling of blowfly neurons with these antisera is due to neuronal callitachykinins.

Tachykinin- and leucokinin-related peptides in the nervous system of the blowfly: Immunocytochemical and chromatographical diversity

We are interested in the presence and function in insects of neuropeptides related to the vertebrate tachykinins. Hence, we have used antisera raised against the tachykinins substance P and kassinin, and against the insect neuropeptide leucokinin I, for localization studies and immunochemical analysis of related peptides in the nervous system of the blowfly Phormia terraenovae. In radioimmunoassays (with antisera against kassinin and leucokinin I) used in combination with reverse-phase HPLC, it was shown that the antisera recognize immunoreactive material with distinctly different hydrophobic properties and each antiserum appear to detect several forms of immunochemically related peptides. With immunocytochemistry it was shown that the kassinin and leucokinin antisera each reacted with material in a distinct set of neurons. The leucokinin-immunoreactive material is present both in interneurons and in neurosecretory cells, suggesting roles of native leucokinin-like peptides as neuromodulators in the nervous system and as neurohormones acting on peripheral targets. The kassinin immunoreactivity was seen in interneurons, but could not be conclusively localized in neurosecretory cells, possibly indicating a role only within the nervous system.