The SALMFamides: a new family of neuropeptides isolated from an echinoderm.

We have isolated two novel related neuropeptides from the radial nerve cords of the starfishes Asterias rubens and Asterias forbesi. One is an octapeptide with the amino acid sequence Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-NH2 and the other is a dodecapeptide with the amino acid sequence Ser-Gly-Pro-Tyr-Ser-Phe-Asn-Ser-Gly-Leu-Thr-Phe-NH2. The peptides were purified using high performance liquid chromatography (HPLC) and a radioimmunoassay for the molluscan FMRFamide-related neuropeptide, pQDPFLRFamide. Both peptides share minimal sequence identity with members of the family of FMRFamide-like peptides so we have designated them as founder members of a new family, the SALMFamides. We refer to the octapeptide as SALMFamide 1 (S1) and the dodecapeptide as SALMFamide 2 (S2). S1 and S2 are the first neuropeptides identified in species belonging to the phylum Echinodermata.     

Arthropod FMRFamide-related peptides modulate muscle activity in helminths

FMRFamide-related peptides are common to a wide variety of invertebrate species, including helminths and arthropods. In arthropods, five distinct FMRFamide-related peptide subfamilies are recognised: the myosuppressins, extended-FLRFamides, -FMRFamides, -RFamides, and sulfakinins, members of which induce potent and diverse myotropic effects. Whilst >80 FMRFamide-related peptides have been identified in nematodes, only four FMRFamide-related peptides have been characterised from flatworms. The Ascaris suum ovijector/body wall bioassay and the Procerodes littoralis muscle fibre bioassay have proved both reliable and sensitive systems for assessing the functional activities of FMRFamide-related peptides in vitro, and data describing the effects of native FMRFamide-related peptides in these systems are rapidly accumulating. This is the first study to determine the cross-phyla activities of non-native FMRFamide-related peptides in both nematode and flatworm species. In the present study, the effects of 10 arthropod FMRFamide-related peptides (leucomyosuppressin [pQDVDHVFLRFamide], schistoFLRFamide [PDVDHVFLRFamide] and truncated analogues [HVFLRFamide and VFLRFamide], lobster peptide I [TNRNFLRFamide], lobster peptide II [SDRNFLRFamide], manducaFLRFamide II [GNSFLRFamide], manducaFLRFamide III [DPSFLRFamide], calliFMRFamide 4 [KPNQDFMRFamide] and perisulfakinin [EQFDDY(SO(3)H)GHMRFamide]), representing the five subfamilies, were examined on the body wall and ovijector of the parasitic porcine nematode, A. suum and dispersed muscle fibres from the free-living turbellarian, P. littoralis. The muscle activity of the ovijector was found to be modulated significantly by each of the arthropod FMRFamide-related peptides tested; the effects were concentration-dependent, reversible and repeatable. All but one (perisulfakinin) of the 10 arthropod FMRFamide-related peptides examined modulated significantly the activity of A. suum body wall muscle. In addition, all of the arthropod FMRFamide-related peptides examined induced potent concentration-dependent contractions of P. littoralis muscle fibres. These results reveal similarities in the ligand requirement(s) between FMRFamide-related peptide receptors within the Phyla Arthropoda, Nematoda and Platyhelminthes, and indicate significant receptor promiscuity, which highlights the potential of FMRFamide-related peptide receptors as legitimate targets for novel endectocidal agents.     

Regulation of Drosophila FMRFamide neuropeptide gene expression.

Physiologically important peptides are often encoded in precursors that contain several gene products; thus, regulation of expression of polypeptide proteins is crucial to transduction pathways. Differential processing of precursors by cell- or tissue-specific proteolytic enzymes can yield messengers with diverse distributions and dissimilar activities. FMRFamide-related peptides (FaRPs) are present throughout the animal kingdom and affect both neural and gastrointestinal functions. Organisms have several genes encoding numerous FaRPs with a common C-terminal structure but different N-terminal amino acid extensions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide contained in the Drosophila FMRFamide gene. To investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish among the three neuropeptides. We have previously reported the distribution of SDNFMRFamide and DPKQDFMRFamide. In this article, we describe TPAEDFMRFamide expression. TPAEDFMRFamide antisera stain cells in embryonic, larval, pupal, and adult thoracic and abdominal ganglia. In addition, TPAEDFMRFamide-immunoreactive material is present in a lateral protocerebrum cell in adult. Thus, TPAEDFMRFamide antisera staining of neural tissue is different from SDNFMRFamide or DPKQDFMRFamide. In addition, TPAEDFMRFamide antisera stain larval, pupal, and adult gut, while SDNFMRFamide and DPKQDFMRFamide do not. TPAEDFMRFamide immunoreactivity is present in cells stained by FMRFamide antisera. Taken together, these data support the conclusion that TPAEDFMRFamide is differentially processed from the FMRFamide polypeptide protein precursor and may act in both neural and gastrointestinal tissue.     

Native and heterologous neuropeptides are cardioactive in Drosophila melanogaster

Nine neuropeptides isolated from Drosophila melanogaster and five neuropeptides, previously isolated from the CNS of Limulus with antisera to FMRFamide-related peptides, were tested for their effects on the myogenic heart of Drosophila melanogaster. Of the native peptides, TDVDHVFLRF-NH(2) (Dromyosuppressin), DPKQDFMRFamide, and PDNFMRFamide significantly slowed the heart. Of the Limulus peptides, DEGHKMLYFamide (LP1) increased heart rate significantly, GHSLLHFamide (LP2) and PDHHMMYFamide (LP3) decreased the heart's rate, while DHGNMLYFamide (LP4) and GGRSPSLRLRFamide (LP5) had no effect at the concentrations we employed. Dromyosuppressin, DPKQDFMRFamide, and PDNFMRFamide from Drosophila, and LP2 and LP3 from Limulus, which belong to a novel group of peptides structurally unrelated to FMRFamide, are among only a very few substances from within the general group of neuropeptides and neurohormones known to slow the heart of Drosophila, and as such offer an important tool for investigating the molecular mechanisms underlying the control of the pacemaker.     

Diurnal regulation of sphingolipids in blood

Key homeostatic functions are regulated in a diurnal manner and a miss-alignment of such rhythms is believed to contribute to the pathophysiology of several diseases. Signaling sphingolipids (SLs) in plasma such as sphingosine 1-phosphate control lymphocytic trafficking, vascular reactivity and platelet activity, physiological functions all of which display a diurnal rhythm themselves. However, the rhythmicity of SL metabolism in plasma and its potential causes have not been sufficiently investigated so far. Therefore, we analyzed blood of mice and healthy adult human subjects by targeted tandem mass-spectrometry at different time points. In order to investigate the influence of the synchronizing hormone melatonin, we compared melatonin proficient C3H/HeN wildtype mice (C3H) with melatonin receptor-1/2 double knockout mice (MT1/2−/−) and melatonin deficient C57BL/6J mice. We found a strong upregulation of plasma S1P with the beginning of the light period in C3H but not in MT1/2−/− or C57BL/6J mice. Accordingly, our study revealed an upregulation of sphingosine 1-phosphate (S1P d18:1) and sphinganine 1-phosphate (S1P d18:0) with the beginning of the light period in humans. Furthermore, plasma S1P d18:1 and S1P d18:0 were inversely correlated with the respective concentrations in platelets, pointing to a possible involvement of platelet SL metabolism. In humans, the diurnal rhythm of SLs was not associated with changes of SL-binding proteins or counts of cellular SL sources. Overall, this study indicates a physiological rhythmicity of plasma and platelet SL metabolism, likely mediated by melatonin, with potentially important implications for physiological diurnal rhythms and the regulation of SL metabolism and its functions.     

Immunoaffinity-based mass spectrometric characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis

The tetrapeptide, FMRFamide, was first discovered in 1977 in the molluscan nervous system and was found to affect the contractile force of molluscan cardiac muscle and other muscles [1]. Since then, numerous FMRFamide-related peptides (FaRPs) have been reported in both invertebrate and vertebrate species [2], [3], [4], [5], [6], [7], [8] and [9]. We have previously reported the detection and identification of numerous FaRPs in Cancer borealis pericardial organs (POs), one of the major neurosecretory structures in the crustaceans [2] and [3]. Here, we have developed two immunoaffinity-based methods, immunoprecipitation (IP) and immuno-dot blot screening assay, for the enrichment of FaRPs in C. borealis POs. A combined mass spectrometry (MS)-based approach involving both matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-QTOF MS/MS) is used for a more comprehensive characterization of the FaRP family by utilizing high mass accuracy measurement and efficient peptide sequencing. Overall, 17 FMRFamide-related peptides were identified using these two complementary immuno-based approaches. Among them, three novel peptides were reported for the first time in this study.     

Isolation and sequencing of two FMRFamide-related peptides from the gut of Locusta migratoria L

Two FMRFamide-related peptides (FaRPs) have been isolated and sequenced from the whole gut of Locusta migratoria L. Peptides were extracted from 500 locust whole guts and separated using reversed-phase high performance liquid chromatography (RP-HPLC). Fractions containing FMRFamide-like immunoreactive (FLI) material were identified using radioimmunoassay (RIA). Sequencing of fractions, using tandem mass spectrometry (MALDI-TOF MS/MS), revealed the myosuppressin previously isolated from the locust CNS, SchistoFLRFamide (PDVDHVFLRFamide), and a novel extended RFamide (LWENLRFamide). The isolation of SchistoFLRFamide from midgut tissue supports the hypothesis that this myosuppressin is released locally from FLI processes over the gut and/or from endocrine-like midgut cells to play a role in the regulation of digestion.     

Production of antisera to growth hormone-releasing factor: usefulness in radioimmunoassay and passive immunization

We have produced two antisera (R-1 & R-2) to human growth hormone-releasing factor (GRF) [1-44] NH2. Both antisera can be used for human GRF radioimmunoassay (RIA) at a final dilution of 1:50000. The antiserum R-2 was specific for the C-terminal amidated sequence of human GRF-44 and selectively recognized GRF [1-44] NH2 but not GRF [1-44] OH or GRF [1-40] OH. The antiserum R-1 also significantly bound 125I-rat GRF [1-43] OH at a final dilution of 1:5000 and enabled us to establish RIA for rat GRF. In both RIA systems, intra- and inter-assay coefficients of variation at 50% inhibition were 8 and 12%, respectively. A median effective dose was 90-120 pg in human GRF RIA and 250-300 pg in rat GRF RIA. Utilizing the RIA, we demonstrated that the hypothalamic GRF content in rats which received monosodium glutamate during the neonatal period was less than 20% of that of controls. However, the hypothalamic GRF content was not altered in rats made hypothyroid by methimazole administration, another condition known to greatly impair GH secretion. An iv administration of the antiserum R-1 significantly suppressed GH release following the injection of antisomatostatin serum. Thus, these antisera can be a useful tool in examining the physiological and/or pathophysiological roles of GRF in human and rat.     

The Pharmacology of Nematode FMRFamide-related Peptides

FMRFamide-related peptides (FaRPs) are the largest known family of invertebrate neuropeptides. Immunocytochemical screens of nematode tissues using antisera raised to these peptides have localized extensive FaRP-immunostaining to their nervous systems. Although 21 FaRPs have been isolated and sequenced from extracts of free-living and parasitic nematodes, available evidence indicates that other FaRPs await discovery. While our knowledge of the pharmacology of these native nematode neuropeptides is extremely limited, reports on their physiological activity in nematodes are ever increasing. All the nematode FaRPs examined so far have been found to have potent and varied actions on nematode neuromuscular activity. It is only through the extensive pharmacological and physiological assessment of the tissue, cell and receptor interactions of these peptidic messengers that an understanding of their activity on nematode neuromusculature will be possible. In this review, Aaron Maule and colleagues examine the current understanding of the pharmacology of nematode FaRPs.     

Identification, characterization and localization of thyrotropin-releasing hormone precursor peptides in perinatal rat pancreas

The sequence of rat hypothalamic prepro TRH, deduced from its complementary DNA, contains five TRH progenitor sequences and six cryptic sequences separated by paired basic amino acid residues. We have utilised antisera against two synthetic peptides corresponding to sequences within proTRH, [Tyr53] preproTRH (53-74), part of the amino terminal leader sequence of proTRH and [Cys 74,83] preproTRH-(75-82), representing a TRH progenitor sequence flanked by cysteine residues (pCC10) in radioimmunoassays (RIA) to identify and chromatographically characterize proTRH derived peptides in extracts of rat perinatal pancreas and to localize these peptides immunohistochemically. Two forms of immunoreactive pYT22 (ipYT22) were observed, similar in size to ipYT22 seen in extracts of adult rat brain. By RIA immunoreactive pCC10 was detectable in neonatal but not fetal pancreas. However, immunohistochemical double staining of both fetal and neonatal rat pancreas colocalized both ipYT22 and ipCC10 with immunoreactive insulin in the B-cell of the developing Islets of Langerhans. These findings indicate that the B-cell of the perinatal pancreas synthesizes TRH from a prohormone encoded by a mRNA similar to that present in adult rat hypothalamus.     

Expression of preproVIP-derived peptides in the human gastrointestinal tract: a biochemical and immunocytochemical study

In order to study biosynthetic processing of the precursor for vasoactive intestinal peptide (preproVIP) in the human gut we have developed antisera against the five functional domains of the precursor molecule: preproVIP 22-79, peptide histidine methionine (PHM), preproVIP 111-122, VIP and preproVIP 156-170. The antisera were used to quantify and characterize VIP-precursor peptides by radioimmunoassay (RIA) together with high-pressure liquid Uchromatography (HPLC) and to examine their cellular localization and colocalization by immunocytochemistry. All five peptides were expressed but not in equimolar amounts as expected from the amino acid sequence of the precursor. However, the ratios between them were fairly constant throughout the gastrointestinal tract. The only exceptions were the lower concentrations of PHM and preproVIP 111-122 in the gastric antrum which could be explained by the presence of PHV (the C-terminally extended form of PHM which includes preproVIP 111-122) in large concentrations in this region. It was also found that the C-terminal lysine residue of preproVIP is not removed during processing. Immunocytochemically all preproVIP-derived peptides were shown in neuronal elements. They had a similar distribution throughout the gut suggesting coexistence, a finding which was supported by doublestaining. The findings indicate that differences in the posttranslational processing of preproVIP exist in subpopulations of neurons in the human gut.     

Mass spectrometric analysis of single identified neurons of an insect

The combination of retrograde labelling with dextran-tetramethylrhodamine and MALDI-TOF mass spectrometry was used to analyse for the first time the peptidome of a series of morphologically identified single neurosecretory cells of an insect. Eight postero-lateral cells of the metathoracic ganglion of the American cockroach, Periplaneta americana, were used to demonstrate that: (1) the complete dissection procedure can be documented and (2) the mass spectrometric analysis of the dissected somata results in highly reproducible mass spectra. In total, 21 FMRFamide-related peptides were detected in each of the postero-lateral cells which release their neurosecretions via thoracic perisympathetic organs. Direct analysis of these neurohemal organs confirmed the co-storage of FMRFamide-related peptides. Two additional abundant peptides from thoracic perisympathetic organs which were not detectable in the postero-lateral cells were characterized using ESI-Q-TOF MS/MS. De novo sequencing yielded two related peptides (FERL/IEamides) without any similarity with known peptide families of insects.     

Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) secreted by cells in culture

Transforming growth factor beta (TGF-beta), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF-beta 1 and TGF-beta 2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF-beta 1 and TGF-beta 2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF-beta 1 and TGF-beta 2 in complex biological fluids. Using these assays we show that both TGF-beta 1 and TGF-beta 2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF-beta 1 or TGF-beta 2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of TGF-beta is independently regulated.     

The quantitative determination of 2'-deoxycytidine-5'-triphosphate in cell extracts by radioimmunoassay

A radioimmunoassay (RIA) capable of quantitating dCTP in femtomolar amounts in cell extracts has been developed, and applied to human fibroblast cell lines and L5178Y mouse lymphoma lines. Cross reactivity of the antibody with CTP, though low (2.7%) has necessitated pre-RIA removal of CTP by either boronate affinity gel chromatography or sodium periodate oxidation. Fractions from the boronate gel column or aliquots of NaIO4-treated cell extract are quantitated directly by the RIA. Recovery of extracted dCTP standard taken through the entire procedure is quantitative and results are reproducible. Due to the high sensitivity of the quantitation step, dCTP can be accurately measured in relatively small numbers of cells--about 10(4) cells.     

Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2.

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.     

Quantification and partial characterisation of regulatory peptides in the liver fluke, Fasciola hepatica, from different mammalian hosts.

1. Extracts of the liver fluke, Fasciola hepatica from three different hosts (cow, sheep, rat) have been subjected to radioimmunoassay using antisera to 6 mammalian regulatory peptides. 2. Immunoreactivity was measured to pancreatic polypeptide, substance P, peptide histidine isoleucine and gastrin-releasing peptide. Levels of each peptide varied considerably in flukes from different hosts. 3. Reverse-phase HPLC of rat and sheep fluke extracts revealed three molecular forms of tachykinin immunoreactivity and single peaks of pancreatic polypeptide and peptide histidine isoleucine immunoreactivity. No GRP-immunoreactivity was detected by RIA of HPLC fractions.     

The ovijector of Ascaris suum: multiple response types revealed by Caenorhabditis elegans FMRFamide-related peptides

Caenorhabditis elegans possesses 22 FMRFamide-like peptide (flp) genes predicted to encode 60 different FMRFamide-related peptides with a range of C-terminal signatures. Peptides from five flp genes (1, 6, 8, 9 and 14) are known to modulate the ovijector of Ascaris suum in vitro. This study examines the physiological effects of peptides from the remaining 17 flp genes such that the variety of FMRFamide-related peptide-induced ovijector response types can be delineated. Five categories of response were identified according to the pattern of changes in contractile behaviour and baseline tension. Peptides encoded on 16 flp genes (1, 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 20) had qualitatively similar inhibitory (response type 1) actions, with the lowest activity thresholds (1 nM) recorded for peptides with FIRFamide or FLRFamide C-terminal signatures. Peptides encoded on four flp genes (2, 18, 19 and 21), and on the A. suum afp-1 gene, had excitatory actions on the ovijector (response type 2), with PGVLRFamides having the lowest activity threshold (1 nM). An flp-2 peptide (LRGEPIRFamide) induced a transient contraction of the ovijector (activity threshold, 10nM) that was designated response type 3. Response type 4 comprised a transient contraction followed by an extended period of inactivity and was observed with peptides encoded on flp-5 (AGAKFIRFamide, APKPKFIRFamide), flp-8 (KNEFIRFamide) and flp-22 (SPSAKWMRFamide). SPSAKWMRFamide was the most potent peptide tested with an activity threshold of 0.1 nM. A single peptide (AMRNALVRFamide; activity threshold 0.1 microM), encoded on flp-11, induced response type 5, a shortening of the ovijector coupled with an increase in contraction frequency. Although most flp genes encode structurally related peptides that trigger one of the five ovijector response types, flp-2 and flp-11 co-encode FMRFamide-related peptides that induce distinct responses. Within the ovijector of A. suum FaRPs play a complex role involving at least five receptor subtypes or signalling pathways.     

Neurotransmitters and neuromodulators controlling the anterior byssus retractor muscle of Mytilus edulis.

1. The anterior byssus retractor muscle (ABRM) of Mytilus edulis is innervated by at least two kinds of nerves, excitatory and relaxing nerves. The principal neurotransmitters released from these nerves have been shown to be acetylcholine and serotonin, respectively. 2. Some other monoamines, such as dopamine and octopamine, and various peptides, such as FMRFamide-related peptides, Mytilus inhibitory peptides, SCP-related peptides and a catch-relaxing peptide, may also be involved in the regulation of the muscle as neurotransmitters or neuromodulators. 3. The ABRM seems to be typical of invertebrate muscles controlled by multiple neurotransmitters and neuromodulators.     

Identification of Epstein-Barr virus terminal protein 1 (TP1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera.

The terminal proteins TP1 and TP2 are putative products of Epstein-Barr virus (EBV) genes expressed during the latent cycle of the virus. They are predicted to code for 53- and 40-kilodalton integral membrane proteins. We used the baculovirus Autographa californica nuclear polyhedrosis virus as an expression vector to produce TP1 in large amounts in insect cells. The DNA sequences used to express TP1 originated from a TP1 cDNA derived from an M-ABA/CBL1 cDNA library. Rabbit antisera raised against procaryotic TP1 fusion proteins recognized a monomer and a dimer of the recombinant TP1 protein in the infected insect cells. Immunofluorescence studies of living insect cells showed that the recombinant protein is located in the plasma membrane. The insect cells infected with the recombinant baculovirus producing TP1 provided a test system to screen human antisera for TP1 antibodies. A total of 168 human EBV-positive and EBV-negative antisera were studied. TP1 antibodies were detected only in sera from nasopharyngeal carcinoma patients (16 out of 42). Rabbit antiserum raised against the recombinant TP1 protein expressed in the baculovirus system specifically recognized a protein of about 54 kilodaltons in the lymphoblastoid cell lines M-ABA and M-ABA/CBL1 and in the Burkitt's lymphoma cell lines BL18 and BL72. This protein could be located in the total membrane fraction of M-ABA cells and is upregulated by treating the cells with 12-O-tetradecanoylphorbol-13-acetate.     

Neuronal co-localization of different isoforms of tachykinin-related peptides (LemTRPs) in the cockroach brain

Seven isoforms of tachykinin-related peptides (TRPs) have been isolated from the brain of the cockroach Leucophaea maderae. These peptides (LemTRP-1, 2, and 5-9) share the C-terminal sequence GFX(1)GX(2)Ramide (where X(1) and X(2) are variable residues). In order to determine the neuronal distribution of several of these LemTRP isoforms, we raised antisera to their variable N-termini. Antisera to LemTRP-1, 2, 3, 7, and 8 were utilized for immunocytochemistry on cryostat sections of the L. maderae brain. As expected, the gut peptide LemTRP-3 was not detected in the brain, and the antisera to LemTRP-1, 2, and 7 labeled the same sets of neurons in different regions of the brain. These neurons could also be labeled with antisera raised to the more conserved C-termini of LemTRP-1 and the locust TRP LomTK-I. The antiserum to LemTRP-8 predominantly labeled a set of neurons distinct from that seen with any other N- or C-terminus-directed antisera, suggesting that it recognizes epitope(s) other than known insect TRPs. Our findings indicate that at least three of the LemTRPs are always co-localized in neurons of the L. maderae brain. We have also been able to show that LemTRP-2, which is an N-terminally extended form (17-mere) of LemTRP-1 with a dibasic putative cleavage site, is transported throughout the processes of the neurons in the same manner as LemTRP-1 and 7. Thus, LemTRP-2 may be released with the other shorter LemTRPs. This is the first investigation of LemTRP distribution in the cockroach central nervous system utilizing antisera to native peptides.