Jürgen Rapus

Myoinhibitory neuropeptides in the American cockroach

A large number of myostimulatory neuropeptides from neurohaemal organs of the American cockroach have been described since 1989. These peptides, isolated from the retrocerebral complex and abdominal perisympathetic organs, are thought to be released as hormones. To study the coordinated action of these neuropeptides in the regulation of visceral muscle activity, it might be necessary to include myoinhibitors as well, however, not a single myoinhibitory neuropeptide of the American cockroach has been described so far. To fill this gap, we describe the isolation of LMS (leucomyosuppressin) and Pea-MIP (myoinhibitory peptide) from neurohaemal organs of the American cockroach. LMS was very effective in inhibiting phasic activity of all visceral muscles tested. It was found in the corpora cardiaca of different species of cockroaches, as well as in related insect groups, including mantids and termites. Pea-MIP which is strongly accumulated in the corpora cardiaca was not detected with a muscle bioassay system but when searching for tryptophane-containing peptides using a diode-array detector. This peptide caused only a moderate inhibition in visceral muscle assays. The distribution of Pea-MIP in neurohaemal organs and cells supplying these organs with Pea-MIP immunoreactive material, is described. Additionally to LMS and Pea-MIP, a member of the allatostatin peptide family, known to exhibit inhibitory properties in other insects, was tested in visceral muscle assays. This allatostatin was highly effective in inhibiting spontaneous activity of the foregut, but not of other tested visceral muscles of the American cockroach.

Periviscerokinin (Pea-PVK): A novel myotropic neuropeptide from the perisympathetic organs of the American cockroach

A myotropic neuropeptide was isolated from extracts of 1000 abdominal perisympathetic organs of males of the cockroach, Periplaneta americana. This peptide, termed periviscerokinin, has excitatory actions on the hyperneural muscle of Periplaneta americana. After peptide sequence analysis and mass spectrometry, the structure of this peptide was confirmed by chemical synthesis and bioassay to be Gly-Ala-Ser-Gly-Leu-Ile-Pro-Val-Met-Arg-Asn-NH2. This sequence is different from the other known myotropic peptides in insects. The threshold concentration for stimulatory effects of the synthetic peptide on the isolated hyperneural muscle was about 10(-9) M, suggesting a physiological role as a neurohormone.

Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach.

Different pyrokinin isoforms were identified from major neurohemal organs of the American cockroach. During their isolation they were recognized by bioassay using a hyperneural muscle preparation that is sensitive to pyrokinins. All structures were elucidated by sequence analysis and mass spectrometry. The primary structures of the novel peptides isolated from the retrocerebral complex are LVPFRPRL-NH2 (designated Pea-PK-3) and DHLPHDVYSPRL-NH2 (designated Pea-PK-4). A pyrokinin, labeled Pea-PK-5, was isolated from abdominal perisympathetic organs. Structural analysis of this peptide yielded the sequence GGGGSGETSGMWFGPRL-NH2. The threshold concentrations of the identified pyrokinins for an eliciting effect on contractions of the hyperneural muscle preparations differed dramatically. This indicates that the different distribution of pyrokinin-isoform observed in neurohemal organs may be associated with different functions. This is the first report of a differential distribution of peptide-isoforms in the neurohemal organs of insects.

Isolation of Periviscerokinin-2 from the Abdominal Perisympathetic Organs of the American Cockroach, Periplaneta americana

Using the isolated hyperneural muscle as bioassay, a novel myotropin was isolated from the abdominal perisympathetic organs of Periplaneta americana. This is the second neuropeptide identified from insect perisympathetic organs. Peptide sequence analysis and mass spectrometry yielded the following structure: Gly-Ser-Ser-Ser-Gly-Leu-Ile-Ser-Met-Pro-Arg-Val-NH2. This peptide, named periviscerokinin-2, was confirmed to be amidated by chemical synthesis, bioassay, and comparison of retention times between native and synthetic peptides. A highly specific antiserum was used to determine sites of synthesis in the abdominal ganglia. Besides periviscerokinin-1, periviscerokinin-2 is the only putative myotropic neurohormone from the abdominal perisympathetic organs that is effective in the nanomolar range. This confirms the hypothesis that the neurohormonal system of the ventral nerve cord is remarkably different from that of the brain.

Octopamine-like immunoreactive neurones in locust genital abdominal ganglia

Using a well characterized anti-serum, the distribution of octopamine-like immunoreactive neurones is described in the locust seventh abdominal (A7) and terminal ganglia (TG), which are associated with genital organs. Apart from 4 paired ventral somata occasionally observed in the TG, all labelled cells could be identified as efferent dorsal- and ventral unpaired median (DUM/VUM) neurones by virtue of the characteristic large size and position of their somata, projections of their primary neurites in DUM-cell tracts, and bifurcating axons which arise from dorsal T-junctions and enter peripheral nerves. For the examined ganglia our data indicate that the whole population of efferent DUM and VUM-cells, defined here as progeny of the segment specific unpaired median neuroblast with peripheral axons, are octopaminergic, and that equal numbers of these cells occur in both sexes: 8 in A7 and 11 in TG. Sex-specific differences are probably restricted to the axonal projections of 5 octopamine-like immunoreactive DUM-somata in A7, and 5 in TG, which in females project into their segment specific sternal nerves, but in males into the genital nerve of the TG. Numerous intersegmentally projecting octopamine-like immunoreactive fibres traverse both ganglia. The majority probably stem from previously described octopamine-like immunoreactive neurones in the thoracic and suboesophageal ganglia.

Allatostatins from the retrocerebral complex and antennal pulsatile organ of the American cockroach: structural elucidation aided by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

The occurrence of allatostatins in retrocerebral complexes and antennal pulsatile organs of the American cockroach, Periplaneta americana, was investigated. Previously, molecular cloning of the P. americana allatostatin gene had predicted 14 peptides of this family [Ding et al., Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata. Eur J Biochem 1997;234:737-746], however, only two forms had been identified by peptide isolation procedures [Weaver et al., Identification of two allatostatins from the CNS of the cockroach Periplaneta americana: novel members of a family of neuropeptide inhibitors of insect juvenile hormone biosynthesis. Comp Biochem Physiol 1994;107(C):119-127]. Using an extract of only 200 corpora cardiaca/corpora allata, we have found that at least 11 allatostatins occur in the retrocerebral complex. These peptides were already separated from other substances of the crude extract in the first HPLC step with heptafluorobutyric acid as organic modifier, and subsequently identified by MALDI-TOF mass spectrometry. Moreover, we have demonstrated the occurrence of nearly all allatostatins, including the cleavage product of Pea-AST-2 (LPVYNFGL-NH2), in antennal pulsatile organs of males and females. Allatostatins are predominant neuropeptides in these organs. Additionally, only two other known peptides could be identified in these organs by mass screening: proctolin and leucomyosuppressin. The function of allatostatins in antennal pulsatile organs remains unclear. We assume a release into the hemolymph via the ampullac, which could act as neurohemal release sites. The method described for the identification of allatostatins is a very fast method for neuropeptide screening in neurohemal tissues.

Small sets of putative interneurons are octopamine-immunoreactive in the central nervous system of the pond snail,Lymnaea stagnalis

An antibody raised against conjugated octopamine was applied to map octopamine-containing neurons in the central nervous system of the pond snail Lymnaea stagnalis. A small number of octopamine-like immunoreactive neurones occurs in all ganglia, but the pleural ones. The neurons are located either in small clusters or occur individually. Major concentrations of octopamine-immunoreactive neurons can first of all be found in the buccal, cerebral and pedal ganglia. Varicose arborizations were observed in the neuropiles, but peripheral projections of labelled elements could not be traced. We suggest that a set of octopaminergic interneurons would exist in the Lymnaea brain. Mapping of octopamine-immunoreactive neurons given may also facilitate physiological investigations on octopaminergic neurotransmission in the gastropod nervous system.

Taurine-like immunoreactivity in octopaminergic neurones of the cockroach, Periplaneta americana (L.)

Taurine (2-aminoethanesulphonic acid) is reported to interact with the octopaminergic system. The distribution of taurine-like immunoreactivity (-LIR) in relation to octopamine-like immunoreactive dorsal unpaired median (DUM) neurones was investigated with the aim of revealing possible colocalization of these two neuromediators. The specificity of the anti-taurine serum used was demonstrated by dot blot immunoassay and by use of preabsorption controls. There was no crossreactivity with octopamine. The specificity of the octopamine antiserum employed has been described elsewhere. Taurine-LIR could be demonstrated in large dorso-median cells in the suboesophageal and the mesothoracic ganglion as well as in the abdominal ganglia. In addition taurine-LIR is distributed in numerous other regions of the ganglia. A comparison of the immunostaining for taurine and octopamine indicates that several of the taurine-like immunoreactive (-LI) neurones are probably members of the octopamine-immunoreactive DUM cell population. These taurine-LI neurones resemble octopamine-LI DUM cells in soma position and size as well as in the projections of their primary neurites. Colocalization of octopamine-LIR and taurine-LIR within the same neuronal element could be shown by alternate immunostaining of consecutive sections. It is probable that all octopamine-LI DUM neurones also exhibit taurine-LIR, and the possible physiological significance of this coexistence is discussed.

A comparative immunocytochemical study using an antiserum against a synthetic analogue of the corpora cardiaca peptide Pea-CAH-I (MI, neurohormone D) of Periplaneta americana

Abstract.An antiserum against the octapeptide Pea-CAH-I, a member of the adipokinetic hormone/red pigment-concentrating hormone family, has been produced for immunocytochemical staining in insects and various other invertebrate species. The anti-Pea-CAH-I serum stains the glandular corpora cardiaca cells of those insect species that synthesize identical or structurally similar peptides. In the corpora cardiaca of species producing peptides with a different C-terminus, these cells remain unstained. Pea-CAH-I-like immunoreactivity has also been found in neurons of the central nervous system of all invertebrate orders studied. The antiserum recognizes the C-terminal sequence Pro-Asn-Trp-NH2 of the Pea-CAH-I molecule as established by enzyme immunoassay. The widespread Pea-CAH-I-like immunoreactivity in all nervous systems of the studied animals probably does not reflect the presence of Pea-CAH-I but the occurrence of peptides carrying similar epitopes.

Octopamine in the central nervous system of Oligochaeta: an immunocytochemical and biochemical study

Abstract.The distribution of octopamine (OA)-like immunoreactive neurons was investigated, and the concentration of OA was assayed by high-performance liquid chromatography in the central nervous system of Oligochaeta species, Lumbricus terrestris, Eisenia fetida, and Lumbricus polyphemus. OA-like immunoreactive nerve cells were found in all parts of the central nervous system; certain regions of the neuropil of the ganglia were densely innervated by immunoreactive fibers. Altogether 96–102 OA-like immunoreactive neurons were detected in the cerebral ganglion, 18 in the subesophageal ganglion, and 14 in the segmental ganglia of 2nd–5th and 40th–45th body segments of Lumbricus terrestris; the relevant numbers of neurons in Eisenia were 88–98, 20–22, and 6, respectively. The sizes of OA immunoreactive-like cells showed great variability according to their anatomical localization. High-performance liquid chromatography assay revealed the presence of OA in each investigated part of the central nervous system, showing concentration values between 8.6 and 16.7 pmol/mg wet weight in the three species. The concentration of the OA precursor tyramine was significantly lower in the central nervous system of Eisenia (<0.5 pmol/mg wet weight) than in that of both Lumbricus species (0.67–2.0 pmol/mg wet weight). The metabolism of 3H-tyrosine revealed that tyramine and OA were synthesized by the enzymes tyrosine-decarboxylase and tyramine-β-hydroxylase, respectively. Thus, OA appears to have a regulatory role in the central nervous system of Oligochaeta.