C. Tomas Lundquist

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-NH2 and GLGNNAFVGVR-NH2. 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.

Insect tachykinin-like peptide: Distribution of leucokinin immunoreactive neurons in the cockroach and blowfly brains

Antisera were raised against leucokinin I, a cockroach myotropic neuropeptide with some resemblance to vertebrate tachykinins. These antisera were used for immunocytochemical mapping of neurons and neurosecretory cells in the brains of a cockroach and a blowfly species. The leucokinin immunoreactive cells are distinct from neurons that can be labeled with antisera against vertebrate type tachykinins. It is suggested that leucokinin-like peptides may have roles as neurohormones and neuromodulators in the insect nervous system.

Characterization of Actions of Leucophaea Tachykinin-Related Peptides (LemTRPs) and Proctolin on Cockroach Hindgut Contractions

The nine Leucophaea Tachykinin-Related Peptides (LemTRP 1-9) isolated from the midgut and brain of the cockroach, Leucophaea maderae, all induced increases in spontaneous contractions of the L. maderae hindgut. Synthetic LemTRP 1 and 3-9, were equally potent in inducing contractions of the hindgut. More than seven of the nine C-terminal residues of the closely related locust peptide locustatachykinin I (LomTK I) are required for full activity of the peptide on the L. maderae hindgut. Proctolin, a well characterized myostimulatory neuropeptide, was shown to be more potent than LemTRPs. LemTRP 1 and proctolin did not have synergistic actions in potentiating the amplitude and tonus of contractions of the L. maderae hindgut. Several differences could be seen in actions of LemTRP 1 and proctolin. In contrast to proctolin, LemTRP 1 could not override the inhibitory action of 10(-9) M of the myoinhibitory peptide leucomyosuppressin. Spantide I, an antagonist of the mammalian tachykinin receptors, at a concentration of 5 microM, blocked the response to LemTRP 1, but not to proctolin. The competitive proctolin receptor antagonist [alpha-methyl-L-tyrosine2]-proctolin blocked the action of both proctolin and LemTRP 1 when applied at 1 microM, whereas cycloproctolin had no antagonist action on either peptide. Verapamil, a blocker of voltage gated Ca2+-channels, and the less specific Ca2+-channel blocker Mn2+, abolished the action of LemTRP 1, but not of proctolin. The results obtained indicate that LemTRPs act on receptors distinct from those of proctolin. Double label immunocytochemistry revealed that all LomTK-like immunoreactive fibers impinge on the proctolinergic fibers in the hindgut. This finding and the inhibitory actions of Ca2+-channel blockers on TRP responses and of the proctolin receptor antagonist on both peptides, may suggest that the LemTRP receptors are not on the hindgut muscle fibers but on the terminals of the proctolinergic neurons. Thus, LemTRPs may induce release of proctolin on the hindgut. An alternative is that LemTRPs act by mechanisms clearly distinct from those of proctolin.

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 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.

Tachykinin-related neuropeptide in the crayfish olfactory midbrain

Abstract Immunoreactivity indicative of tachykinin-related peptide (TRP) was detected in the olfactory midbrain of the crayfish Pacifastacus leniusculus when using an antiserum to the insect neuropeptide locustatachykinin I (LomTK-I). A monoclonal antibody to the mammalian tachykinin substance P was shown in double-labeling experiments to label structures in the crayfish brain identical to those labeled with the LomTK antiserum. Within the midbrain LomTK-like immunoreactive (LomTK-LI) material was observed in a limited population of neuronal somata and their varicose processes. A single pair of large interneurons gave rise to numerous varicose LomTK-LI processes innervating a cluster of cell bodies (cluster 10) as well as the olfactory neuropils. The latter neuropil was also innervated by a population of LomTK-LI globuli cells with cell bodies in cluster 9. Radioimmunoassay (RIA), utilizing the LomTK antiserum, and reverse-phase high-performance liquid chromatography (HPLC) were used to partially characterize the immunoreactive material in extract of the portion of the midbrain that houses the olfactory (OL) and accessory (AL) lobes and cell clusters 9 and 10 on the one hand, and in extract of the remaining parts of the brain on the other. Approximately the same amounts of LomTK-LI material were observed for the two extracts. RIA showed that the immunoreactive material of both extracts diluted roughly in parallel to synthetic LomTK-I and HPLC analysis of the extracts revealed immunoreactive material in both tissues which eluted with retention times in the range of synthetic LomTK-I and LomTK-II. These results suggest that TRPs similar to LomTKs are present in the olfactory midbrain of Pacifastacus. The distribution of immunolabeled neuronal structures suggests that in the crayfish, peptide(s) closely related to insect TRPs may act as a neuroactive substance released from nerve fibers in olfactory neuropil areas and at certain neuronal cell bodies.

An aminoisobutyric acid-containing analogue of the cockroach tachykinin-related peptide, LemTRP-1, with potent bioactivity and resistance to an insect angiotensin-converting enzyme.

Nine tachykinin-related peptides (TRPs), designated LemTRP-1-9, were recently isolated from the cockroach, Leucopheae maderae. To obtain a LemTRP resistant to endo- and exoprotease-mediated hydrolysis, we synthesized a peptide with one of the carboxy terminus residues substituted for a sterically hindered aminoisobutyric acid (Aib) and with the amino terminus blocked with a pyroglutamate. The Aib-containing analogue of the nonapeptide LemTRP-1 (Aib-LemTRP-1) thus has the sequence pGlu-Ala-Pro-Ser-Gly-Phe-Leu-Aib-Val-Arg-NH2. This analogue was shown to be resistant to hydrolysis by recombinant angiotensin-converting enzyme (ACE), from Drosophila melanogaster. Endogenous LemTRP-1 on the other hand was rapidly hydrolysed by ACE at the Gly7-Val8 bond, resulting in a single heptapeptide. The Aib-LemTRP-1 has about the same potency as LemTRP-I in inducing contractions of the L. maderae hindgut muscle. It was also tested in intracellular recordings for ability to induce firing of action potentials in dorsal unpaired median (DUM) neurons in the metathoracic ganglion of the locust Locusta migratoria. The Aib-containing analogue was nearly as active as LemTRP-1 and the natural ligand locustatachykinin I. LemTRP-1 and Aib-LemTRP-1 had the same transient time course of action on the cockroach hindgut. This suggests that peptide degradation is not likely to be the cause of the transient action of TRPs.

Autoradiographic localization of 125I-galanin binding sites in the blowfly brain

The localization of porcine galanin (pGAL) binding sites in the brain of the blowfly Phormia terraenovae was investigated by autoradiography using the following radioiodinated ligands: pGAL 1-29 (two isoforms), pGAL 15-29 and rat (r) GAL 1-29. The different porcine radioligands bound specifically with the following intensity: 125I-[Tyr26]-pGAL15-29 > > 125I-[Tyr26]-pGAL1-29 > > 125I-[Tyr9]-pGAL1-29. With rat galanin 125I-[Tyr9]-rGAL1-29 no specific binding could be shown. In addition, displacement of 125I-[Tyr26]-pGAL1-29 was tested with pGAL 1-29, pGAL 1-22 and pGAL 15-29 (at 0.1 nM-1 microM). A gradual displacement was achieved with increasing concentrations of pGAL 1-29 and pGAL15-29, whereas no displacement with pGAL 1-22 was detected. The results indicate that the C-terminal portion of pGAL is important for binding in the blowfly. The pGAL binding sites were localized in synaptic neuropils of the central body, the antennal lobes, the optic lobes, the pars intercerebralis and the subesophageal ganglion, all of which contain GAL-like immunoreactive neural processes.

Galanin message-associated Peptide-like immunoreactivity in the nervous system of the blowfly: distribution and chromatographic characterization.

Galanin message‐associated peptide (GMAP) is a flanking peptide in mammalian preprogalanin located C‐terminally of galanin (GAL). GMAP‐like immunoreactive (LI) material in the brain of the blowfly Phormia terraenovae was analysed by radioimmunoassay combined with reversed‐phase high‐performance liquid chromatography and immunocytochemistry and compared to GAL‐LI material. A sensitive radioimmunoassay, developed against a species‐conserved portion of mammalian GMAP (synthetic porcine GMAP(19–41)amide), was applied to serially diluted blowfly head extracts. High‐performance liquid chromatography combined with radioimmunoassay showed that the GMAP‐LI material eluted as several different components with one major component coeluting with the synthetic GMAP fragment. One GMAP‐LI peak co‐eluted with a GAL‐LI component of the extract. By immunocytochemistry it was shown that a distinct set of GMAP‐LI neurons and neurosecretory cells is present in the blowfly brain and thoracico‐abdominal ganglion. About 150 GMAP‐LI cell bodies were found in the brain, distributed in the protocerebrum, tritocerebrum and suboesophageal ganglion. Several hundred GMAP‐LI cell bodies were detected in the medulla of the optic lobe. In the fused thoracico‐abdominal ganglion there are about 70 GMAP‐LI cell bodies distributed in a segmental fashion. Several of the GMAP‐LI neurons also contain GAL‐LI material whereas some do not. In addition, there are GAL‐LI neurons that do not react with the GMAP antiserum. Some of the GMAP‐LI interneurons and neurosecretory cells could be traced in detail enabling a resolution of putative sites of action of the peptide.

INSECT TACHYKININ-RELATED NEUROPEPTIDES : DEVELOPMENTAL CHANGES IN EXPRESSION OF CALLITACHYKININ ISOFORMS IN THE CENTRAL NERVOUS SYSTEM AND INTESTINE OF THE BLOWFLY, CALLIPHORA VOMITORIA

We have analyzed the relative distribution of tachykinin-related peptides (TRPs) in extracts of adult brains, thoracico-abdominal ganglia, and midguts and of the larval central nervous system of the blowfly Calliphora vomitoria using high performance liquid chromatography (HPLC) in combination with radioimmunoassay (RIA). The RIA employed antisera to the insect TRPs, locustatachykinin I (LomTK I) and callitachykinin II (CavTK II). For identification of the two known blowfly tachykinins we monitored the retention times of synthetic CavTK I and CAVTK II as a reference. With the CavTK II antiserum, all assayed tissues displayed two immunoreactive HPLC fractions with exactly the same retention times as synthetic CavTK I and CavTK II, respectively. An additional immunoreactive fraction eluting earlier than the reference peptides was detected in the adult midgut extract. When assaying the HPLC fractions with antiserum to LomTK I, we obtained the same patterns of immunoreactivity except that now the early eluting material was detectable in all the adult extracts. In addition, in the larval central nervous system, a third major immunoreactive component was displayed using the LomTK RIA and a fourth detected with the CavTK II RIA. We conclude that CavTK I and II are present at a ratio of about 1:1 in all assayed tissues and that two or three additional unidentified tatchykinin-immunoreactive peptides may exist. One of these was seen in the adult tissues; the others appear to be specific for the larval central nervous system (CNS). The RIA was also utilized to determine the total amount of CavTK-immunoreactive material in adult brain, thoracic-abdominal ganglia, and midgut as well as in larval CNS and intestine. The adult CNS contained about seven times more CavTK-immunoreactive material than the larval CNS, and the adult midgut contained 15 times more than the larval intestine. Correlated with these RIA results, many fewer CavTK immunoreactive endocrine cells were labeled in the larval midgut and fewer neurons in the larval CNS than in the Corresponding tissues of adults. Arch. Insect Biochem. Physiol. 34:475–491, 1997.