In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.     

昆虫激肽的生理功能及机制

昆虫激肽是一类高度保守的小分子神经活性物质,自其从马德拉蟑螂脑中分离得到至今,人们在多种无脊椎动物体内均发现了这一激肽家族成员。它们具有促进昆虫后肠收缩、马氏管扭动、原尿分泌,调节血淋巴量和水盐平衡,使马氏管跨膜电位去极化,抑制昆虫体内消化酶释放、幼虫体重增长等功能。然而,天然的昆虫激肽很容易被蛋白酶所降解,因此须对其进行结构改造及构效关系研究,以开发出更有潜力的假肽和非肽模拟抗酶解昆虫激肽类似物,对今后实现环境友好型害虫防治策略具有重要意义。     

Primary structures of hypertrehalosaemic neuropeptides isolated from the corpora cardiaca of the cockroaches Leucophaea maderae, Gromphadorhina portentosa, Blattella germanica and Blatta orientalis and of the stick insect Extatosoma tiaratum assigned by tandem fast atom bombardment mass spectrometry

Hypertrehalosaemic peptides were isolated by reversed-phase high-performance liquid chromatography from corpora cardiaca of four species of cockroaches (Leucophaea maderae, Gromphadorhina portentosa, Blattella germanica, and Blatta orientalis) and one stick insect species (Extatosoma tiaratum), and their primary sequences were assigned by collision-induced decomposition tandem fast atom bombardment mass spectrometry (FABMS/CID/MS). The members of the cockroach families Blaberidae (L. maderae and G. portentosa) and Blattellidae (B. germanica) contained an identical decapeptide (Glu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-ThrNH2), whereas the member of the cockroach family Blattidae (B. orientalis) had two octapeptides (Glu-Val-Asn-Phe-Ser-Pro-Asn-TrpNH2 and Glu-Leu-Thr-Phe-Thr-Pro-Asn-TrpNH2). The structure of the stick insect hypertrehalosaemic compound was assigned as a decapeptide (Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH2). The respective synthetic peptides elevated blood carbohydrates in their respective acceptor species. The results are discussed in the light of family-specificity of members of the adipokinetic hormone/red pigment-concentrating hormone family.     

The interactions between juvenile hormone (JH), lipophorin, vitellogenin, and JH esterases in two cockroach species

For the cockroach species Leucophaea maderae and Periplaneta americana two major juvenile hormone (JH)-binding proteins have been identified: lipophorin (Lp) and vitellogenin (Vg). Each of these macromolecules binds JH with an approximate affinity of K(d) of 10 nM. In Leucophaea the concentration of Lp is augmented by JH during vitellogenesis at the same time when Vg is induced de novo. The circulating levels of each of Lp and Vg at mid-vitellogenesis are in the 10 microM range. Similar values have been determined for Periplaneta. Total JH concentrations (bound and free) can be as high as micromolar in Leucophaea. However, because of the large quantities of the two major JH-binding proteins and their high affinity for JH, we can assume that the amount of free (unbound) JH in circulation is extremely low (the actual values are not know).The JH esterases (JHEs) of the hemolymph in both cockroach species have been isolated by anion exchange chromatography. The JHEs of Leucophaea bound to the anion exchange resin more tightly than the JHE of Periplaneta. The V(max) of the Leucophaea esterases fluctuated by a factor of 2 to 3 during vitellogenesis. The K(m) values for the two distinct esterases of Leucophaea were similar (about 0.15x10(-6) M). On the other hand, k(cat) of the JHEs for Leucophaea at ovulation time was two to three times higher than earlier during vitellogenesis, i.e. 23.30 min(-l) compared to 6.20 min(-1). The JHE of Leucophaea is shown to bind JH III with high affinity: K(d)=3x10(-9) M. However, since there are only very small amounts of JH available for degradation (due to the binding to Lp and Vg), the quantitative removal of JH from circulation, and this includes the release of bound JH, is indeed slow, with a measured half-life of 6-8 h. Classical kinetic assumptions are not met in conditions where the enzyme concentrations exceed by far that of the available substrate. Nonetheless, we attempted to determine the initial velocity of JH hydrolysis under natural conditions, i.e. for undiluted hemolymph, by measuring the initial velocities of JH hydrolysis in serially diluted hemolymph and extrapolating to zero dilution. For in vivo conditions we estimated an initial velocity of JH hydrolysis of <0.1 fmol microl hemolymph(-1) min(-1), i.e. four to five orders of magnitude lower than that measured at substrate saturation in vitro.     

Innervation of the foregut of the cockroach Leucophaea maderae and inhibition of spontaneous contractile activity by callatostatin neuropeptides

Abstract. The innervation of the gut of the cockroach Leucophaea madera (F.) has been studied by means of wholemount immunocytochemistry with antisera raised against Leu-callatostatin, a cockroach allatostatin homologue identified from neuropeptide isolation and gene studies in the blowfly Calliphora vomitoria. Leu-callatostatin-imunoreactive neurones in the brain, with axon trajectories in the stomatogastric nervous system, innervate the foregut and midgut. Neurones in the last abdominal ganglion supply the hindgut and the midgut via the proctodeal nerve. In addition to a rich callatostatin-immunoreactive nerve supply, the midgut, including the midgut caeca, contain numerous callatostatin-immunoreactive endocrine cells. Physiological studies show that the spontaneous contractile activities of the foregut, but not the hindgut, are inhibited by callatostatin neuropeptides. Leu-callatostatin 3 was the most potent of the range of Leu-and Met-callatostatins tested, with a dose-dependent response between 10^-13 and 10^-7 M. This is similar to the results obtained with the previously identified myoinhibitory peptide of L. maderae , leucomyosuppressin. However, this peptide, with a different type of structure to the allatostatins, inhibits both foregut and hindgut motility equally. Experiments with a series of analogues of the Met-callatostatins showed that the free acid (as opposed to the carboxyamidated peptide) and N-terminally truncated peptides were inactive. These morphological and physiological results are thought to be representative of the, as yet unidentified, naturally occurring allatostatin homologues of L. maderae. This family of peptides should be added to the increasing list of insect gut myoinhibitory substances.     

Evidence for two vitellogenin-related genes in Leucophaea maderae: the protein primary structure and its processing

We previously reported a cDNA for vitellogenin (Vg) from the cockroach, Leucophaea maderae (Lm). In the present study, we identified another cDNA encoding a second Vg (Vg2) having stretches of amino acid sequences different from the first one, Vg1, reported earlier. The complete nucleotide sequence of Vg2 consisted of 5,915 bp, which encoded a primary protein of 1,911 residues including a 16-residue putative signal peptide. The regions different in both Vg precursors (Pro-Vg1 and pro-Vg2) were four in number, and two, relatively longer, existed at the carboxy terminal. The presence of two Vg-related cDNAs was confirmed by sequencing of RT-PCR products generated using primers designed based on the common sequences flanking the regions different in amino acid sequences. Both forms were transcribed since they could be amplified on mRNA from fat bodies of different individual females. Southern blot analysis of digested genomic DNA revealed the existence of two Vg-related genes in L. maderae indicating that each Vg cDNA originated from a separate gene. Also, the immunoblot analysis using antibodies generated against peptides unique to both Vg1 and Vg2 probed the same antigen in the same individual, suggesting LmVg to be a product coded by two different Vg precursors. Both Vg primary products showed 96% similarity at an amino acid level. Compared to other insect Vgs, Vg2 showed a slightly higher (1-2%) similarity than Vg1. We previously reported, based on amino-terminal sequence analysis, that L. maderae pro-Vg was cleaved into four subunit polypeptides (112-, 100-, 92-, and 55-kD), which were deposited in the egg as four respective vitellin (Vn) polypeptides. We show now based on immunoblot analysis that the 112-kD polypeptide is further cleaved, near the C-terminus, to an 87-kD polypeptide before it is secreted into the hemolymph. Both the L. maderae Vgs were compared with each other and with other insect Vgs and the processing pattern is discussed.     

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.     

Oral toxicity to flesh flies of a neurotoxic polypeptide.

An insect selective neurotoxic polypeptide from venom of the scorpion Androctonus australis (AaIT, M(r) 8,000) was shown to cross the midgut of the flesh fly Sarcophaga falculata, using assays of oral toxicity, column chromatography, and microscopic autoradiography of the native and radioiodinated toxin. AaIT induced paralysis of flies within 1-2 h after oral administration, with a lethal dose (LD50) of 10 micrograms/100 mg of body weight. Oral toxicity was about 0.14% of toxicity by injection. Hemolymph collection 70-85 min after feeding flies with [125I]AaIT showed that 5% of ingested radioactivity appeared in hemolymph. Most of this represented degradation products, but included about 0.3% of the chromatographically intact toxin. In contrast, hemolymph of identically treated lepidopterous larvae (Manduca, Helioverpa [= Heliothis]) contained degradation products but no intact toxin. [125I]AaIT was shown to cross the midgut of Sarcophaga through a morphologically distinct segment of the midgut previously shown to be permeable to a cytotoxic, positively charged polypeptide of similar molecular weight. These results suggest that Sarcophaga midgut contains a morphologically and functionally distinct segment that transports small peptides, and that employment of neurotoxic polypeptides for insect control may be feasible. Activity might be greatly improved through modification and metabolic stabilization of active peptides.     

Isolation, identification and synthesis of locustamyoinhibiting peptide (LOM-MIP), a novel biologically active neuropeptide from Locusta migratoria.

A novel peptide termed locustamyoinhibiting peptide (LOM-MIP) was isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structure of this nonapeptide has been determined Ala-Trp-Gln-Asp-Leu-Asn-Ala-Gly-Trp-NH2. LOM-MIP suppresses the spontaneous contractions of the hindgut and oviduct of Locusta migratoria and of the hindgut of Leucophaea maderae. This novel peptide is, however, structurally different from leucomyosuppressin, a hindgut suppressing peptide isolated from Leucophaea maderae heads. LOM-MIP has a Gly-TrpNH2 carboxy-terminal in common with APGWamide, a penis retractor muscle inhibiting peptide isolated from the snail, Lymnea stagnalis. In addition, it shows carboxy-terminal sequence similarities with locust AKH II which ends in AGWamide. No sequence similarities were found with other vertebrate or invertebrate peptides. Synthetic LOM-MIP showed biological as well as chemical characteristics indistinguishable from those of native LOM-MIP.     

cDNA cloning of an adult male putative lipocalin specific to tergal gland aphrodisiac secretion in an insect (Leucophaea maderae).

Lma-P22 is a cuticular surface protein specific to the tergal gland secretion of Leucophaea maderae adult males which is ingested by females just before copulation. The complete Lma-P22 cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. The recombinant protein expressed in Escherichia coli was recognized by an anti-Lma-P22 antibody. Northern blot analysis indicates that the corresponding mRNA is transcribed only in the epidermis of male tergites. Sequence analysis indicated that Lma-P22 deduced protein belongs to the lipocalin family. Lipocalins are extracellular proteins which carry hydrophobic compounds and some of them can bind sexual pheromone in vertebrates. Lma-P22 is the first example of a lipocalin-like protein involved in insect sexual behavior.     

Captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfly Neobellieria bullata.

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata. In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE. Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal-induced trypsin peak in the midgut and elevated levels of protein meal-induced yolk polypeptides in the hemolymph. The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. Arch.     

Myotropic effect of helicokinins,tachykinin-related peptides and Manduca sexta allatotropin on the gut of Heliothis virescens (Lepidoptera: Noctuidae)

Different insect neuropeptides (helicokinins, tachykinin-related and allatoregulating peptides) were investigated with regard to their myostimulatory effects using whole-gut preparations isolated from fifth instar Heliothis virescens larvae. The experiments demonstrated that representatives of all three peptide families are able to induce and amplify gut contractions in this species in a dose-dependent manner. Structure-activity studies (alanine scan, D-amino acid scan and truncated analogues) with the helicokinin Hez-K1 supported the finding, that the core sequence for biological activity of kinins is the amidated C-terminal pentapeptide (FSPWG-amide). Similar investigations with insect tachykinin isolated from Leucophaea madera (Lem-TRP1) revealed that the minimum sequence evoking a physiological gut response in H. virescens is the amidated hexapeptide (GFLGVR-amide), which represents the conserved amino acid sequence for Leucophaea TRPs in general. The peptide concentration causing a half-maximal gut contraction (EC(50)) for Lem-TRP1 was about 26 nM. Although the potency of Lem-TRP1 was 9-fold lower compared with Hez-KI (EC(50): 3 nM), the maximal tension of the gut obtained with Lem-TRP1 was 1.7-fold higher compared with Hez-KI. The EC(50) of Manduca sexta allatotropin (Mas-AT; 79 nM) was of lowest potency among all three peptides tested. In a pharmacological study, co-incubation experiments with Lem-TRP1, Hez-KI or Mas-AT and compounds interfering with signal transduction pathways were employed to investigate the mode of action of the myotropic effects of these peptides. Cadmium and the protein kinase C (PKC) inhibitor tamoxifen attenuated the contractile effects of all three peptides tested. The data suggest that in the gut muscle of H. virescens the myotropic peptides bind to G-protein-coupled receptors that cause contraction by promoting the entry of extracellular calcium mediated by a PKC involved pathway.     

Hemolymph and tissue-bound peptidase-resistant analogs of the insect allatostatins

While neuropeptides of the allatostatin family inhibit in vitro production of juvenile hormone, which modulates aspects of development and reproduction in the cockroach, Diploptera punctata, they are susceptible to inactivation by peptidases in the hemolymph, gut, and bound to internal tissues. Patterns of peptidase cleavage were investigated in two allatostatin analogs in which sterically bulky components were incorporated into the active core region to block peptidase attack. The results were used to design and synthesize the first pseudopeptide analog of an insect neuropeptide resistant to degradation by both hemolymph and tissue-bound peptidases. This pseudotetrapeptide allatostatin mimetic analog represents a valuable tool to neuroendocrinologists studying mechanisms by which the natural peptides operate and the physiological consequences of challenging an insect with an allatostatin that is not readily degraded via peptidase enzymes. Disruption of critical physiological processes modulated by neuropeptides such as the allatostatins via peptidase-resistant mimetic analogs could form the basis for novel pest insect management strategies in the future.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)⁶]- and [D-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH₂)⁶-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Isolation and partial characterization of five myotropic peptides present in head extracts of the cockroach Leucophaea maderae

Five peptides were isolated by reverse-phase HPLC from head extracts of the cockroach Leucophaea maderae. Four of the peptides were inactivated by aminopeptidase M (APM). The inability of APM to digest the fifth peptide suggests a blocked NH2-terminus. Four of the peptides were inactivated by carboxypeptidase Y (CPY). The activity of the fraction which would have contained proctolin was decreased by about 20%. The complete deactivation of proctolin by CPY indicated that a second peptide, co-eluting with proctolin but refractory to CPY digestion, was responsible for 80% of the biological activity in that fraction. Concentrations of the peptides necessary to produce a threshold response from the isolated cockroach hindgut ranged from 0.009 to 0.083 head equivalents/ml.     

The folliculostatins of two dipteran insect species, their relation to matrix proteins and prospects for practical applications

The first insect folliculostatic peptide was isolated from vitellogenic ovaries of the mosquitoAedes aegypti. This decapeptide directly inhibits trypsin biosynthesis in the gut, and indirectly ovarian development. The factor was named Trypsin Modulating Oostatic Factor or TMOF by its discoverers. From the fleshfly Neobellieria bullata 2 folliculostatins have been isolated, the hexapeptide Neb-TMOF and the 19-mer Neb-colloostatin. The available data suggest that at least 2 of the 3 folliculostatins originate from matrix (like) proteins present in the ovary, a hitherto unknown source of hormones. Furthermore, one of the folliculostatins (Neb-TMOF) is a potent inhibitor of ecdysone biosynthesis by larval ring glands of fleshflies. The discovery of the dipteran folliculostatins, which do not show any resemblance to inhibins of vertebrates, may significantly contribute to a better understanding of the hormonal control of growth in insects and perhaps, in other animals as well. None of the 3 folliculostatins is blocked at its N- or C-terminus. This, in combination with the pleiotropy of their effects and the narrow species specificity make such peptides prime candidates for, testing their potential in insect pest control by means of molecular biological methods.     

Lom-AG-myotropin: a novel myotropic peptide from the male accessory glands of Locusta migratoria

A myotropic peptide, termed Lom-AG-myotropin, was isolated from extracts of 4400 accessory gland complexes of males of the locust, Locusta migratoria; the following sequence was derived: Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH2. This sequence is completely different from all presently known myotropic peptides from Locusta or other insects. The Lom-AG-myotropin is active on the oviduct and hindgut of Locusta migratoria and Leucophaea maderae. The stimulatory activity is, in both insects, 1000 times greater on the oviduct than on the hindgut, suggesting a specificity for the oviduct.     

Locustatachykinin III and IV: two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family

Two myotropic peptides termed locustatachykinin III and IV were isolated from 9000 brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structures of Lom-TK III and IV were established as amidated decapeptides: Ala-Pro-Gln-Ala-Gly-Phe-Tyr-Gly-Val-Arg-NH2 (Lom-TK III) and Ala-Pro-Ser-Leu-Gly-Phe-His-Gly-Val-Arg-NH2 (Lom-TK IV). The locustatachykinins were synthesized and shown to have chromatographic and biological properties identical with those of the native materials. They stimulate visceral muscle contractions of the oviduct and the foregut of Locusta migratoria and of the hindgut of Leucophaea maderae. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence similarity is greater with the fish and amphibian tachykinins (up to 40%) than with the mammalian tachykinins. In addition, the intestinal and oviducal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. Both chemical and biological similarities of vertebrate and insect tachykinins substantiates the evidence for a long evolutionary history of the tachykinin peptide family.     

Tachykinin-related peptide precursors in two cockroach species

Tachykinins and tachykinin-related peptides (TKRPs) play major roles in signaling in the nervous system and intestine of both invertebrates and vertebrates. Here we have identified cDNAs encoding precursors of multiple TKRPs from the cockroaches Leucophaea maderae and Periplaneta americana. All nine LemTKRPs that had been chemically isolated in earlier experiments could be identified on the precursor of L. maderae. Four previously unidentified LemTKRPs were found in addition on the precursor. The P. americana cDNA displayed an open reading frame very similar to that of L. maderae with 13 different TKRPs. MALDI-TOF mass spectra from tissues of both species confirms the presence of all the TKRPs encoded on the precursor plus two additional peptides that are cleavage products of the N-terminally extended TKRPs. A tissue-specific distribution of TKRPs was observed in earlier experiments at isolation from brain and midgut of L. maderae. Our data do not suggest a differential gene expression but a different efficacy in processing of LemTKRP-2 and Lem/PeaTKRP-3 in the brain and intestine, respectively. This results in a gut-specific accumulation of these extended peptides, whereas in the brain their cleavage products, LemTKRP-1 and LemTKRP-3(11-19), are most abundant. Mass spectrometric analysis demonstrated the occurrence of the different TKRPs in single glomeruli of the tritocerebrum and in cells of the optical lobe.     

Comparative pharmacological actions of leucokinins V-VIII on the visceral muscles of Leucophaea maderae

1. Leucokinins V-VIII (Lem-K-V to VIII) did not activate visceral muscles of the cockroach Leucophaea maderae uniformly as a group but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 2-20% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100- to 1000-fold less sensitive than the hindgut, and each of the former required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, did not respond to any of these peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. These results suggest that nonsynaptic receptors for the peptides exist in visceral muscle.