Proctolin, its presence in and action on the oviduct of an insect

Proctolin has been isolated from oviduct extracts of Leucophaea maderae by HPLC. Quantitative bioassay with the hindgut of L. maderae demonstrated a proctolin titre of 0.93 +/- 0.15 ng/oviduct. Exposure to proctolin produced three changes in the spontaneous contractile activity of the oviduct: an increase in muscle tonus, an increase in the amplitude and frequency of phasic contractions. The sensitivity of the oviduct to proctolin was compared with the hindgut and foregut organ preparations from the same insect. Oviducts were responsive to proctolin in a calcium-free medium and the peptide also appeared to facilitate the reentry of calcium after depleted preparations were returned to normal levels of external calcium.     

Comparative actions of the neuropeptides leucopyrokinin and periplanetin CCI on the visceral muscle systems of the cockroaches Leucophaea maderae and Periplaneta americana

1. The effects of two structurally-related peptides, leucopyrokinin (LPK) and periplanetin CC-I (CCI), on contractile activities of visceral muscle systems were compared in the two cockroaches from which these peptides were originally isolated.2. LPK elicited consistent proctolin-like responses on the hindgut, foregut, oviduct and heart of the Madeira cockroach, Leucophaea maderae, with increases in both amplitude and frequency of contraction. CCI, on the other hand, elicited a mostly tonic response on these tissues.3. For the American cockroach, Periplaneta americana, the responses elicited by LPK and CCI were tonic in nature.4. With the exception of the response of the L. maderae hindgut and heart to LPK, threshold levels for either LPK or CCI on all other tissues of both roaches were considerably higher (10–100 times greater) than those for proctolin on the same tissues.5. The maximum response to any concentration of LPK or CCI on the foregut and oviduct of L. maderae and that on the foregut and hindgut of P. americana never reached more than 60% of the maximum contraction achieved with proctolin.     

The effect of proctolin analogues and other peptides on locust oviduct muscle contractions

The locust oviduct bioassay system was used to assess the ability of a variety of peptides to induce oviducal contractions. Proctolin analogues were three orders of magnitude less potent than proctolin. Proctolin supra-analogue and Arg-Tyr-Leu-Ala-Thr demonstrated high activity. Perhaps the most significant finding was the discrepancy between the high binding capacity of the proctolin analogue Arg-Tyr-Ser-Pro-Thr and its relatively low myotropic activity. This observation argues for a crucial role for the leucine residue in activating the proctolin receptor. Several other myotropic peptides were tested for their effect on oviduct contractions. FMRFamide caused contractions at doses several orders of magnitude higher than proctolin. The FLRFamide leucomyosuppressin inhibited proctolin-induced contractions. In addition, myomodulin and catch relaxing peptide caused oviducal contractions at low concentrations. The enkephalins had no effect when applied alone but potentiated proctolin-induced oviduct contractions. The mechanism of the potentiation is not known. The data argue for the presence of several binding sites on the oviduct membrane.     

Proctolin's role in neurally evoked contractions of the locust oviducts

The effects of proctolin (RYLPT) on neurally evoked contractions of locust oviduct muscle were studied to examine the role of proctolin as a cotransmitter. Increasing the number of stimuli in a burst (from one to 30 stimuli) resulted in an increase in amplitude of contraction of locust oviduct muscle. Proctolin was capable of increasing the amplitude of neurally evoked contractions at lower-stimulus regimes (one- and two-stimulus bursts) but did not do so at higher-stimulus regimes (five- and 10-stimulus bursts). The effects of proctolin were dose dependent within the one- and two-stimulus regimes, with thresholds at 10⁻⁹ M and maxima at 2.5 × 10⁻⁸ M. Addition of proctolin increased the basal tonus and size of a postcontraction relaxation of the oviduct muscle in a dose-dependent manner during all stimulus regimes. However, the effect of proctolin on basal tonus and the postcontraction relaxation was much less at the higher stimulus regimes. Previously, several proctolin analogues have been tested for their ability to antagonize proctolin-induced contractions of the oviduct muscle. Since proctolin is proposed to be a cotransmitter at this neuromuscular junction, one of these analogues, cycloproctolin, was used to antagonize proctolin's effects on neurally evoked contractions. In the presence of the antagonist, the maximum amplitude induced by application of proctolin was decreased by 22.7%, while the proctolin-induced increase in basal tonus was decreased by 45.8%. Finally, the maximum increase in the size of the postcontraction relaxation caused by proctolin was lowered by 32.0%. The results of the present study show that exogenously applied proctolin is an excitant of the oviduct muscle at lower, rather than higher, stimulus regimes, and this latter inaction may be due to the corelease of endogenous proctolin during increased neural stimulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 139–150, 1997     

Identification of proctolin in the central nervous system of the horseshoe crab, Limulus polyphemus

A proctolin-like peptide was isolated from the prosomal CNS of the chelicerate arthropod, Limulus, and purified using size exclusion, ion exchange and high performance liquid chromatography. Coincident bioassay (cockroach hindgut) and radioimmunoassay were employed to identify fractions which contained proctolin-like material. Proctolin-like activity coeluted with synthetic proctolin with all three chromatographic techniques employed. When applied to either the Limulus heart or hindgut preparations, purified Limulus proctolin produced excitatory responses which were indistinguishable from those produced by the synthetic peptide. Purified samples of the Limulus proctolin-like peptide were subjected to Edman degradation and tandem mass spectrometry and the amino acid sequence of the Limulus peptide was determined to be identical to that of cockroach proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH). The presence of proctolin in the Limulus CNS and its biological action on the isolated heart and hindgut suggest a physiological role for this peptide in the regulation of cardiac output and hindgut motility.     

Structure function analysis of an arthropod peptide hormone: proctolin and synthetic analogues compared on the cockroach hindgut receptor

Proctolin is a pentapeptide (arg-tyr-leu-pro-thr) found in nervous tissues throughout the phylum Arthropoda. Initially described as a peptidergic neuromuscular transmitter, it now appears that proctolin is a major arthropod neurohormone modulating nervous activity, muscle tonus and contractile force. Structure-function studies with synthetic analogues demonstrate diverse peptides which retain agonistic activity, but few exhibit a high degree of affinity for the cockroach hindgut receptor compared with proctolin (Kdapp = 2 x 10(-8) M). High affinity agonists (Kdapp less than or equal to 10(-7) M) are limited to [phe2]-proctolin, [lys1]-proctolin and specific N-terminal additions. In this regard the hindgut receptor differs in its ligand specificity from that reported for the locust extensor tibia receptor. Using the analogue studies to predict sequences which may act as agonists, we have examined the known vertebrate peptide hormones for proctolin-like sequences. A possible relationship between vasoactive intestinal peptide, proctolin and erythrophore concentrating hormone is critically evaluated.     

Effects of FMRFamide-related peptides and morphine on the isolated foregut of the locust schistocerca gregaria

1. Morphine and YAGFMamide were the most effective potentiators of 5-hydroxytryptamine (5-HT)-induced relaxation of the isolated foregut.2. Morphine had no effect on proctolin-induced tissue contraction which was inhibited by YGGF-Mamide and YFMRFamide.3. The differing potency of FaRPs and morphine to potentiate 5-HT effects and reduce proctolin responses suggests that there are two separate FaRP receptor sub types.4. This proposal is supported by the observation that, while naloxone (10⁻⁵ M) is a relatively potent antagonist of FaRP induced inhibition of proctolin contraction, it has less effect on FaRP-induced potentiation of 5-HT-induced relaxation.     

Comparative biological activities of potent analogues of alpha-melanotropin. Effect of nonaromatic and para substituted aromatic amino acids at position 7

Several alpha-melanotropin (alpha-MSH) analogues with para substituted aromatic and nonaromatic amino acids in the 7-position of the hormone were prepared and their melanotropic activities determined in the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. D and L-Phe(p-NO2), D- and L-Tyr, D- and L-Ala, and Gly were substituted in the 7-position. The use of substituted D or L-aromatic amino acids in the 7th position of the central Ac-[Nle4]-alpha-MSH4-11-NH2 fragment resulted in a loss in potency relative to the corresponding phenylalanine-containing analogue. The loss in potency cannot be due entirely to steric hindrance at the melanophore receptor, since nonaromatic amino acids substituted in the 7th position of this octapeptide fragment also generally led to a loss in biological activity. We reported previously that replacement of phenylalanine-7 by its D enantiomer led to a marked increase in potency in each fragment analogue tested. Analogues containing other D amino acids in the 7th position also were more potent than their L amino acid-containing analogues with one exception: Ac-[Nle4, Ala7]-alpha-MSH4-11-NH2 was more potent than Ac-[Nle4, D-Ala7]-alpha-MSH4-11-NH2 in the frog skin bioassay. Replacement of phenylalanine-7 by glycine resulted in a large decrease in potency in both bioassays, illustrating the importance of the side chain group, in this position of alpha-MSH, to biological potency of the hormone.     

Synthesis and opioid activity of 2-substituted dynorphin A-(1-13) amide analogues.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.     

Proctolin: a review with emphasis on insects

The distribution, physiological role, mode of action, and pharmacology of the pentapeptide neuroregulator proctolin are reviewed, with special emphasis on insects. Whereas proctolin is distributed extensively throughout arthropods, its presence in molluscs, annelids, or chordates is not well established. In the arthropods, proctolin acts as a neuromodulator and possibly as a neurohormone. It does not appear to function as a conventional neurotransmitter. Two model proctolinergic systems are highlighted: motor control of the visceral muscles of the locust oviduct and of the skeletal muscles of the locust ovipositor. In these preparations proctolin is a cotransmitter acting to enhance neuromuscular transmission and muscular contraction. The mode of action of proctolin is not well understood, although the second messengers cAMP, phosphatidyl inositol, and calcium have been implicated in various systems. Pharmacologically, the proctolin receptor has been examined with structure/activity studies, and the effects of a variety of amino acid substitutions and deletions of the pentapeptide are described. It is unfortunate that no specific antagonists of the proctolin receptor appear to be available and that no receptor-binding studies have been reported. The prospects are good for advances in our understanding of modulatory mechanisms, since proctolin appears to be emerging as the model for studies of this type.     

The role of proctolin and glutamate in the excitation-contraction coupling of insect visceral muscle

Proctolin (1 X 10(-10) to 1 X 10(-9) M) had a minimal effect on the spontaneous and evoked electrical events of the hindgut of the cockroach Leucophea maderae. Spontaneous action potentials and contractile activity stopped when the hindgut was exposed to 2 mM Mn2+. Eighty per cent of the response of the hindgut to glutamate was blocked by manganese, but only 35% of the response to proctolin. Hindguts were responsive to proctolin in a calcium-free medium, but not to glutamate. Moreover, proctolin appeared to facilitate the reentry of calcium after depleted preparations were returned to normal levels of external calcium. The results offer evidence for two calcium transmembrane channels in insect visceral muscle.     

Peptide conformations--49(1): synthesis and structure-activity relationships of side chain modified peptides of cyclo(-D-Pro-Phe-Thr-Lys-Trp-Phe.)

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe2, Lys4, and Phe6 of cyclo(-D-Pro1-Phe2-Thr3-Lys(Z)4-Trp5-Phe6-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys4.     

Myotropic activity and immunolocalization of selected neuropeptides of the burying beetle Nicrophorus vespilloides (Coleoptera: Silphidae)

Burying beetles (Nicrophorus sp.) are necrophagous insects with developed parental care. Genome of Nicrophorus vespilloides has been recently sequenced, which makes them interesting model organism in behavioral ecology. However, we know very little about their physiology, including the functioning of their neuroendocrine system. In this study, one of the physiological activities of proctolin, myosuppressin (Nieve? MS), myoinhibitory peptide (Trica-MIP-5) and the short neuropeptide F (Nicve-sNPF) in N. vespilloides have been investigated. The tested neuropeptides were myoactive on N. vespilloides hindgut. After application of the proctolin increased hindgut contraction frequency was observed (EC50 value was 5.47 x 10-8 mol/L). The other tested neuropeptides led to inhibition of N. vespilloides hindgut contractions (Nicve-MS: IC50 = 5.20 x 10~5 mol/L;Trica-MIP-5: IC50 = 5.95 x 10-6 mol/L;Nicvc-sNPF: IC50 = 4.08 x 10-5 mol/L). Moreover, the tested neuropeptides were immunolocalized in the nervous system of N. vespilloides. Neurons containing sNPF and MIP in brain and ventral nerve cord (VNC) were identified. Proctolin-immunolabeled neurons only in VNC were observed. Moreover, MIP-immunolabeled varicosities and fibers in retrocerebral complex were observed. In addition, our results have been supplemented with alignments of amino acid sequences of these neuropeptides in beetle species. This alignment analysis clearly showed amino acid sequence similarities between neuropeptides. Moreover, this allowed to deduce amino acid sequence of N. vespilloides proctolin (RYLPTa), Nicve-MS (QDVDHVFLRFa) and six isoforms ofNicve-MIP (Nicve-MIP-1一 DWNRNLHSWa;Nicve-MIP-2—AWQNLQGGWa;Nicve-MIP-3—AWQNLQGGWa;Nicve-MlP-4—AWKNLNNAGWa;Nicve-MIP-5—SEWGNFRGSWa;Nicve-MIP-6— DPAWTNLKGIWa;and Nicve-sNPF—SGRSPSLRLRFa).     

Halogenated tryptophan derivatives disrupt essential transamination mechanisms in bloodstream form Trypanosoma brucei

Amino acid metabolism within Trypanosoma brucei, the causative agent of human African trypanosomiasis, is critical for parasite survival and virulence. Of these metabolic processes, the transamination of aromatic amino acids is one of the most important. In this study, a series of halogenated tryptophan analogues were investigated for their anti-parasitic potency. Several of these analogues showed significant trypanocidal activity. Metabolomics analysis of compound-treated parasites revealed key differences occurring within aromatic amino acid metabolism, particularly within the widely reported and essential transamination processes of this parasite.     

New Proctolin Analogues: Synthesis and Biological Investigation in Insects

We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X¹-Tyr-Leu-Pro-Thr-OH, where X¹ = D-Arg (I), N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn(iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) and D-Lys(Nic) (IX). In analogues I–IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) or homo-Arg (compound VII). Analogues I–IX were evaluated for myotropic activity on the in vitro heart preparation of Tenebrio molitor, whereas peptides II, V, and VII–IX were tested for contractile activity on the isolated foregut of locust Schistocerca gregaria. Peptide II and III showed full cardiotropic activity in T. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.     

Systematic evaluation of the metabolic to mitogenic potency ratio for B10-substituted insulin analogues

Background

Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio.

Methodology/Principal Findings

A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed.

Conclusions/Significance

Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.     

Endomorphin 2 analogues containing Dmp residue as an aromatic amino acid surrogate with high mu-opioid receptor affinity and selectivity

To investigate the effectiveness of a 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid surrogate, endomorphin 2 (EM(2): Tyr-Pro-Phe-Phe-NH(2)) analogues were prepared, in which the constitutive aromatic amino acids (Tyr(1), Phe(3), or Phe(4)) were replaced by Dmp or its isomer, D-Dmp. Replacement of Phe(3) by Dmp increased the affinity over 10-fold for both mu- and delta-opioid receptors, without affecting receptor selectivity. In contrast, replacement of Phe(4) considerably reduced the mu-receptor affinity and selectivity. These data indicated that the Dmp-substitution of Phe(3), but not Phe(4), in EM(2) is favorable for improving mu-receptor specificity. Inversion of the chirality of the substituted Dmp residue resulted in marked decrease in the mu-receptor affinity. Replacement of Tyr(1) by Dmp yielded an analogue that exhibited only a limited decrease in mu-receptor affinity and GPI potency, despite the lack of a phenolic hydroxyl group at the N-terminal residue. In contrast, D-Dmp(1)- or Phe(1)-substitution of Tyr(1) resulted in a significant decrease in mu-receptor affinity and GPI potency. These results suggested that the Dmp residue can mimic Tyr(1), which is one of the critical structural elements of opioid peptides.     

Quantification of cockroach allatostatin-like peptide and its myotropic effects in males of the earwig Euborellia annulipes

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to establish a competitive enzyme‐linked immunosorbent assay for quantification of allatostatin‐like peptides in the hindgut of the adult male earwig, Euborellia annulipes. Hindguts of 0‐day males contained significantly more allatostatin‐positive material than those of 8‐day males fed on catfood. However, males starved for the first 8 days of adult life had significantly higher levels of allatostatin‐positive material than those of either 0‐day or of 8‐day fed males. Hindguts from 0‐day old males exhibited lower spontaneous motility in vitro than those from 8‐day males. Hindguts from males at both ages responded to allostatin with reversible, dosage‐dependent decreases in hindgut motility, and responded to proctolin with reversible, dosage‐dependent increases in hindgut motility. When both allatostatin and proctolin were applied to hindgut preparations simultaneously and in equal concentrations, the response varied with the stage of the male. Starvation enhanced hindgut motility and abolished the response to allatostatin, but not to proctolin. These results indicate the presence of material similar to cockroach allatostatins in male earwigs, and that the levels change with age and physiological stage. Furthermore, such peptides may indeed be regulatory neuropeptides and could modulate hindgut contraction. There was an increase in sensitivity to exogenous allatostatin in the hindgut during development from day 0 to day 8 in feeding males, but a loss in sensitivity in response to starvation; sensitivity to exogenous proctolin also increased with age, but such responsiveness was not diminished by starvation.     

Structural requirements at the N-terminus of urotensin II octapeptides

Urotensin II is the latest of a growing list of peptides exhibiting potent cardiovascular effects. It is an extremely potent vasoconstrictor in primates; its excretion is elevated in hypertensive patients thus suggesting therapeutic potential for urotensin II analogues, particularly receptor antagonists. In the present study, a number of interesting structural features pertaining to the N-terminus of urotensin II have been evaluated for binding to cloned human and rat urotensin II receptors and functional effects on rat upper thoracic aorta smooth muscle preparations. Shortened octapeptides retained full binding affinities and functional activities, did not require a free N-terminal amino group, and could tolerate an amidated C-terminus. The N-terminal Asp residue present in the octapeptides did not require a negatively charged side chain, merely one which contained a hydrogen bond acceptor CO group which could be present at a variety of positions on the side chain. The side chain could be constrained into a trans-olefinic configuration with full retention of potency, but potency was lost in the cis configuration. N-terminal aromatic amino substituted with polar groups such as OH and NO₂ also resulted in high affinity analogues. Overall, the correlation between binding affinities for the human and rat receptors was quite good. These findings could be of value in the development of more potent urotensin II receptor antagonists based on the previously identified somatostatin antagonist octapeptides which we have recently found, function as relatively weak urotensin II antagonists.     

A comparison of prostaglandin F2alpha and three 16-aryloxy analogues on the isolated rabbit jejunum

Three 16-aryloxy analogues of PGF2alpha are potent, full agonists on the isolated rabbit jejunum. Their actions are more prolonged than that of PGF2alpha, and radioactive tracer studies with one of the analogues reveal a slower wash-out of the analogue compared to PGF2alpha, under superfusion conditions. During the prolonged contractile response diminished responses to PGF2alpha were obtained: this effect was investigated in terms of receptor desensitization. The actions of these analogues were also investigated on the isolated guinea-pig ileum and the rabbit oviduct in vivo.