Peptidergic regulation of the Limulus midgut

1. The morphology and innervation of the midgut (intestine) in the horseshoe crab, Limulus polyphemus was investigated. The organization of this tissue was examined with routine histology. Radioimmunoassay, immunohistochemistry and high performance liquid chromatography were employed to detect, localize and identify peptidergic innervation of the midgut. The actions of synthetic and native proctolin-like and FMRFamide-like peptides were compared on the isolated midgut preparation. 2. Levels of proctolin and FMRFamide were determined in extracts of Limulus midgut tissue using radioimmunoassay. High levels of proctolin-like immunoreactivity (69.5 +/- 11.3 ng/g) were detected, while levels of FMRFamide-like immunoreactivity (0.8 +/- 0.2 ng/g) were less. Proctolin levels were equally distributed, while the levels of FMRFamide-like immunoreactivity exhibited an anterior bias. 3. Proctolin- and FMRFamide-like immunoreactivities in the Limulus midgut were localized with immunohistochemistry. Proctolin- and FMRFamide-immunoreactive elements were detected in intestinal nerve branches and individual fibers running along the surface of the midgut in whole-mount preparations. In sectioned tissue, staining for these peptides was observed throughout the midgut, typically associated with muscle bands and fibers. Only a few immunoreactive cell bodies were observed. 4. Proctolin, and several FMRFamide-like peptides produced distinct and opposing actions on the isolated Limulus midgut preparation. Proctolin elicited contracture and rhythmic contractions of this tissue, while FMRFamide and N-terminally extended analogs of FLRFamide relaxed gut tension. FMRFamide-like peptides partially reversed the excitatory actions of proctolin. 5. Proctolin- and FMRFamide-like peptides in Limulus midgut extracts were partially characterized with high performance liquid chromatography. One peak of proctolin-like activity was detected on a linear gradient of 18 to 31.5% acetonitrile. The native proctolin-like peptide produced excitatory actions on the isolated midgut preparation which were indistinguishable from those produced by synthetic proctolin. Several peaks of FMRFamide-like bioactivity (Busycon radula protractor muscle assay) were detected with a linear gradient of 5 to 30% acetonitrile. Fractions from two distinct peaks produced FMRFamide-like inhibitory effects on the isolated Limulus midgut preparation. These findings suggest a role for proctolin-like and FMRFamide-like peptides as regulators of intestinal motility in Limulus.     

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.     

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.     

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.     

Transgenic expression in tobacco of a poly-proctolin construct leading to production of the bioactive peptide

Short peptides can be expressed in plants using synthetic genes encoding multiple copies of the peptide spaced by dibasic endoproteolytic cleavage sites. A synthetic gene encoding an array of repeated copies of proctolin, a very well characterized insect myotropic peptide, spaced by Arg residues, was synthesized and expressed in tobacco plants. The successful production of bioactive proctolin from the precursor in transgenic plants was demonstrated by immunoblot, HPLC, mass spectrometry and a bioassay based on the contraction of isolated cockroach hindgut. These results suggest that in planta, as in animals and yeasts, endopeptidases of the serine proteases family may be involved in precursor processing.     

Partial characterization and isolation of earwig `allatostatins'': biological activities in earwigs and cockroaches

Allatostatins are a family of neuropeptides first isolated from the cockroach, Diploptera punctata, that inhibit juvenile hormone production in that species (but do not do so in earwigs), and inhibit hindgut muscle contractions in some insects, including the earwig, Euborellia annulipes. We examined whether material from earwig brains is similar to cockroach allatostatins biochemically, immunologically and physiologically. Brain extracts from adult female earwigs were separated by high performance liquid chromatography (HPLC), followed by radioimmunoassay using antibodies to cockroach allatostatin (Dip-AST). Fractions that co-eluted with cockroach allatostatins were immunoreactive, and at least two peaks of immunoreactivity were detected. Material from each peak at 10 nM Dip-AST equivalents inhibited juvenile hormone biosynthesis in vitro by corpora allata of 2-day virgin D. punctata cockroaches; 1 nM was less effective, and non-immunoreactive fractions failed to inhibit juvenile hormone biosynthesis. Both crude and Sep-Pak (Waters) purified extracts of brains of earwigs containing 1 nM Dip-AST equivalents failed to suppress hindgut contractions in vitro of 2-day earwigs and of brooding female earwigs. In contrast, 1 nM cockroach allostatin 1 (Dip-AST 7) reversibly inhibited hindgut contractions in vitro. These results suggested the presence of another brain factor, such as proctolin, that counteracts the inhibitory effects of Dip-AST. In support of this hypothesis, proctolin stimulated hindgut contractions in vitro at 1 nM; the effects of equal concentrations of allatostatin and proctolin varied with the stage of the female. Furthermore, HPLC-separated fractions that co-eluted with cockroach allatostatin and were immunoreactive with antibodies to Dip-AST suppressed hindgut contractions in vitro of 2-day female earwigs. Finally, crude brain extracts of earwigs suppressed earwig juvenile hormone biosynthesis in vitro in glands of low, but not in glands of high, activity. Thus, earwig brain extract after HPLC separation has Dip-AST-like material that inhibits cockroach corpora allata and suppresses earwig hindgut contractions. Sep-Pak-extracted earwig brain material, however, does not inhibit earwig gut contraction. Although synthetic Dip-AST 7 does not inhibit juvenile hormone synthesis by earwig corpora allata, there is heat-stable material in earwig brain extract that does have this action.     

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.     

Isolation of proctolin,a myotropic peptide,from Periplaneta americana

The isolation of a myotropic substance, proctolin, associated with the innervation of the viscera of the cockroach, Periplaneta americana, is reported. Proctolin, previously described as a neurotransmitter or neurohormone, is a small basic peptide with an estimated molecular weight of 500 to 700. The proctodeal (hindgut) muscles of P. americana contract at threshold levels of about 0·5 ng/ml, equivalent to about 10⁻⁹ M proctolin. Approximately 180 μg proctolin were isolated from 125 kg of whole cockroaches. The isolated peptide was homogeneous on paper and thin-layer chromatography and on high-voltage paper electrophoresis at pH 3·5 and 6·4.     

Proctolin: A possible releasing factor in the corpus cardiacum/corpus allatum of the locust

The corpus cardiacum (CC) and corpus allatum (CA) of the locust, Locusta migratoria, contain intense proctolin-like immunoreactivity (PLI) within processes and varicosities. In contrast, in the cockroach, Diploptera punctata, although a similar staining pattern occurs within the CC, PLI appears absent within the CA. The possible role of proctolin as a releasing factor for adipokinetic hormone (AKH) and juvenile hormone (JH) was investigated in the locust. Proctolin caused a dose-dependent increase in AKH I release (determined by RP-HPLC) from the locust CC over a range of doses with threshold above 10(-8)M and maximal release at about 10(-7)M proctolin. Isolated glandular lobes of the CC released greater amounts of AKH I following treatment with proctolin and in these studies AKH II was also released. Confirmation of AKH I release was obtained by injecting perfusate from incubated CCs into locusts and measuring hemolymph lipid concentration. Perfusate from CC incubated in proctolin contained material with similar biological activity to AKH. Proctolin was also found to significantly increase the synthesis and release of JH from locust CA, with the increase being greatest from CAs that had a relatively low basal rate of JH biosynthesis (<35 pmol h(-1) per CA). In contrast, proctolin did not alter the synthesis and release of JH from the cockroach CA. These results suggest that proctolin may act as a releasing factor for AKHs and JH in the locust but does not act as a releasing factor for JH in the cockroach.     

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.     

A proctolin-like peptide and its immunocytochemical localization in the Colorado potato beetle,Leptinotarsa decemlineata

Summary Evidence is presented that neurons in the adult Colorado potato beetle contain a proctolin-like substance. By use of immunocytochemical methods the location of immunoreactive neurons in the central and stomatogastric nervous systems is described. No such neurons were found in the proto- and deutocerebrum or optic lobe. Few immunoreactive neurons are present in the tritocerebrum and numerous proctolin-immunoreactive neurons occur in all ventral ganglia and in the frontal ganglion. Two groups of neurosecretory cells in the suboesophageal ganglion contain a proctolin-immunoreactive substance. In these cells this material is co-localized with a bovine pancreatic polypeptide/FMRF amide-like substance and with a vasopressin/vasotocin/oxytocin-like substance. Proctolin-immunoreactive axon terminals were found on the musculature of the fore- and hindgut and of the vas deferens, and on some segmental muscles. Furthermore, proctolin-immunoreactive neurosecretory axon terminals were found in the corpus cardiacum. The proctolin-like substance may therefore function both as a neurotransmitter/neuromodulator and as a neurohormone. The presence of a proctolin-like substance was also demonstrated with a sensitive bioassay. On fractionation of extracts of the nervous systems of Leptinotarsa decemlineata with high performance liquid chromatography most of the proctolin-like bioactive material comigrated with authentic proctolin. This shows that a proctolin-like substance in this insect is very similar to, if not identical with, the known pentapeptide proctolin.     

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.     

Cockroach oviducts: The presence and release of octopamine and proctolin

The oviducts of the cockroach, Periplaneta americana, contain octopamine (determined by radioenzymatic assay) and proctolin (determined from proctolin-like bioactivity following separation on reversed-phase high performance liquid chromatography). Octopamine content is 5–10-fold greater than proctolin content. Both substances are released in a calcium-dependent manner following depolarisation with high potassium saline. This method resulted in the release of 14% of the total store of octopamine, but only 0.3% of the total store of proctolin. The results are discussed in relation to the recent demonstration of octopamine-containing and proctolin-containing neurones associated with locust oviducts.     

[des-Arg1]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg¹]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg¹]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg¹]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg¹]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg¹]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.     

The association of proctolin with a ventral abdominal muscle of Locusta migratoria

The association of proctolin with the external ventral protractor muscle of the VIIth abdominal segment (M234) of Locusta migratoria was investigated using immunohistochemistry and RP-HPLC in conjunction with the sensitive locust oviduct bioassay. Immunohistochemistry of whole-mount tissues revealed two proctolin-like immunoreactive axons in N2B_2b1 (the nerve branch which innervates M234) as well as immunoreactive processes and varicosities on the surface of M234. Immunogold staining of M234 demonstrated that the proctolin-like immunoreactivity was present in electron-dense granules in its motor terminals. A material indistinguishable from proctolin and with proctolin-like bioactivity co-eluted with authentic proctolin on two different RP-HPLC systems. Bath application of proctolin at concentrations greater than 10^-11 M resulted in a dose-dependent increase in the neurally-evoked fast twitch amplitude and duration of M234. Concentrations greater than 10^-9 M resulted in a dose-dependent increase in basal tonus of M234. These results indicate that proctolin, or a peptide very similar to proctolin, is present in the motor innervation of M234 and acts as a cotransmitter and/or neuromodulator at this typical fast skeletal muscle.Abbreviations M234 external ventral protractor muscle of the Vllth abdominal segment - RP-HPLC reverse-phase high pressure liquid chromatography - TFA trifluoroacetic acid     

Comparison of the myotropic activity of position-2 modified analogues of proctolin on the hindgut of Periplaneta americana and the oviduct of Locusta migratoria

The largest series of position-2 modified proctolin analogues to have been examined to date were tested for their ability to mimic the basal contraction induced by proctolin on hindgut of the cockroach, Periplaneta americana, and oviduct of the locust, Locusta migratoria. Twelve analogues of proctolin (Arg-Tyr-Leu-Pro-Thr), differing in the substituent (H, OMe, OEt, OPr, F, Cl, Br, I, NO(2), NH(2), N(3), Me) located at the para-position of the aromatic amino acid, caused dose-dependent contractions of both tissues at concentrations quite similar to proctolin. Seven showed greater or equal potency on the hindgut but, with one exception, they were less active on the oviduct than proctolin. The rank order of potency of the analogues depends on the tissue, lending more support to the notion that insects have more than one type of proctolin receptor. No relationship was observed between myoactivity and lipophilic, steric, electron donating or electron withdrawing properties of the substituents at the para-position of the aromatic amino acid. This may be the result of more than one sub-type of proctolin receptor on the specific tissue with differing structural requirements for optimum activity.     

Proctolin: a peptide transmitter candidate in insects.

The slow, striated muscles of the proctodeum (hindgut) of the cockroach, $\text{Periplaneta americana}$ (L.), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from $\text{Periplaneta}$ recently isolated and identified. The graded contractions evoked by repetitive nerve stimulation were simulated by 5-HT and proctolin at threshold concentrations of about 10⁻⁷ and 10⁻⁹ M respectively; responses to glutamate (∼10⁻⁴ M) were not similarly graded. The 5-HT receptors are distinct from other receptors, including the post-synaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide. Proctolin was fully active on TTX-treated or surgically denervated muscle indicating that the proctolin receptors are located on the muscle fibre membrane. Tyramine, at threshold levels 5×10⁻⁸ M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the 5-HT and glutamate responses. Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate. Pharmacologically, the proctolin and post-synaptic receptors appear to be identical and distinct from the glutamate and 5-HT receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosectory granules in addition to electron-lucent, synaptic vesicles.     

The vagina muscles of the blood‐sucking insect Rhodnius prolixus as a model for exploring the physiological effects of proctolin

Proctolin is a neuroactive pentapeptide first isolated from the cockroach Periplaneta americana in which it has an excitatory effect on contractions on visceral muscles of the hindgut. Subsequently, proctolin is reported in a wide variety of invertebrates, and considerable efforts have been made to determine its mode of action. Its primary role appears to be that of a neuromodulator rather than a classical neurotransmitter, and it may also serve as a neurohormone, depending on the muscles examined. The present study identifies the vagina muscles of the blood‐sucking insect Rhodnius prolixus (Stål) as a proctolinergic system. Physiological doses of proctolin generate prolonged contractions that closely mimic the effects of motor nerve stimulation. This preparation is convenient and robust, warranting its use as an experimental system to further understand the role of proctolin in the regulation of muscle contractions in insects. Moreover, these muscles are innervated by an identifiable inhibitory component providing a means to investigate the interaction between proctolin excitation and neural inhibition.     

Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae

A peptide which stimulates the contractile activity of the cockroach hindgut was isolated from head extracts of the cockroach, Leucophaea maderae. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated gas-phase peptide sequencer. The structure determined (pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2) was synthesized and shown to be both chemically and biologically identical with the natural product.     

Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).