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.     

Characterization of proctolin binding sites on locust oviduct membranes

The binding of [³H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a K_d of 400 ± 82 nM and a B_max of 23.7 ± 6.7 pmol/mg protein. The second had a K_d of 2.4 ± 0.2 μM and a B_max of 96.3 ± 16.7 pmo/mg protein.

Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [³]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed.     

A review of the involvement of proctolin as a cotransmitter and local neurohormone in the oviduct of the locust,Locusta migratoria

The pentapeptide proctolin, originally identified in the cockroach, has been shown to be widely distributed in many insects and to have a broad range of physiological functions. In the oviduct of the locust, Locusta migratoria, proctolin's role as a neurotransmitter/neuromodulator has been well documented; however, a neurohormonal role in the locust is less certain. This review will examine the various roles of proctolin in locust oviduct contraction and will present evidence that a substance chromatographically, immunologically and physiologically indistinguishable from proctolin is present in the hemolymph of the locust, L. migratoria. This material is concentrated in the plasma, rather than the hemocytes, and is present at concentrations ranging from 0.1 to 0.2nM. This review extends the role of proctolin in insects, and suggests that proctolin may play a neurohormonal role in the locust.     

Mode of action of proctolin on locust visceral muscle

Proctolin increases the frequency and amplitude of myogenic contractions and results in a sustained contraction of the oviducts of Locusta migratoria. The possible mode of action of proctolin receptors on this visceral muscle has been investigated. Calcium-free saline, containing either 20 mM magnesium ions or 100 μM EGTA, inhibited myogenic contractions, lowered basal tension, and abolished all the effects of proctolin following a 20 min incubation. These effects were reversible upon washing with normal saline. Similar results were obtained with normal saline containing 10 mM cobalt ions. Nifedipine at 50 μM lowered basal tension, abolished myogenic contractions, and reduced the proctolin-induced sustained contraction by 42-62% at 0.5 nM proctolin and by 33-37% at 5 nM proctolin. Similar results were obtained with 100 μM verapamil. Proctolin was still capable of eliciting considerable contractions (25-67% of controls) in preparations depolarized with 100 mM potassium saline. The removal of calcium from the high-potassium saline reversibly abolished the potassium-induced contraction and reversibly blocked the action of proctolin. Nifedipine was ineffective in blocking the action of proctolin in high-potassium saline. Neither cyclic AMP levels nor cyclic GMP levels of the lateral oviducts were elevated by proctolin in the presence of a phosphodiesterase inhibitor. The results indicate that proctolin mediates its effects via an influx of external calcium ions. This calcium appears to enter through two channels, a voltage-dependent channel and a receptor-operated channel. Cyclic nucleotides do not appear to be involved in the action of proctolin in this visceral muscle.     

The modulation of skeletal muscle contraction by FMRFamide-related peptides of the locust

The external ventral protractor muscle of the VIIth abdominal segment, M234, is a skeletal muscle that possesses receptors that recognize a range of FMRFamide-related peptides and application of these peptides results in an increase in the amplitude of neurally evoked contractions with little or no effect on basal tonus. FLRFamide itself has the same biologic activity as the extended peptides, whereas truncation to LRFamide or RFamide results in a peptide with no biologic activity. The receptors recognizing these extended FLRFamides, which include SchistoFLRFamide, seem to be different from the SchistoFLRFamide receptors found on locust oviduct visceral muscle. SchistoFLRFamide and the non-peptide mimetic, benzethonium chloride (Bztc), increase the frequency and amplitude of miniature endplate potentials, increase the amplitude of neurally evoked excitatory junction potentials, and result in a hyperpolarisation of resting membrane potential. Bztc, however, also abolishes the active membrane response that may explain its ability to decrease neurally evoked contractions.     

The oviduct musculature of the horsefly, Tabanus sulcifrons, and its response to 5-hydroxytryptamine and proctolin

ABSTRACT. A delicate lace-like membrane covers the ovaries of Tabanus sulcifrons. These membranes were found to contain muscle fibres that provide the organs with motile properties. The lateral oviducts consist of a single layer of longitudinal muscles that form a structural syncytium by means of an extensive anastomosis of the fibres comprising it. The common oviduct is composed of two muscle layers, an outer sheath of circular muscle and an inner substratum of longitudinal muscle. Both of these layers showed evidence of a structural syncytium. When isolated in saline, the oviduct was spontaneously active and gave a simple phasic pattern of contraction. Such muscle preparations were sensitive to both 5-hydroxytryptamine (5HT) and the insect myotropic peptide, proctolin. Excitation was generally indicated by a rise in muscle tonus or an increase in the frequency and amplitude of individual phasic contractions, or all three characteristics. The threshold for activation with 5HT was variable, ranging from as low as 4 × 10^-9M to 1 × 10^-7M. Proctolin evoked a noticeable increase in the tonus of most oviducts at 10^-10M. However, several preparations responded to as little as 3 × 10^-11M proctolin.     

Growth-inhibition effects of pacifastin-like peptides on a pest insect: the desert locust, Schistocerca gregaria

The main reason for the varying degrees of success of peptidase inhibitors (PI) as biological insecticides is the existence of a poorly understood mechanism, which allows pest insects to compensate for PI present in their diet. To challenge this highly flexible physiological mechanism and to prolong the inhibitory effect of PI on insect growth, a number of measures were taken into account before and during experiments with a notorious pest insect, the desert locust, Schistocerca gregaria: (i) non-plant PI (pacifastin-related inhibitors) were used to reduce the risk of a specific co-evolutionary adaptation of the pest insect, (ii) based on the main types of digestive enzymes present in the midgut, mixtures of multiple PI with different enzyme specificity were selected, allowing for a maximal inhibition of the proteolytic activity and (iii) digestive peptidase samples were taken during oral administration experiments to study compensatory mechanisms. Contrary to larvae fed on a diet containing plant-derived PI, a significant growth impediment was observed in larvae that were fed a mixture of different pacifastin-like PI. Nevertheless, the growth inhibition effect of this PI mixture attenuated after a few days, Moreover, a comprehensive study of the observed responses after oral administration of PI revealed that S. gregaria larvae can adjust their secreted digestive enzyme activities in two distinct ways depending on the composition/concentration of the PI-mixture.     

N-terminal modified analogs of HVFLRFamide with inhibitory activity on the locust oviduct

New N-terminal analogs of the peptide HVFLRFamide, the minimum sequence of the insect myosuppressins capable of inhibiting spontaneous and induced contractions of the locust oviduct, were synthesized and tested for biologic activity on locust oviduct. Most active, as judged by the ability to inhibit proctolin-induced contractions of locust oviduct, was (N(alpha)-acetyl)-HVFLRFamide. D-Pro-HVFLRFamide was also highly inhibitory. Interestingly, low doses of the pentapeptide analog (N(alpha)-imidazoleacrylyl)-VFLRFamide inhibited oviduct contractions. This is the first pentapeptide analog shown to inhibit contractions of locust oviduct, and this result indicates that the alpha-amino group of His is not absolutely required for inhibitory activity. In all cases when His was replaced by a D-amino acid, the analogs were stimulatory, resulting in an increase in basal tonus of the locust oviduct. The results provide further insight into the structural features of the HVFLRFamide molecule that are required for inhibitory activity on locust oviduct muscle.     

Some pharmacological properties of neuromuscular transmission in the oviduct of the locust,Locusta migratoria

The effects of various pharmacological agents on neurally evoked contractions of the visceral muscles of the oviduct of Locusta migratoria have been examined. The pentapeptide, proctolin, at low concentrations (10^?11 M?10^?10 M), induced an increase in the amplitude of neurally evoked contractions and basal tonus, and induced the appearance and increased the frequency of myogenic contractions. Glutamate, at 10^?4 M, produced a small transient contraction which in some preparations was accompanied by a reduction in amplitude of neurally evoked contractions. Octopamine, at 10^?6 M, reduced the amplitude of neurally evoked contractions and also resulted in a relaxation of the muscles. The octopaminergic effects were inhibited by the α-aminergic antagonist phentolamine. Neurally evoked contractions were unaffected by dopamine, 5-HT or the acetylcholine receptor antagonists atropine and hexamethonium. Acetylcholine increased the amplitude of neurally evoked contractions, but only at the high concentration of 10^?3 M. The possible role of proctolin and glutamate as excitatory neuro-transmitters and the inhibitory action of octopamine is discussed.     

Effects of tissue extracts on oviduct contraction in the migratory grasshopper,Melanoplus sanguinipes

The isolated oviduct of the migratory grasshopper, Melanoplus sanguinipes, shows a myogenic, rhythmic pattern of contraction. The pattern of contraction can be modified by treatment with hemolymph or extracts from a variety of tissues from animals of differing age, sex, and mated status. Extracts of oviducts and ventral nerve cord (VNC), as well as hemolymph, from both virgin and mated females were almost always stimulating, though their effect on frequency, amplitude and/or tonus varied. In contrast, whereas extracts of spermatheca and brain from mated females were stimulating, extracts of these tissues from virgin females inhibited contraction. All male material tested (long hyaline tubules (LHT), the male accessory gland complex less the LHT, testes, brain, VNC and hemolymph) stimulated oviduct contraction in a dose-dependent manner, usually enhancing at least two of frequency, amplitude and tonus. However, oviposition-stimulating protein, a purified product of the LHT, provoked only an increase in frequency when applied to the oviduct. LHT extract modulated the effects of virgin female tissue extracts, always in a stimulatory manner.     

Myogenic contractions in locust muscle induced by proctolin and by wasp, Philanthus triangulum, venom

Apparently myogenic slow contractions of the extensor tibiae of Locusta migratoria can be induced by proctolin in a concentration of 10^?10 to 10^?9 mole per liter perfusion fluid. Proctolin in a concentration of 10^?8 mole/l causes a prolonged contraction interrupted by rhythmical relaxations. Higher concentrations of proctolin cause a powerful but irreversible contraction.In some preparations in which proctolin is ineffective in a concentration of 10^?9 mole/l, a short stimulation of nerve 3b can initiate a series of rhythmic contractions. If, however, nerve 3b is stimulated at the peak of such a contraction a rapid relaxation is induced.Administration of the venom of Philanthus triangulum in a concentration which blocks the excitatory and inhibitory neuromuscular transmission, induces similar myogenic contractions. Stimulation of nerve 3b at the peak of such a contraction again causes a relaxation. Similar myogenic contractions can also be induced by administration of a homogenate of a locust.     

Modulatory effects of proctolin on a crab ventilatory muscle

Proctolin enhances nerve-evoked, phasic contractions of a selected respiratory muscle of the shore crab, Carcinus maenas, but has no effect on muscle tonus. Proctolin also increases the work and power output of this muscle. These effects are functionally appropriate in view of previous reports that proctolin stimulates the ventilatory rhythm. They also suggest that proctolin exerts coordinated modulatory control at the central and peripheral levels of the gill ventilatory system. In contrast, serotonin, dopamine and octopamine have no effect on this muscle.     

Proctolin antagonists bind to [(3)H]proctolin binding sites in the locust hindgut

Proctolin caused dose-dependent (1-200 nM) contraction of the isolated hindgut of S. gregaria which was abolished by [alpha-methyl-L-tyrosine(2)]-proctolin (1 microM). In comparison, cycloproctolin (5 microM) reduced the proctolin maximum response by 41%. Hindgut homogenates contained [(3)H]proctolin binding sites with a K(d) value of 660 nM, a B(max) value of 23.8 pmol/mg protein and a Hill coefficient of 0.934. Cycloproctolin (IC(50,) 220 nM; K(i), 204 nM), unlabeled proctolin (IC(50) 680 nM) and [alpha-methyl-L-tryosine(2)]-proctolin (IC(50) 3.1 microM, K(i), 2.9 microM) but not SchistoFLRFamide (1 nM-10 microM) were capable of displacing bound [(3)H]proctolin.     

Dual effects of proctolin on the rhythmic burst activity of the cardiac ganglion

The neuropeptide proctolin has distinguishable excitatory effects upon premotor cells and motorneurons of Homarus cardiac ganglion. Proctolin's excitation of the small, premotor, posterior cells is rapid in onset (5–10 s) and readily reversible (< 3 min). Prolonged bursts in small cells often produce a “doublet” ganglionic burst mode via interactions with large motorneuron burst-generating driver potentials. In contrast to small cell response, proctolin's direct excitatory effects upon motorneuron are slow in onset (60–90 s to peak) and long-lasting (10–20 min). The latter include: (a) a concentration-dependent (10^?9–10^?7M) depolarization of the somatic membrane potential; (b) increases in burst frequency and (c) enhancement of the rate of depolarization of the interburst pacemaker potential. Experiments on isolated large cells indicate: (a) the slow depolarization is produced by a decrease in the resting G_K and (b) proctolin can produce or enhance motorneuron autorhythmicity. A two-tiered non-hierarchical network model is proposed. The differential pharmacodynamics exhibited by the two cell types accounts for the sequential modes of ganglionic burst activity produced by proctolin.     

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.     

Delayed pharmacological effects of proctolin and CCAP on heartbeat in pupae of the tobacco hornworm, Manduca sexta

Abstract. The effects in vivo of cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae of Manduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10⁻⁶ M (calculated from µg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10^-9 − 10^-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10^-8 − 10^-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10⁻⁶ M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptides in vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditions in vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae of M. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions.     

Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

Summary The presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the -adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.Abbreviations BPP Bovine pancreatic polypeptide - BSA Bovine serum albumin - EJP Excitatory junctional potential - FaRPs FMRFamide-related peptides - FLI FMRFamide-like immuno-reactivity - LMS Leucomyosuppressin - RIA Radioimmunoassay - RP-HPLC Reversed-phase high performance liquid chromatography - TFA Trifluoroacetic acid     

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.