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.     

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.     

Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects.

Proctolin, a myotropic substance with potent activity on the proctodeal (hindgut) muscles of the cockroach, $\text{Periplaneta americana}$ (L.), has been identified by the use of Edman degradation and dansylation techniques as Arg-Tyr-Leu-Pro-Thr. Synthesis of the pentapeptide having this sequence and exhibiting the properties of natural proctolin confirmed the structure. Threshold activity on proctodeal muscle occurs at about 10⁻⁹ M proctolin.     

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.     

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.     

The insect neuropeptide proctolin can affect the CNS and the smooth=muscle of mammals

The insect neurotransmitter proctolin can affect different behavioural parameters in rats after intracerebroventricular administration. Proctolin decreased the horizontal exploratory activity and induced a long-lasting analgesia in a hot-plate test. Proctolin lenghthened the passive avoidance latency of rats after post-trial treatment and also in the pre-retention test situation. The self-stimulation rate of rats was significantly diminished after i.c.v. proctolin administration. Proctolin also induced in rat and mouse ileum in vitro a dose-dependent contraction of the gut smooth-muscle that can be blocked by atropine. The results show that an invertebrate neuropeptide (or its fragments) can also be active in mammals. The physiological relevance of the effects observed recently is unknown.     

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.     

Neuromuscular modulation in Limulus by both octopamine and proctolin

Both octopamine and proctolin potentiate nerve-evoked skeletal muscle contractions in the horseshoe crab, Limulus. The threshold concentration for octopamine was 10^?9 to 10^?8M, while for proctolin it was 3 × 10^?9M. Norepinephrine and dopamine produced effects similar to octopamine but at higher thresholds; tyramine and serotonin were ineffective. Octopamine caused significant increases in amplitudes of excitatory postsynaptic potentials (epsps) of muscle fibers, but had little effect on muscle fiber input resistance or membrane potential. Also, octopamine did not affect depolarization of muscle fibers and subsequent contraction due to the direct action of exogenously applied glutamate. These results suggest that octopamine potentiates nerve-evoked contractions primarily by facilitating release of neuromuscular transmitter. At concentrations above 10^?7M, however, octopamine sometimes caused muscle spikes in response to motoneuron stimulation, a finding that suggests that octopamine may also have some postsynaptic action. Proctolin potentiated the muscle contractions evoked by glutamate but had little effect on glutamate-evoked muscle fiber depolarization, muscle fiber input resistance, or membrane potential. Thus, proctolin appears to act directly on skeletal muscle to enhance contractility. The proctolin-induced potentiations of contraction were sometimes accompanied by modest increases in epsp amplitude, so that unlike lobster skeletal and Limulus cardiac neuromuscular preparations, proctolin may have a secondary direct synaptic effect. Both octopamine and proctolin have been found in Limulus cardiac ganglion. This potential access to the hemolymph and the relatively low threshold concentrations needed for physiological action suggest that octopamine and proctolin could function as hormonal modulators of neuromuscular function in Limulus.     

Occurrence of proctolin in six orders of insects

Nine representatives of six orders of insects (Orthoptera, Diptera, Coleoptera, Hemiptera, Hymenoptera, Lepidoptera) were extracted and partially processed by means used in the recent isolation of proctolin, a pentapeptide transmitter candidate in insects. Each insect yielded a substance with pharmacological activity on cockroach proctodeal muscle similar to that of proctolin. Like the responses evoked by proctolin and nerve stimulation, responses to the purified extracts were inhibited by tyramine. All of the active substances behaved as proctolin when subjected to paper chromatography or high voltage paper electrophoresis at pH 6.4 and 3.5. Proctolin appears to be widely and perhaps universally present in the Insecta occurring in most at levels of 2 to 9 μg/kg body weight.     

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     

Electrophysiological studies of the effect of the neuropeptide proctolin on the hyperneural muscle of Periplaneta americana (L.)

The myotropic neuropeptide proctolin is, in additional to its action on proctodaeum and on some other systems, highly effective on the hyperneural muscle of Periplaneta americana and evokes long-term contractions. During this proctolin response the input resistance (R_input) increases by about 25% accompanied by only slight depolarization. These processes require extracellular Ca²⁺ but are still present in Na⁺-free solution.Junction potentials evoked by threshold stimulation of the nerve are not affected by proctolin. Synaptic processes do not seem to be important for the proctolin action on hyperneural muscle. It is more likely that the whole membrane of the muscle fibre serves as target for proctolin. Proctolin reduces the threshold for neurally evoked muscle contractions, the only available route of excitation since the muscle fibres themselves are not electrically excitable.The K⁺-channel blocker 4-aminopyridine may evoke contraction as well as proctolin, but this is only a transitory response. In contrast to proctolin, 4-aminopyridine is still effective after blocking the Ca²⁺-channels by Co²⁺, but the response is smaller. Therefore proctolin seems to be primarily effective via Ca²⁺-channels, whereas 4-aminopyridine exerts its effects via K⁺-channels. The decrease in membrane conductance produced by proctolin could result from a Ca²⁺-dependent reduction of the K⁺-outward current.     

Pharmacological studies of proctolin receptors on foregut and hindgut of blaberus craniifer

Proctolin (Arg-Tyr-Leu-Pro-Thr) and proctolin analogs modified at position 1, 2, or 5 caused dose dependent contractions of Blaberus fore- and hindgut. The varying contractile effects between both tissues revealed the possible presence of receptor subtypes as identified by [GABA¹]-proctolin. A single population of binding sites (K_d ≈ 100 n ) was deduced from Scatchard analysis. In addition, nanomolar concentrations of proctolin induced a dose-dependent hydrolysis of phosphoinositides (PIns) augmented by GTPγS (1 μ ) on foregut membranes but no accumulation of cAMP. Proctolin induced contractions are likely mediated via a phospholipase C linked to a heptahelical receptor bound to heterotrimeric G-proteins.     

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.     

In vivo-application of anti-proctolin-antiserum affects antennal flight posture in crickets

Tethered crickets flying in a wind tunnel adopt a characteristic posture in which the antennae are pointed in parallel and anteriorly into the headwind. Although the firing rates of antennal motoneurons are largely reduced after the start of a flight sequence, the associated postural changes of the antennae are small. It is hypothesised that proctolin, which is present in antennal motoneurons, stabilises the prolonged antennal forward position. To test this hypothesis, proctolin was blocked by anti-proctolin antiserum injections into one antennal base in otherwise intact behaving crickets. The antiserum quickly led to prolonged backward deflections of the treated antennae in 65% of cases. It then took more than one hour for the deflected posture to revert to a normal flight posture. It appears that proctolin is necessary to produce muscle tension large enough to hold the antennae in a forward position and to compensate for the headwind drag. Proctolin, therefore, acts to generate force with reduced electrical activity of motoneurons and muscles.     

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.     

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.     

Effects of leucomyosuppressin on the excitation-concentration coupling of insect Leucophaea maderae visceral muscle

1. Leucomyosuppressin (LMS) inhibited neurally evoked contractions of the hindgut of the cockroach Leucophaea maderae. The threshold for this inhibition of LMS was in the range of 1 × 10⁻¹⁰ M.2. LMS caused a sharp reduction in both l-glutamate and proctolin induced contractions. Dose-response profiles of the effect of LMS (held constant at 10⁻⁸M) on variable amounts of proctolin showed an inhibitory effect at 10⁻⁹ M proctolin and below, but at 5 × 10⁻⁹ M proctolin and above, LMS caused no inhibition.3. Potassium (158 mM) depolarized hindguts treated with LMS (10⁻⁸ M) showed a marked reduction (76% ± 2.1) in the proctolin (10⁻⁸ M) response.4. When calcium depleted preparations were returned to normal calcium levels (2 mM) in the presence of proctolin (10 ⁻⁸ M) a contraction occurred that was 45% ± 4 of the maximum in normal saline solution. However, LMS (10⁻⁸ M) reduced this response to only 28% ± 2 of the maximum.5. Proctolin (10⁻⁸ M) induced contractions in the presence of the manganous ions (2mM) fell to 63% ± 4 of the maximum but on the addition of LMS (10⁻⁸M), such responses fell to only 16% ± 5 of the maximum.6. These results offer evidence for a non-synaptic site of action for LMS and a perturbation of key calcium dependent events in the excitation-contraction coupling sequence of visceral muscle by this peptide.     

Interaction between octopamine and proctolin on the oviducts of Locusta migratoria

The biogenic amine octopamine and the pentapeptide proctolin are two important neuroactive chemicals that control contraction of the oviducts of the African locust Locusta migratoria. The physiological responses and signal transduction pathways used by octopamine and proctolin have been well characterized in the locust oviducts and this therefore provides the opportunity to examine the interaction between these two pathways. Octopamine, via the intracellular messenger adenosine 3',5'-cyclic monophosphate (cyclic AMP), inhibits contraction of the oviducts, while proctolin, via the phosphoinositol pathway, stimulates contraction. We have examined the physiological response of the oviducts to combinations of octopamine and proctolin and also looked at how combinations of these affect one of the main intracellular mediators of the octopamine response, namely cyclic AMP. It was found that application of octopamine to the oviducts led to a dose-dependent reduction in tonus of the muscle and also a decrease in the amplitude and frequency of spontaneous phasic contractions. Octopamine-induced relaxation was enhanced in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Octopamine was also able to inhibit proctolin-induced contractions of the oviducts in a dose-dependent manner. A 10(-9) M proctolin-induced contraction was inhibited by 83% in the presence of 10(-5) M octopamine, and was completely inhibited in the presence of 10(-5) M octopamine plus 5x10(-4) M IBMX. Octopamine led to a dose-dependent increase in cyclic AMP content as measured by radioimmunoassay. In the presence of 10(-9) M proctolin, this octopamine-induced increase in cyclic AMP was reduced by as much as 60%. Proctolin also caused a dose-dependent decrease in the cyclic AMP elevation produced by 5x10(-6) M octopamine. These results indicate that octopamine and proctolin can antagonize each other's physiological response when added in combination, and that proctolin is able to modulate the response of the oviducts to octopamine by influencing cyclic AMP levels.     

Inositol phospholipid hydrolysis may mediate the action of proctolin on insect visceral muscle

The formation of inositol phosphates in response to the neuropeptide proctolin was studied in locust oviducts. Glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate were identified in the locust oviducts using anion-exchange chromatography. Proctolin stimulated the release of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate during a 5-min incubation. In the presence of lithium ions the effects of proctolin were enhanced, with elevations of 98%, 42%, and 45% of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, respectively. Physiologically the effects of proctolin upon muscular contraction of locust oviducts were mimicked by the active phorbol ester, phorbol 12-myristate 13-acetate, and by the diacylglycerol analogue, 1-oleoyl-2-acetylglycerol. The inactive phorbol ester, 12-myristate 13-acetate 4-O-methyl ether, was without effect. The effects of the active phorbol ester and the diacylglycerol analogue were calcium-dependent requiring micromolar concentrations of calcium. The results indicate that the locust oviducts possess proctolin receptors that are linked to phosphatidylinositol metabolism and that inositol phospholipid hydrolysis may mediate the physiological action of proctolin.     

Proctolin in the brain and ganglia of Triatoma infestans (Hemiptera: Reduviidae)

The distribution of proctolin in the central nervous system of the hemipteran bug, Triatoma infestans, was studied by immunohistochemistry using the sensitive avidin‐biotin technique combined with nickel salt intensification of the reaction product. Proctolin was present in cells and fibers of the brain and ganglia. In the brain, protocerebral proctolin‐immunoreactive cell bodies were found in the pars intercerebralis, the optic lobes, and the lateral soma rind. The deutocerebrum showed positive somata in relation to the antennal motor center and the tritocerebrum had intense immunoreactive fibers but few positive cells. Proctolin‐immunoreactive cell bodies of different sizes were observed in the subesophageal ganglion. Large cell bodies were found mainly rostrally and beaded positive processes were present around the ventral border of the esophageal foramen and in the rostrolateral neuropil of this ganglion. Small‐ to medium‐sized positive somata were found in the posterior part of the prothoracic ganglion; some of these cells were sending immunoreactive processes to the central neuropil. The meso‐metathoracic‐abdominal ganglionic mass showed positive cells in all the neuromeres, where some of them were large and had thick immunoreactive granules. The results show that the labeling pattern of proctolin‐like immunoreactivity in Triatoma i. appears to be widespread and unique for its central nervous system. It is suggested that proctolin may serve neuroendocrine, integrative, and motor functions in the brain of T. infestans. J. Morphol. 240:39–47, 1999. © 1999 Wiley‐Liss, Inc.