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.     

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     

A peptide action in a lobster neuromuscular preparation

The neuropeptide proctolin causes a sustained contraction of the opener muscle of the dactyl of the lobster walking leg. This substance acts directly on the muscle at concentrations as low as 10^?10M. The contraction is dependent on extracellular calcium. Neither a significant depolarization nor a detectable change in the input resistance accompanies the response. No presynaptic action of proctolin is indicated; excitatory and inhibitory junctional potential sizes and the frequency of spontaneous miniature excitatory junctional potentials are unaffected.     

Experimental studies upon a bundle of tonic fibres in the locust extensor tibialis muscle

The bundle of tonic fibres situated at the proximal end of the locust metathoracic extensor tibialis muscle is innervated by the dorsal unpaired median neurone (DUMETi) as well as by the slow excitatory (SETi)) and common inhibitor (CI) neurones. It is not innervated by the fast excitatory neurone (FETi).These fibres contract spontaneously and rhythmically. The myogenic rhythm can be modified by neural stimulation.Spontaneous slow depolarizing potentials resembling the pacemaker potentials of insect cardiac muscle were demonstrated in these fibres.The actions of glutamate on the tonic muscle fibres are not compatible with its being a specific excitatory transmitter. Glutamate can stimulate weak contractions of the muscle, but this action is inhibited when chloride ions are removed from the saline.10^?6 M Octapamine hyperpolarizes the tonic fibre membrane. Octopamine, GABA and glutamate all inhibit the myogenic contractions and reduce the force of the neurally evoked contractions.The tonic muscle is very responsive to proctolin. At 5 × 10^?11 M proctolin enhances the force and increases the frequency of myogenic contractions. At 10^?9 M it depolarizes the muscle membrane potential, and at that and higher concentrations it causes the muscle to contract. At 2 × 10^?7 M proctolin induces contractures which resemble those evoked by sustained high-frequency neural stimulation. Iontophoretic experiments show that proctolin receptors occur at localized sites on the tonic fibre membrane.     

The mechanism of block of glutamate synapses by dipicolinic acid

The effect of dipicolinic acid (2,6-pyridine dicarboxylic acid) on the mealworm neuromuscular junction was studied using conventional microelectrode recording techniques. Dipicolinic acid (10^?5-10^?3 M) added to the bathing solution reversibly blocked neuromuscular transmission. The depolarization in response to iontophoretically applied L-glutamate (glutamate potential) was not affected by dipicolinic acid even when the neurally evoked excitatory postsynaptic potential (EPSP) was totally abolished. Focal extracellular recordings from single synaptic sites revealed that in the presence of 1 x 10^?4 M dipicolinic acid the presynaptic spike was unchanged, but the quantal content for evoked transmitter release was reduced. The calcium-dependent action potential elicited by direct stimulation of the muscle fiber was not impaired by dipicolinic acid. These results suggest that dipicolinic acid interferes with the transmitter-releasing mechanism from the presynaptic terminal.     

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.     

Peptide actions on oviduct contractions in the large pine weevil,Hylobius abietis

Abstract The peptides proctolin, crustacean cardioactive peptide (CCAP) and FMRFamide, which are known to modulate insect muscle contractions, were assayed for their action on oviduct contractions in Hylobius abietis. A video microscopy technique and computer‐based method of data acquisition and analysis were used to investigate the effects of theses peptides on spontaneous contractions of continuously perfused oviducts. All three peptides tested stimulate spontaneous contraction activity of the pine weevil oviduct, increasing the frequency and amplitude of phasic contractions in a dose‐dependent manner. Proctolin is more potent as a stimulator than CCAP. For proctolin a threshold response of oviduct muscles is at concentration of peptide 10^?11–10^?10 mol/L and for CCAP at concentration range 10^?10–10^?9 mol/L. FMRFamide exerts a weak stimulatory effect on the oviduct, and at higher concentrations of the peptide (above 10^?8 mol/L). The peptides exert different responses on oviduct contractions and they may play a role as functional regulators in such processes as egg movement and oviposition.     

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.     

Neuromuscular transmission in an insect visceral muscle

The electrical properties of the muscles of locust oviduct have been examined using intracellular recordings. The muscle cells are both dye and electrically coupled. They possess a wide array of spontaneous electrical activity ranging from slow oscillations of membrane potential to action potentials. In addition to possessing spontaneous electrical activity, certain regions of the oviduct are under motor control. The amplitude of evoked excitatory junction potentials (EJPs) increased step wise revealing innervation from a maximum of three motor units. These EJPs underwent summation and facilitation, and reached a critical threshold at which point the membrane revealed an active response. Bath applied glutamate, aspartate, proctolin, and octopamine were tested for their ability to alter resting potential and EJPs. L-glutamate (1.6 X 10(-5) M and above) produced a dose-dependent depolarization of membrane potential accompanied by a reduction in amplitude of EJPs. Although L-aspartate resulted in similar effects, the concentrations required were higher than those for glutamate. Proctolin (6.3 X 10(-11) M-6.0 X 10(-9) M) resulted in a dose-dependent depolarization but had little or no effect on amplitude of EJPs. Application of D, L-octopamine (3.2 X 10(-5) M-1.7 X 10(-4) M) induced a small hyperpolarization and a reduction in amplitude of EJP. It is suggested that contractions of locust oviduct appear to be regulated by a combination of a classical neurotransmitter such as glutamate, along with the neuromodulators octopamine and proctolin.     

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.     

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.     

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.     

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.     

The effects of some putative transmitters and biogenic amines on uropod abductor muscle in the crayfish Procambarus clarkii and Cambaroides japonicus

The effects of some putative transmitters and biogenic amines were examined on the uropod ventral abductor exopodite (AbdExV) muscle in two crayfish species Procambarus clarkii and Cambaroides japonicus. Bath application of L-glutamate to the AbdExV muscle caused sustained contract while gamma-aminobutyric acid (GABA) depressed the nerve-evoked contraction of the muscle. Acetylcholine (ACh) had no effect on both the resting tension and the nerve-evoked contraction. Iontophoresis of L-glutamate and GABA onto the surface of the muscle fiber further confirmed that glutamate and GABA are the possible excitatory and inhibitory transmitters respectively at the neuromuscular junction of AbdExV muscle. Bath application of 5-hydroxytryptamine (5-HT) and octopamine (Oct) caused enhancement of the nerve-evoked contraction but dopamine (DA) had no effect on both the resting tension and the nerve-evoked contraction.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

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     

Biogenic amines modulate synaptic transmission between identified giant interneurons and thoracic interneurons in the escape system of the cockroach

In the escape system of the cockroach, Periplaneta americana, a population of uniquely identifiable throacic interneurons (type A or TI_As) receive information about wind via chemical synapses from a population of ventral giant interneurons (vGIs). The TI_As are involved in the integration of sensory information necessary for orienting the animal during escape. It is likely that there are times in an animal's life when it is advantageous to modify the effectiveness of synaptic transmission between the vGIs and the TI_As. Given the central position of the TI_As inthe escape system, this would greatly alter associated motor outputs. We tested the ability of octopamine, serotonin, and dopamine to modulate synaptic transmission between vGIs and TI_As. Both octopamine and dopamine significantly increased the amplitude of vGI-evoked excitatory postsynaptic potentials (EPSPs) in TI_As at 10^?4?10^?2 M, and 10^?3 M, respectively. On the other hand, serotonin significantly decreased the vGI-evoked EPSPs in TI_As at 10^?4?10^?3 M. These results indicate that octopamine, serotonin, and dopamine are capable of modulating the efficacy of transmission of important neural connections within this circuit. © 1992 John Wiley & Sons, Inc.     

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.     

Agonistic action of synthetic analogues of quisqualic acid at the insect neuromuscular junction

One hundred twenty analogues of quisqualic acid were synthesized and assayed on the neuromuscular junction of larva of the mealworm, Tenebrio molitor. Two new agonists for amino acid receptors, L-glutamic acid N-thiocarboxyanhydride (L-GANTA) and DL-hydantoinpropionic acid (DL-HPA), were discovered in this study. L-GANTA and DL-HPA produced muscle membrane depolarization, accompanied by a reduction of the muscle input resistance. The amplitude of excitatory postsynaptic potentials was decreased in the presence of L-GANTA and DL-HPA. The apparent dissociation constants obtained from dose-depolarization plots were 7 x 10^?4 M for L-GANTA and 9 x 10^?4 M for DL-HPA. Some structural constraints imposed on agonists at amino acid receptors on insect muscle were discussed.     

Control of the alary muscles of locust dorsal diaphragm

ABSTRACT. The heartbeat in whole, intact, adult Locusta migratoria R.F. was characterized by a regular rate but apparently irregular amplitude. Cutting segmental nerves often eliminated apparent amplitude fluctuations, and electrically shocking a segmental nerve in the whole animal evoked apparent amplitude changes corresponding to the shocks. Saline-perfused tissue preparations showed that the apparent amplitude fluctuations could be duplicated by segmental nerve stimulation, and that the fluctuations were due largely to contractions of the alary muscles of the dorsal diaphragm which shifted the position of the heart chamber without a change in volume. The alary muscles are each multi-terminally innervated by one motor axon. Neurally-evoked postsynaptic potentials facilitated and summated, and the diaphragm muscles began visibly contracting at stimulation rates as low as 2 Hz. Stimulation at higher frequencies caused greater depolarization of the muscle fibres with no indication of electrically-excited responses. The alary muscles were insensitive to perfusion with acetylcholine, l -glutamate, l -aspartate, dopamine, octopamine, noradrenaline, proctolin, 5-hydroxytryptamine, or gamma aminobutyric-acid in saline at concentrations up to 10^-3M. Larval or adult brain extracts of Locusta at 10 μg/μl and diluted 1:5 in saline caused uniform contractions of the alary muscle preparation, while perfusion of skeletal muscle extracts produced no response.