The presence and distribution of crustacean cardioactive peptide in the central and peripheral nervous system of the stick insect, Baculum extradentatum

Crustacean cardioactive peptide (CCAP)-like immunoreactivity was localized and quantified in the central and peripheral nervous system of the Vietnamese stick insect, Baculum extradentatum, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). The brain, frontal ganglion, suboesophageal ganglion and ventral nerve cord displayed neurons and processes with CCAP-like immunoreactivity. The brain, in comparison to the other parts of the central nervous system, contained the greatest amount of CCAP (167 +/- 18 fmol), and showed CCAP-like staining in neurons, neuropil regions and the central complex. There were also CCAP-like varicosities and processes associated with the corpus cardiacum. The alimentary canal of B. extradentatum contained CCAP with the largest amount localized in the midgut (1110 +/- 274 fmol CCAP equivalents). The midgut contained numerous endocrine-like cells which stained positively for CCAP, whereas the foregut and hindgut revealed an extensive network of CCAP-like immunoreactive axons and varicosities. Based on physiological assays, the hindgut of the stick insect was found to be sensitive to CCAP, showing dose-dependent increases in contractions with threshold at 10(-10) M CCAP and maximal response at 5 x 10(-7) M CCAP. There were negligible quantities of CCAP in the oviducts and no CCAP-like immunoreactivity was associated with the oviducts. CCAP had no effect on spontaneous contractions of the oviducts. The presence of CCAP in the central nervous system, the stomatogastric nervous system, the corpus cardiacum and the alimentary canal, suggest broad ranging roles for CCAP in B. extradentatum.     

The association of crustacean cardioactive peptide with the spermatheca of the African migratory locust, Locusta migratoria

Crustacean cardioactive peptide (CCAP)-like immunoreactivity was identified in neurons of the VIIIth abdominal ganglion and in axons in the nerves that project to the spermatheca of 3-4 week old adult female locusts. In addition, lightly stained CCAP-like immunoreactive processes were localized over the spermathecae. The amount of CCAP in the spermathecal tissue was quantified using an enzyme-linked immunosorbent assay (ELISA) performed on extracts of the whole spermatheca, and on its constituent parts, namely the sperm sac, coiled duct and straight duct. The spermatheca contains 920+/-273 fmol (mean+/-SE) of CCAP equivalents, with the majority localized in the coiled duct. There are age-related differences in the amount of CCAP present in the spermathecae with less content in spermathecae from 1 to 5 day old and greater content in spermathecae from 3 to 4 week old adults. There was also no difference in CCAP content of spermathecae in mated and virgin 3 to 4 week old adults. Reversed phase-high performance liquid chromatography (RP-HPLC) followed by ELISA further confirmed the presence of CCAP-like material in extracts of locust spermathecae. Physiological assays demonstrated that CCAP increased the basal tonus and frequency of spontaneous contractions of the spermatheca, with thresholds between 10(-10) and 10(-9)M and maxima at 10(-7)M CCAP. CCAP also increases the amplitude of neurally evoked contractions with a threshold less than 10(-11)M and a maximum of 10(-7)M CCAP. The present study suggests that CCAP acts as a neuromodulator/neurotransmitter at the spermathecal visceral tissue of female Locusta migratoria.     

The effects of crustacean cardioactive peptide on locust oviducts are calcium-dependent

The role of calcium as a second messenger in the crustacean cardioactive peptide (CCAP)-induced contractions of the locust oviducts was investigated. Incubation of the oviducts in a calcium-free saline containing, a preferential calcium cation chelator, or an extracellular calcium channel blocker, abolished CCAP-induced contractions, indicating that the effects of CCAP on the oviducts are calcium-dependent. In contrast, sodium free saline did not affect CCAP-induced contractions. Co-application of CCAP to the oviducts with preferential L-type voltage-dependent calcium channel blockers reduced CCAP-induced contractions by 32-54%. Two preferential T-type voltage-dependent calcium channel blockers both inhibited CCAP-induced oviduct contractions although affecting different components of the contractions. Amiloride decreased the tonic component of CCAP-induced contractions by 40-55% and flunarizine dihydrochloride decreased the frequency of CCAP-induced phasic contractions by as much as 65%, without affecting tonus. Flunarizine dihydrochloride did not alter the proctolin-induced contractions of the oviducts. Results suggest that the actions of CCAP are partially mediated by voltage-dependent calcium channels similar to vertebrate L-type and T-type channels. High-potassium saline does not abolish CCAP-induced contractions indicating the presence of receptor-operated calcium channels that mediate the actions of CCAP on the oviducts. The involvement of calcium from intracellular stores in CCAP-induced contractions of the oviducts is likely since, an intracellular calcium antagonist decreased CCAP-induced contractions by 30-35%.     

Evidence for crustacean cardioactive peptide-like innervation of the gut in Locusta migratoria

Hindguts from female Vth instar larvae, young adults (1-2 days) and old adults (>10 days) are equally sensitive to the crustacean cardioactive peptide (CCAP), with changes in contraction occurring at a threshold concentration of 10(-9)M and maximal responses observed at concentrations ranging between 10(-7) and 5x10(-6)M. An immunohistochemical examination of the gut of Locusta migratoria with an antiserum raised against CCAP revealed an extensive network of CCAP-like immunoreactive processes on the hindgut and posterior midgut via the 11th sternal nerve arising from the terminal abdominal ganglion. Anterograde filling of the 11th sternal nerve with neurobiotin revealed extensive processes and terminals on the hindgut. Retrograde filling of the branch of the 11th sternal nerve which innervates the hindgut with neurobiotin revealed two bilaterally paired cells in the terminal abdominal ganglion which co-localized with CCAP-like immunoreactivity. Results suggest that a CCAP-like substance acts as a neurotransmitter/neuromodulator at the locust hindgut.     

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     

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.     

Functional hypervariability and gene diversity of cardioactive neuropeptides

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.     

Occurrence of crustacean cardioactive peptide (CCAP) in the nervous system of an insect,Locusta migratoria

Summary Using a radioimmunoassay developed for the determination of crustacean cardioactive peptide (CCAP), immunoreactive material was detected in extracts of locust nervous tissue. Serial dilutions of a brain extract gave a displacement curve parallel to the CCAP standard curve. One locust nervous system was calculated to contain approximately 1.4 pmol CCAP-like material.In order to investigate whether the immunoreactive substance was similar or identical to the crustacean neuropeptide, isolation and complete characterization was carried out using 800 locust nervous systems. The isolation procedure consisted of pre-purification of the crude extract on a Sep-Pak cartridge, affinity chromatography on a column which was prepared by coupling of anti-CCAP antibody to CNBr-activated Sepharose, and reversed phase high performance liquid chromatography (HPLC). In the HPLC-profile immunoreactivity was confined to a single peak which co-chromatographed with authentic CCAP. The peptide was carboxymethylated and analyzed in an automated gas-phase sequencer. Its amino acid sequence, is identical to that of CCAP fromCarcinus maenas.Synthetic CCAP was tested on the isolated locust hindgut in vitro. The peptide proved to be a potent enhancer of gut contractions, with a significant effect being observable at concentrations of 10^–10 M. It is concluded that in the locust CCAP may function as a myotropic peptide.     

Patterns of oviducal motility in the cow during the oestrous cycle

Microtransducers sensitive to changes in internal diameter were chronically implanted in the oviducts of 5 dairy cows. Motility patterns were recorded throughout 9 oestrous cycles. Cyclic variations in patterns of motility were observed and compared with circulating concentrations of plasma progesterone. Luteal-phase motility patterns were of low amplitude and frequency. The frequency and amplitude of motility increased 3-5 days before behavioural oestrus. This activity consisted primarily of longitudinal muscle contractions, with an interspacing of circular muscle activity occurring during oestrus. Patterns of activity after oestrus were similar to those before oestrus, with activity decreasing 3-5 days after oestrus. Transducers implanted bilaterally in 2 animals permitted observation of asynchronous patterns between right and left oviducts. Preliminary data suggested a higher level of activity in the oviduct ipsilateral to the active ovary. These variations may be due to a local effect, possibly mediated by the functional ovary or the ovum.     

Spontaneous and neurally evoked contractions of visceral muscles in the oviduct of Locusta migratoria

The oviducts of Locusta migratoria are innervated by a pair of nerves which arise from, the seventh abdominal ganglion. A distinctive network of striated muscle fibres occurs in the oviducts. The lateral oviducts and common oviduct consist of an inner circular layer of muscle and an outer longitudinal layer of muscle. At the junction of the lateral and common oviduct an additional thin longitudinal layer is found adjacent to the basement epithelium. The oviducts contracted spontaneously when isolated from the central nervous system. These myogenic contractions took the form of peristaltic contractions in the lateral oviduct, and intermittent phasic-like contractions of the posterior regions of the lateral oviduct and the common oviduct. These phasic-like contractions were associated with individual complex potentials recorded extracellularly from the muscle fibres. In locusts that had been interrupted in the process of egg laying, there were large-amplitude action potentials, firing in a bursting pattern, in the oviducal nerves. These large action potentials were absent in locusts that had not been egg-laying. These action potentials were associated with both bioelectric potentials and mechanical events in the posterior region of the lateral oviduct and the common oviduct. Electrical stimulation of the oviducal nerve mimicked the effects of spontaneous action potentials, resulting in the appearance of monophasic potentials and contractions. The contractions were graded and dependent upon both frequency and duration of stimulation. It is concluded that the oviducts of Locusta are both myogenic and neurogenic. The role of these contractions in oviposition is discussed.     

The presence and specificity of crustacean cardioactive peptide (CCAP)-immunoreactivity in gastropod neurons

CCAP-like immunoreactivity was detected in central neurons with small and medium diameters in both Helix and Lymnaea CNS. The intensity of immunoreactivity showed seasonal changes with a maximum intensity during spring. The overwhelming majority of nerve cell bodies exhibiting CCAP immunoreactivity is located in the cerebral and parietal ganglia of both Helix and Lymnaea. The neurons of pleural and buccal ganglia were devoid of CCAP-immunoreactivity. Following preabsorbtion of CCAP antibody in 1:15000 dilution with 10(-3) M CCAP or CCAP-related peptide (Helix -CCAP), immunoreactivity could not be observed in neurons, demonstrating the specificity of the antibody to CCAP-related molecules in both Helix and Lymnaea.     

A neurohormonal role for serotonin in the control of locust oviducts

Serotonin increases the frequency and amplitude of spontaneous contractions and leads to an increase in the basal tonus of the locust oviducts. These effects were dose-dependent and were seen on both the non-innnervated and innervated portion of the oviducts. Vertebrate type serotonin agonists and antagonists were used and the profile shows that the receptors on the non-innervated and innervated portion of the oviducts are more similar to 5-HT3 receptors than to either 5-HT1 or 5-HT2 receptors. No serotonin was found associated with the oviducts or the innervation to the oviducts using immunohistochemistry and HPLC coupled to electrochemical detection, suggesting a neurohormonal role for serotonin in the control of locust oviducts.     

Turnover of protein and diacylglycerol components of lipophorin in insect haemolymph

Lipophorin, radiolabelled in the protein or diacylglycerol moiety, was purified from adult locusts injected previously with [¹⁴C]protein hydrolysate or sodium[1-¹⁴C]palmitate. The radiolabelled lipophorin was injected into adult male locusts and haemolymph samples taken periodically to determine the rate of disappearance of radioactivity from the haemolymph. Lipophorin was also purified from locusts that had been injected four days previously with ([¹⁴C]protein)-lipophorin to demonstrate that the radioactivity observed in the haemolymph at this time is due to radiolabelled lipophorin. The results indicate that the half-life of the protein component of lipophorin in resting insects is about 5–6 days whereas that of the diacylglycerol component is only about 2–3 hr.The results are consistent with the hypothesis that lipophorin functions as a “reusable shuttle” to transport a variety of lipid classes between sites of absorption, storage and utilisation.     

Excitatory interactions between antagonistic motor neurones underlying locust kicking and jumping during maturation after the adult moult

There is a change in the synaptic connections between motor neurones that underlie locust kicking and jumping during maturation following the adult moult. The fast extensor tibiae (FETi) motor neurone makes monosynaptic excitatory connections with flexor tibiae motor neurones that have previously been implicated in maintaining flexor activity during the co-contraction phase of jumping, in which energy generated by the muscles of a hind leg is stored. The amplitude of the FETi spike decreases when repetitively activated, and this decrement is larger in locusts immediately following the adult moult than in mature locusts. The decrement in␣the FETi spike is correlated with a greater decrease in the amplitude of the flexor excitatory postsynaptic potential (EPSP) in newly moulted locusts and in turn with the failure of these locusts to kick or jump. The results presented here indicate that the developmental change in the connections between the motor neurones contributes to the change in behaviour following the moult. Accepted: 28 April 1997     

Neural inhibition of egg-laying in the locust, Locusta migratoria

The role of the oviducal nerves during egg-laying in Locusta migratoria has been examined. Section of the oviducal nerves did not inhibit egg-laying in any observable way. Electrical stimulation of the oviducal nerves resulted in a contraction of the common and lower lateral oviducts which propelled ovulated eggs up towards the ovaries. Recordings from oviducal nerves using chronically implanted electrodes showed that electrical activity was low during actual egg-laying, but high at times when egg-laying was not occurring (i.e. during digging behaviour, or following interruption of egg-laying). During these periods of high activity recurrent bursts of action potentials occurred. Similar patterns of electrical activity were recorded in semi-intact preparations using suction electrodes applied to exposed oviducal nerves of locusts which had been interrupted during the process of egg-laying. High frequency bursts of activity were recorded simultaneously from both left and right oviducal nerves.It is concluded that one function of the oviducal nerves is to inhibit egg-laying at inappropriate times, by inducing contractions of the oviducts which propel eggs back towards the ovaries. These nerves therefore provide a physiological basis for part of the adaptive ovipositional activities of locusts.     

Evidence for octopaminergic modulation of an insect visceral muscle

Two dorsal unpaired median neurons (DUMOV1 and DUMOV2) lying in the posterior region of the VIIth abdominal ganglion of Locusta migratoria have axons which project to the muscles of the oviducts. This study reports the presence of octopamine within isolated DUMOV cell bodies, as well as in the oviducal nerve and innervated oviducal muscle. Individual cell bodies were pooled and found to contain about 0.34 pmol of octopamine per cell body giving an approximate value of 1.27 mM octopamine. Octopamine is concentrated within the area of oviducal muscle which receives DUMOV axons. Pharmacological studies reveal that the amplitude of neurally-evoked contractions of the oviducal muscle is reduced in a dose-dependent manner by octopamine, with threshold lying between 5 X 10(-10) M and 7 X 10(-9) M. The receptors for this response show a specificity for octopamine and synephrine, with an order of potency being octopamine = synephrine greater than metanephrine greater than tyramine greater than dopamine. The presence of octopamine throughout this neural pathway, coupled with the demonstration of octopaminergic modulation of muscular contraction, supports the hypothesis that octopamine serves a physiological role in this visceral system.     

Pharmacological profile of octopamine and 5HT receptors on the lateral oviducts of the cockroach,Periplaneta americana

The effects of the amines 5HT and octopamine on the myogenic activity of Periplaneta americana (L.) oviducts and the pharmacological profile of octopamine and 5HT receptors on the lateral oviducts have been determined. Application of 5HT to the oviducts resulted in a dose-dependent increase in basal tonus and amplitude of contractions. Antagonist studies revealed that the 5HT receptor on the cockroach oviduct most resembles the mammalian 5HT₂ receptor. Application of octopamine resulted in a decrease in basal tonus and had a biphasic effect on the amplitude of contractions, being stimulatory at low doses and inhibitory at higher ones. The inhibitory effects of octopamine appear to be mediated via cAMP and are blocked by antagonists which indicate that the octopamine receptor is of the octopamine-2 type. © 1995 Wiley-Liss, Inc.     

Circadian regulation of the lark RNA‐binding protein within identifiable neurosecretory cells

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA‐binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)‐containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA‐binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly‐emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA‐binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 14–29, 2000     

Circadian regulation of the lark RNA-binding protein within identifiable neurosecretory cells

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability.     

Neuromuscular block in locust skeletal muscle caused by a venom preparation made from the digger wasp Philanthus triangulum F. from Egypt.

A preparation from P. triangulum F., made by extracting abdomens and purified by Sephadex filtration, does not affect potassium ion-induced contractions of the retractor unguis muscle of S. gregaria, but the reduction of the glutamate contractions is at least as pronounced as the effect on the neurally-evoked twitch. Glutamate potentials are affected at a lower venom dose than are the neurally evoked excitatory postsynaptic potentials (EPSPs). The half-decay-time of the glutamate potentials starts to decrease just before the decrease in amplitude is initiated.In the retractor unguis muscle the resting plasma membrane is slightly depolarized at high venom concentrations, but this effect cannot explain the effects on neuromuscular transmission. It is concluded that the venom preparation of P. triangulum affects the glutamate or transmitter-induced transient permeability change, possibly by blocking the open ion-channels.