Eclosion hormone

EVOLUTIONARY HOMOLOGS

Eclosion hormone structure

How important is the C-terminus of Bombyx EH for biological activity? A molecule lacking the four amino acids at the C-terminus is 100-fold less active than native EH. By contrast, a peptide lacking the six N-terminal amino acids retain full activity. Thus, the C-terminus plays an important part in the biological function of EH. This domain could potentially function in binding to a receptor in the target cells. Another notable feature is the conservation of the six cysteine residues. These form intramolecular bonds. The positions of these disulfide bonds has been determined in Manduca and Bombyx EH (Kataoka, 1992 and Kono, 1990).

The locations of the three disulfide bonds of eclosion hormone (EH) isolated from Manduca sexta were assigned by sequence analysis of thermolysin fragments and by comparison of a key heterodimeric fragment to regiospecifically synthesized parallel and antiparallel isomers. The complete structure of Manduca EH is a 62-residue peptide that has three disulfide bonds between Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49 (Kataoka, 1992).

The cDNAs encoding eclosion hormone in the silkworm Bombyx mori were isolated and sequenced. The pre-EH molecule contains a 26-amino acid signal peptide and a 62-amino acid mature EH. A leucine residue at the carboxyl terminal of EH had not been detected directly by the peptide analysis. Primer extension and Northern hybridization analyses reveal that 0.9 kb mRNA is transcribed with a 66-nucleotide non-translated sequence at the 5'-end region. In situ hybridization shows that the EH gene is expressed in two pairs of nuerosecretory cells in the brain of 5th instar larva (Kamito, 1992).

Recombinant silkworm eclosion hormone was produced for the first time in yeast, which was transformed with a shuttle plasmid containing a construct coding a signal peptide and the mature sequence of the silkworm eclosion hormone. Successfully transformed yeast process recombinant silkworm eclosion hormone I (EH-I) was transported to periplasm at a concentration of 60 micrograms per liter of culture. The biological activity of the purified recombinant silkworm eclosion hormone exhibits the ED50 value of 0.2 ng, which is the same as that of the authentic hormone isolated from the silkworm brain (Hayashi, 1990).

A gene encoding eclosion hormone (EH) from the silkworm Bombyx mori was chemically synthesized, inserted into a secretion vector and expressed in Escherichia coli, leading to the production of biologically active EH. Sequence analysis of cystine-containing peptides in a thermolysin digest of this EH established the locations of 3 disulfide bonds in the molecule. The 6 residues at the NH2-terminus are dispensable but 4 residues at the COOH-terminal play an important role in EH activity (Kono, 1990).

The EH-encoding gene of the tobacco hornworm Manduca sexta was isolated by using a designed 72-mer oligonucleotide probe. Sequence analysis of this gene and its corresponding cDNA show that the EH gene is 7.8 kilobases and consists of three exons. Exon I is totally nontranslated; exon II contains a 26-amino acid signal peptide and amino acids 1-4 of the EH peptide, and exon III encodes the remainder of the peptide. The EH gene is present in a single copy per haploid genome and transcribes an 0.8-kb mRNA that is expressed in larval, diapausing pupal, and developing adult brains but not in the ventral nerve cord or in nonneural tissues. In situ hybridization shows that the EH gene is expressed in two pairs of ventromedial neurosecretory cells in brains of both larvae and developing adults (Horodyski, 1989).

Eclosion hormone and Ecdysis triggering hormone

A successful ecdysis in insects requires the precise coordination of behaviour with the developmental changes that occur late in a molt. This coordination involves two sets of endocrine cells: the peripherally located Inka cells, which release ecdysis triggering hormone (ETH), and the centrally located neurosecretory neurons, the VM neurons, which release eclosion hormone (EH). These two sets of endocrine cells mutually excite one another: EH acts on the Inka cells to cause the release of ETH. ETH, in turn, acts on the VM neurons to cause the release of EH. This positive-feedback relationship allows the Inka cells and the VM neurons to be the peripheral and central halves, respectively, of a decision-making circuit. Once conditions for both halves have been satisfied, their reciprocal excitation results in a massive EH/ETH surge in the blood as well as a release of EH within the central nervous system. This phasic signal then causes the tonic activation of a distributed network of peptidergic neurons that contain crustacean cardioactive peptide (Ewer, 1997).

In insects, the shedding of the old cuticle at the end of a molt involves a stereotyped sequence of distinct behaviors. Two peptides, ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (CCAP), elicit the first two motor behaviors, the pre-ecdysis and ecdysis behaviors, respectively. Exposing isolated abdominal ganglia to ETH results in the generation of sustained pre-ecdysis bursts. By contrast, exposing the entire isolated CNS to ETH results in the sequential appearance of pre-ecdysis and ecdysis motor outputs. Previous research has shown that ETH activates neurons within the brain that then release eclosion hormone within the CNS. The latter elevates cGMP levels within target neurons and increases the excitability of a group of neurons containing CCAP. The ETH-induced onset of ecdysis bursts is always associated with a rise in intracellular cGMP within these CCAP neurons. CCAP immunoreactivity decreases centrally during normal ecdysis. Isolated, desheathed abdominal ganglia respond to CCAP by generating rhythmical ecdysis bursts. These ecdysis motor bursts persist as long as CCAP is present and can be reinduced by successive application of the peptide. CCAP exposure also actively terminates pre-ecdysis bursts from the abdominal CNS, even in the continued presence of ETH. Thus, the sequential performance of the two behaviors arises from one modulator activating the first behavior and also initiating the release of the second modulator. The second modulator then turns off the first behavior while activating the second (Gammie, 1997).

Three insect peptide hormones, eclosion hormone (EH), ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (CCAP), have been implicated in controlling ecdysis behavior in insects. This study examines the interactions among these three peptides in the regulation of the ecdysis sequence. Using intracellular recordings, it has been found that ETH is a potent activator of the EH neurons, causing spontaneous action potential firing, broadening of the action potential and an increase in spike peak amplitude. In turn, electrical stimulation of the EH neurons or bath application of EH to desheathed ganglia results in the elevation of cyclic GMP (cGMP) levels within the Cell 27/704 group (which contains CCAP). This cGMP production increases the excitability of these neurons, thereby facilitating CCAP release and the generation of the ecdysis motor program. Extracellular recordings from isolated nervous systems show that EH has no effect on nervous systems with an intact sheath. In desheathed preparations, in contrast, EH causes only the ecdysis motor output. The latency from EH application to ecdysis is longer than that after CCAP application, but shorter than that when ETH is applied to a whole central nervous system. These data support a model in which ETH causes pre-ecdysis behavior and at higher concentrations stimulates the EH neurons. EH release then facilitates the onset of ecdysis by enhancing the excitability of the CCAP neurons (Gammie, 1999).

Ecdysis, or molting behavior, in insects requires the sequential action of high levels of ecdysteroids, which induce accumulation of ecdysis-triggering hormone (ETH) in Inka cells, followed by low levels of ecdysteroids, permissive for the onset of the behavior. High ecdysteroid levels suppress the onset of the behavioral sequence by inhibiting the development of competence to secrete ETH. In pharate pupae of Manduca sexta, Inka cells in the epitracheal glands normally develop competence to secrete ETH in response to eclosion hormone (EH) 8 h before pupation. Injection of 20-hydroxyecdysone (20E) into precompetent insects prevents this acquisition of competence, but does not affect EH-evoked accumulation of the second messenger cyclic GMP. Precompetent glands acquire competence in vitro after overnight culture, and this can be prevented by the inclusion of 20E at concentrations greater than 0.1 microg ml(-1)in the culture medium. Actinomycin D completely inhibits the acquisition of competence, demonstrating that it is dependent on transcriptional events. Cultured epitracheal glands become refractory to the inhibitory effects of 20E in the acquisition of competence at least 3 h earlier than for Actinomycin D, indicating that 20E acts on an early step in a sequence of nuclear events leading to transcription of a structural gene. These findings suggest that declining ecdysteroid levels permit a late event in transcription, the product of which is downstream of EH receptor activation and cyclic GMP accumulation in the cascade leading to ETH secretion (Kingan, 2000).

Downstream effector pathway of eclosion hormone

During ecdysis EH induces an increase in cyclic GMP (cGMP). Using antibodies against this second messenger, it has been shown that this increase is confined to a network of 50 peptidergic neurons distributed throughout the CNS. Increases appeared 30 min after EH treatment, spread rapidly throughout these neurons, and are extremely long lived. This response is synaptically driven, and does not involve the soluble, nitric oxide (NO)-activated, guanylate cyclase. Stereotyped variations in the duration of the cGMP response among neurons suggest a role in coordinating responses having different latencies and durations (Ewer, 1994).

Additional studies were carried out to test whether nitric oxide is involved in increases in cGMP caused by the activity of eclosion hormone. In Manduca, the eclosion hormone-stimulated increase in cGMP is independent of either nitric oxide or carbon monoxide. In addition, a wide variety of inhibitors of lipid metabolism block the eclosion hormone-stimulated cGMP increase. This supports the hypothesis that the activation of the gyanylate cyclase is mediated by a lipid messenger. Eclosion hormone stimulates an increase in the levels of inositol(1,4,5)trisphosphate. The time course of this increase is consistent with the hyposthesis that eclosion hormone stimulation of a phospholipase C is an early event in the cascade that results in an increase in cGMP. Receptor-mediated lipid hydrolysis is often mediated by G protein-coupled receptors. Experiments using pertussis toxin show that the eclosion hormone-stimulated increase in cGMP is not mediated by a pertussis toxin-sensitive G protein (Morton, 1995b)

Eclosion hormone stimulates the formation of two intracellular second messengers, cGMP and inositol(1,4,5)trisphosphate in the abdominal ganglia of Bombyx mori. In order to elucidate the intracellular signaling pathway involving these second messengers, the eclosion hormone-mediated signal transduction was studied using saponin-treated abdominal ganglia. Eclosion hormone activates nitric oxide synthase. The eclosion hormone-induced cGMP increase is inhibited by various enzyme inhibitors: these include NG-nitro-arginine, a nitric oxide synthase inhibitor (EGTA), a calcium chelating reagent (W-5), a calmodulin inhibitor and compound 48/80 (a phospholipase C inhibitor). The inositol(1,4,5)trisphosphate stimulates the formation of cGMP in the Bombyx abdominal ganglia. Based on these findings a pathway is proposed: the signal initially triggered by eclosion hormone and eclosion hormone receptor complex induces activation of phospholipase C, which produces inositol(1,4,5)trisphosphate. Inositol(1,4,5)trisphosphate increases intracellular Ca2+, followed by subsequent activation of nitric oxide synthase through the formation of Ca(2+)-calmodulin complex (note: contradictory evidence above in Ewer [1994]). The reaction product, nitric oxide, acts on soluble guanylate cyclase to stimulate cGMP formation that induces the ecdysis behavior in Bombyx pharate adults (Shibanaka, 1994).

Eclosion hormone stimulates phosphatidylinositol (PtdIns) hydrolysis in abdominal ganglia isolated from Bombyx mori at a specific stage in adult development. In the presence of EH, incubation of abdominal ganglia from silkworm pharate adults leads to an increase in formation of inositol(1,4,5)trisphosphate, but this increase takes place transiently, maximum increase being observed 30 s after the addition of EH. PtdIns hydrolysis is stimulated by exogenous EH in a dose-dependent fashion and is completely abolished by the phospholipase C inhibitors: neomycin and compound 48/80. The EH-induced PtdIns hydrolysis develops in parallel with the EH-induced eclosion behaviour during development of the adult. These results suggest that the EH-stimulated PtdIns hydrolysis plays an important role in EH-mediated signal transduction during adult development of B. mori (Shibanaka, 1993).

The signal transduction of the peptide Eclosion hormone in the silkworm Bombyx mori appears to be mediated via the second messenger cyclic GMP throughout the life cycle. Injection of 8-bromo-cGMP induces the ecdysis behavior in pharate adults with similar latency to eclosion hormone-induced ecdysis; the moulting occurs 50-70 min after the injection. The potency of 8Br-cGMP is 10(2) fold higher than that of cGMP; the efficacy is increased by the co-injection of the phosphodiesterase inhibitor IBMX. In silkworm pupal ecdysis, both Eclosion hormone and 8Br-cGMP induce the molting behavior in a dose-dependent manner. The adult development of the ability to respond to 8Br-cGMP takes place concomitantly with the response to Eclosion hormone. Both the developmental time courses are shifted by a shift in the light and dark cycles. Accordingly, the sensitivities to the peptide and cyclic nucleotide develop correspondingly with shifts in circadian rhythm of light and dark. Thus throughout the silkworm life cycle, eclosion hormone is effective to trigger the ecdysis behavior; cGMP plays a crucial role as the second messenger in the eclosion hormone-mediated signal transduction (Shibanaka, 1991).

The neuropeptide eclosion hormone acts directly on the nervous system of the tobacco hornworm Manduca sexta to trigger ecdysis behavior at the end of each molt. The action of eclosion hormone is mediated via the intracellular messenger cyclic GMP. No stimulation of guanylate cyclase is seen in homogenized nervous tissue, suggesting that eclosion hormone does not directly stimulate a membrane-bound form of guanylate cyclase. Nitric oxide synthase inhibitors, N-methylarginine and nitroarginine, have no effect on eclosion hormone-stimulated cyclic GMP levels. By contrast, 4-bromophenacyl bromide, an inhibitor of arachidonic acid release, and nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, almost completely abolish the eclosion hormone-stimulated cyclic GMP increase. It is hypothesized that eclosion hormone receptors are coupled to a lipase, activation of which causes the release of arachidonic acid. Either the arachidonic acid directly stimulates the soluble guanylate cyclase or further metabolism of arachidonic acid yields compounds that activate guanylate cyclase (Morton, 1992).

Two phosphoproteins, both with an apparent molecular weight of 54 kDa, are described in the CNS of the tobacco hornworm, Manduca sexta. Their phosphorylation is regulated by the neuropeptide Eclosion hormone, and the second messenger cGMP. The phosphoprotein thus have been named the EGPs (Eclosion hormone- and cGMP-regulated phosphoproteins). Although cAMP is more effective than cGMP at stimulating the phosphorylation of the EGPs in CNS homogenates, cGMP is more effective in the intact CNS. Since cGMP mediates the action of EH, this strongly suggests that cGMP is the second messenger that stimulates the phosphorylation of the EGPs in vivo. The EGPs can only be phosphorylated in vitro during discrete time periods in the life of Manduca. During the larval and pupal molts, the EGPs can first be phosphorylated just prior to ecdysis. Their ability to be phosphorylated is correlated with the time when the insect is sensitive to EH. This close temporal correlation suggests that the ability to phosphorylate the EGPs determines when the insect can first respond to EH. During adult development, the EGPs first appeared 6 d before sensitivity to EH, suggesting that at this stage other factors may also be involved in the regulation of sensitivity. For the ecdyses of all 3 stages, EH appears to stimulate the phosphorylation of the EGPs at ecdysis. The EGPs are found in all regions of the prepupal nervous system, but only in the abdominal and pterothoracic ganglia of the developing adult. Fractionation of nervous system homogenates by ultracentrifugation reveals that one of the EGPs is present only in the pellet fraction, whereas the other is approximately equally distributed between pellet and supernatant. Furthermore, the EGPs in the pellet fraction can be partially solubilized with detergents and high salt concentrations (Morton, 1988a).

Two proteins (the EGPs) in the CNS of the tobacco hornworm Manduca sexta are phosphorylated by the action of the neuropeptide eclosion hormone (EH), which triggers ecdysis behavior. The onset of sensitivity to EH requires prior exposure to the steroid hormone 20-hydroxyecdysone (20-HE). A series of steroid removal and replacement experiments indicates that the EGPs are also regulated by 20-HE with a time course that is similar to that seen for the 20-HE regulation of behavioral sensitivity to EH. This suggests that the steroid regulation of EH sensitivity is due to the regulation of the EGPs. The appearance of the EGPs requires not only the presence of 20-HE, but also its subsequent removal. The appearance of the EGPs can be blocked in vivo and in vitro by maintaining artificially elevated levels of 20-HE, but only up to a particular time in development. The ending of this steroid-sensitive period occurs 4 hr before the normal appearance of the EGPs, consistent with the hypothesis that the EGPs are synthesized de novo in response to the removal of 20-HE (Morton, 1988b).

The action of Eclosion hormone is mediated by an increase in cGMP and is associated with the phophorylation of two proteins, the EGPs. The ability of insects to responed to EH is developmentally regulated with sensitivity being first seen at about 8 hours prior to the normal time of ecdysis. The EGPs are also first detectable in the CNS at 8 hours prior to ecdysis, suggesting that it is their synthesis which determines EH sensitivity. The protein synthesis inhibitor cyclohexamide was used to study the development of the events leading to pupal ecdysis in Manduca. Protein synthesis is necessary about 10 hours before ecdysis for both the development of EH sensitivity and for the appearance of the EGPs (Morton, 1995a)

Eclosion hormone effects on neural activity

In the moth Manduca sexta, the declining ecdysteroid titer on the final day of the molt from the fourth to the fifth larval instar acts on the ventromedial neurosecretory cells (VM cells) to stimulate the release of eclosion hormone (EH). EH then triggers the motor programs involved in ecdysis behavior. Intracellular recordings made from the VM cells throughout the intermolt and molting stages show no spontaneous action potentials until 0.9 hr before ecdysis (during the expected time of EH release), when 50% of the VM cells fire tonically at rest. This change is associated with a marked reduction in VM cell threshold without alteration of input resistance, resting potential, or synaptic drive. The increase in VM cell excitability is dependent on the declining ecdysteroid titer, because the injection of 20-hydroxyecdysone (20-HE) 11.5 hr before ecdysis significantly delays the expected decrease in VM cell threshold. Since the same steroid treatment given 4.6 hr before ecydysis does not delay the subsequent increase in VM cell excitability, the inhibitory actions of 20-HE appear not to be mediated by a rapid membrane mechanism. The possible involvement of genomic events in the steroid-dependent increase in VM cell excitability was examined using the RNA synthesis inhibitor actinomycin D (AcD). When injected 6.3 hr before ecdysis, AcD blocks EH release without altering the response of the nervous system to exogenous peptide. Such AcD treatments also prevents the increase in VM cell excitability. These results suggest that the declining ecdysteroid titer increases the excitability of the VM cells via a transcription-dependent process (Hewes, 1994).

The effects of removing or disconnecting portions of the central nervous system prior to the time of EH release was examined on the initiation of pre-ecdysis and ecdysis behaviors at the final larval moltof Manduca. The initiation of pre-ecdysis abdominal conpressions at the appropriate time requires the terminal abdominal ganglion but not the brain. The initiation of pre-ecdysis proleg retractions at the appropriate time requires neither the terminal abdominal ganglion nor the brain. The initiation of ecdysis at the appropriate time usually requires the brain but not the terminal abdominal ganglion. Premature pre-ecdysis (but not ecdysis) can be elicited in isolated abdomens by injection of eclosion hormone. Finally, pre-ecdysis behavior performed by brainless larvae is not associated with the normal elevation of eclosion hormone bioactivity in the hemolymph or the normal loss of of EH immunoreactivity from peripheral neurohemal release sites (Novicki, 1996).

Eclosion hormone and programmed cell death

At the end of metamorphosis, the intersegmental muscles of the moth Antheraea polyphemus undergo rapid degeneration in response to the peptide eclosion hormone (EH). Muscle death is preceded by a 22-fold increase in muscle guanosine-3',5'-cyclic monophosphate (cGMP) titers, which peak 60 min after peptide exposure. EH induces a dose-dependent increase in muscle cGMP content with a threshold dose similar to that needed to induce cell death. Exogenous cGMP, but not cAMP, mimic the action of EH. Sodium nitroprusside, a potent stimulator of guanylate cyclase, and methylated xanthines, a class of 3',5'-cyclic-nucleotide phosphodiesterase inhibitors, also induce the selective death of these muscles. It is concluded that an elevation of cGMP level is involved in EH-induced muscle degeneration. The intersegmental muscles become sensitive to EH at the end of adult development in response to the declining titers of the steroid molting hormones, the ecdysteroids. At earlier times, treatment with EH, exogenous cGMP, sodium nitroprusside, or methylated xanthines is ineffective in causing cell death. Nevertheless, treatment with EH at this time results in a marked increase in intersegmental-muscle cGMP. Thus, the onset of physiological responsiveness to the peptide hormone presumably results from biochemical changes distal to the EH receptors and guanylate cyclase (Schwartz, 1984).

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