Origin and release sites of a hormone stimulating oviposition in the stick insect Clitumnus extradentatus

In the stick insect, Clitumnus extradentatus, a hormone stimulating oviposition, produced in type-A neurosecretory cells is located in the thoracic and anterior abdominal ganglia. At the end of the photophase, this neurohormone is stored in neurohaemal areas associated with segmental nerves. During the scotophase, the neurohormone is released into the haemolymph and acts on the genital tract, triggering nocturnal oviposition. The oviposition stimulating hormone is not species-specific.     

Rhythmic release of prothoracicotropic hormone from the brain of an adult insect during egg development

Prothoracicotropic hormone (PTTH) is a brain neurohormone that has been studied for over 80 years. The only known target of PTTH is the prothoracic glands (PGs) of larvae, which synthesize the insect molting hormones (ecdysteroids) and a massive literature exists on this axis. The PGs degenerate around the time of adult emergence, yet presence of PTTH has been reported in the brains of several adult insects. Using an in vitro bioassay system, we confirm that PTTH is present in the adult female brain of Rhodnius prolixus. The material is electrophoretically, immunologically and biologically indistinguishable from larval PTTH. The amount of PTTH in the brain shows a daily rhythm during egg development. We show that brains in vitro release PTTH with a daily rhythm over this period of time. PTTH is released at each scotophase. This is the first report that PTTH is released from the adult brain and functions as a hormone, inviting explanation of its function. Larval PTTH is also known to be released with a daily rhythm, and the clock in the brain controls both larval and adult rhythms. The potential significance of rhythmic PTTH release in female adults is discussed in relation to the regulation of ecdysteroids, egg development and the concept of internal temporal order.     

Bombyxin exhibits an insulin-like response to modification in the N-terminal region of the A chain.

Bombyxin is an insect neurohormone with an insulin-like structure. The N-terminal A chain helix, a region which is considered part of the active site in insulin, is almost identical between the two hormones. Bombyxin analogues with modifications at the N-terminus of the A-chain were synthesized and investigated for their ability to bind to bombyxin-specific receptors. While N-acetylation reduced the affinity to the bombyxin receptor to 18% the removal of glycine (A1) inactivated the hormone completely. Replacement of glycine (A1) by L-amino acids caused a significant loss in activity (11%) while its replacement by D-amino acid resulted in active bombyxin analogues (55%). Comparative CD spectroscopy indicated a change in structure for desGly(A1)bombyxin. Although the insect hormone does not have an insulin-like function it exhibits mammalian insulin-like structural sensitivity for A chain modifications.     

Experimentelle Untersuchungen zum Freisetzungsmechanismus von Neurohormonen nach elektrischer Reizung der Corpora cardiaca von Periplaneta americana in vitro

Releasing of neurohormones by in vitro stimulation of the corpora cardiaca of Periplaneta americana by means of suction electrodes and by simultaneous application of sympathicomimetics and -lytics as well as parasympathicomimetics and -lytics was investigated. The release of neurohormone D by stimulation of N.c.c. I in the presence of atropine in bath-fluid is inhibited. Contrary to this, the presence of eserine stimulates release. Application of reserpine, as well as of the sympathicolytics tolazolin, retinin, and guanethidin, does not influence release of this hormone in connexion with the stimulation of N.c.c. I. These results indicate that the release of neurohormone D by stimulation of N.c.c. I is regulated by cholinergic components. On the other hand, release of the hyperlgykaemic factor by electrical stimulation of N.c.c. II is controlled by adrenergic components. This conclusion results from the increase of the release rate caused by reserpine. Sympathicolytics decreased the release rate. Atropine and eserine do not influence hormone release in connexion with the stimulation of N.c.c. II.     

Pctaire1 phosphorylates N-ethylmaleimide-sensitive fusion protein: implications in the regulation of its hexamerization and exocytosis

Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related family, has recently been shown to be phosphorylated and regulated by Cdk5/p35. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. We performed a yeast two-hybrid screen to identify proteins that interact with Pctaire1. N-Ethylmaleimide-sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was identified as one of the Pctaire1 interacting proteins. We demonstrate that the D2 domain of NSF, which is required for the oligomerization of NSF subunits, binds directly to and is phosphorylated by Pctaire1 on serine 569. Mutation of this phosphorylation site on NSF (S569A) augments its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhances the self-association of NSF. Interestingly, Pctaire1 associates with NSF and synaptic vesicle-associated proteins in adult rat brain. To investigate whether Pctaire1 phosphorylation of NSF is involved in regulation of Ca(2+)-dependent exocytosis, we examined the effect of expressing Pctaire1 or NSF phosphorylation mutants on the regulated secretion of growth hormone from PC12 cells. Interestingly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increases high K(+)-stimulated growth hormone release. Taken together, our findings provide the first demonstration that Pctaire1 phosphorylation of NSF regulates the ability of NSF to oligomerize, implicating an unexpected role of this kinase in modulating exocytosis. These findings open a new avenue of research in studying the functional roles of Pctaire1 in the nervous system.     

The gonadotropin-releasing hormone receptor: signals involved in gonadotropin secretion and biosynthesis

The neurohormone gonadotropin-releasing hormone (GnRH) is a decapeptide which is synthesized in the hypothalamus and released into the hypophysial portal system in a pulsatile manner. GnRH exerts its effect on the anterior pituitary gonadotrophs where it regulates the secretion and synthesis of gonadotropins (luteinizing hormone and follicle-stimulating hormone) through receptor-mediated actions. The GnRH receptor has been characterized and shown to be coupled to the formation of 'second messengers' which participate in signal transduction mechanisms. GnRH stimulation of luteinizing hormone release is a Ca2(+)-dependent process. G protein, phosphoinositide hydrolysis, protein kinase C as well as arachidonic acid and some of its metabolites were identified as possible mediators in the process.     

Exocytosis of neurosecretory granules from the crustacean sinus gland in freeze-fracture

Exocytosis is clearly shown in freeze-fracture preparations to be the mechanism for neurosecretion granule release from axon endings in the crayfish sinus gland. The cytoplasmic leaflet (A-face) of axon ending membrane is characterized by randomly situated depressions representing invaginations of the axolemma, which are in contact with limiting membranes of neurohormone granules in the subjacent cytoplasm. The extracellular leaflet (B-face) of the axolemma at release sites exhibits complementary volcano-shaped protrusions which are cross-fractures through necks of channels formed by invaginating plasma membrane in contact with underlying neurosecretion granules. Structural variation in B-face protrusions is consistent with a spectrum of exocytotic profiles in various stages of formation, and with granules at different stages of passage out of the endings. Evidence in this study suggests that formation of exocytotic structures may begin by alteration of axon membrane structure at the neurosecretory ending-hemolymph interface prior to contact of the neurohormone granules with the axolemma. Limiting membranes of neurosecretory granules exhibit protrusions which appear to interconnect granules adjacent to release sites and to attach granules to the axolemma. Freeze-fracture is clearly shown to be an invaluable tool for monitoring the degree of exocytosis exhibited by sinus glands under normal conditions and under experimental acceleration of hormone release. This technique is capable therefore, of detecting slight increases in numbers of exocytotic profiles much more quickly and accurately than the examination of random thin sections.     

Recent trends in research on induced spawning of fish in aquaculture

Since 1973, the treatment of sexually mature fish with brain hormones (i. e. neurohormones) in order to induce spawning activity has gradually been replacing the hypophysation, although the latter is still widely used. Some brain-hormone analogues have a hightened spawning-inducing effect. Since the discovery that dopamine inhibits gonadotropic hormone release, dopamine antagonists—pimozide or domperidone—are injected before or together with brain-hormone analogues. This double treatment, i. e. the suppression of dopamine inhibition followed by neurohormone stimulation, has become a current technique in aquaculture. The discovery of the pulsatile release of gonadotropic hormone from the pituitary hints at the possibility of using techniques in which exogenous hormones are injected in pulses. In the past few years, induction of spawning through the control of photoperiod and/or temperature has become increasingly important.     

Role of LPXRFamide peptide in the neuroendocrine regulation of reproduction in fish

The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin (GTH) secretion. This review focuses on a family of neuropeptides, LPXRFamide (LPXRFa) peptides, which have been implicated in the regulation of GTH secretion. LPXRFa acts on the pituitary via a G protein-coupled receptor, LPXRFa-R, to enhance gonadal development and maintenance by increasing gonadotropin release and synthesis. Because LPXRFa exists and functions in several fish species, LPXRFa is considered to be a key neurohormone in fish reproduction control. The precursors to LPXRFamide peptides encoded plural LPXRFamide peptides and were highly divergent in vertebrates, particularly in lower vertebrates. Tissue distribution analyses indicated that LPXRFamide peptides were highly concentrated in the hypothalamus and other brainstem regions. In view of the localization and expression of LPXRFamide peptides in the hypothalamo-hypophysial system, LPXRFamide peptide in fish increase GTH release in vitro and in vivo. This review summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of LPXRFa, a newly discovered key neurohormone.     

Production of antibodies to Manduca sexta cerebral neurosecretory cells

Understanding the neuroendocrine regulation of insect development depends upon having antibody probes to the neurohormones involved, which are usually present at trace levels, making antibody generation difficult. This report describes a simple method for producing antibodies specific to cerebral neurosecretory cells (NSC) of the tobacco hornworm, Manduca sexta, and to the neurohormone(s) they produce. The method involves the isolation of only a few hundred NSC somata (～ 0.3 μg of protein) that serve as the immunogen. The cerebral NSC used were the L-NSC III, the prothoracicotropes, that produce the prothoracicotropic hormone (PTTH), the principal neuroendocrine effector of insect molting and metamorphosis. A PTTH-containing extract of microsurgically isolated somata of the L-NSC III was injected intraperitoneally into a Balb/c mouse. The antiserum produced specifically immunostained the L-NSC III in wholemounts of brains from different developmental stages. This antiserum also contained antibodies directed against PTTH, as shown by its ability to inhibit the neurohormone's biological activity in an in vitro prothoracic gland bioassay. Such antiserum can be used to investigate the ontogeny and phylogeny of NSCs. With the hybridoma technique, monoclonal antibodies to individual NSC proteins (PTTH) could be obtained, circumventing the need to purify them for antibody production. This method should be applicable to comparable neurosecretory cell systems in other insect species.     

Receptors and neurohormones in human pituitary adenomas

In order to go further into the pathogenesis of human pituitary adenomas, we studied receptors for neurohormones (thyroliberin, TRH; dopamine, DA; somatostatin, SRIH), for estradiol and epidermal growth factor (EGF) thought to influence hormone secretion and/or cell growth. The following results were obtained: (1) the receptors listed above, with the exception of EGF receptors in the adenomas, are present in normal pituitary tissue and in prolactin (PRL)- and growth hormone (GH)-secreting adenomas; (2) they are functional and their affinities are not different in normal or tumoral tissues; (3) their density is variable and depends on the type of secreting adenoma (GH or PRL), the size of the tumor and the plasma level of the hormone which is secreted, and (4) in nonsecreting adenomas, only TRH receptors are found with characteristics identical to those observed in secreting adenomas. We also showed that TRH is contained in normal and tumoral pituitary tissues. TRH and SRIH are released in vitro from adenomatous cells in large amounts, suggesting their possible synthesis by the pituitary. In both cases a local regulation is observed. TRH release is stimulated in the presence of DA while SRIH is inhibited in the presence of TRH. This neuropeptide release may be implicated in the pituitary hormone regulation through a paracrine or an autocrine mechanism. Thus, the neurohormone receptors found in pituitary adenomas should be dependent on a more complex regulation than it has been envisaged till now.     

Metabolism of juvenile hormone in cultures of ovaries and fat body in the cockroachperiplaneta Americana

Summary The time course of juvenile hormone (JH) metabolism is examined in cultures ofPeriplaneta americana fat body and ovaries in medium containingManduca sexta carrier protein or cockroach hemolymph. In the absence ofM. sexta carrier protein or cockroach hemolymph, both tissues extensively catabolize exogenous [³H]JH in the medium. Addition of the carrier protein or hemolymph to the culture system prevents the hydrolysis of the hormone in the medium. Within the tissues JH is degraded whether or not carrier protein or hemolymph is present which suggests that the protective role of these molecules is exclusively extracellular. Incubation of [³H]JH with medium preconditioned with tissue results in destruction of the hormone. This suggests that the fat body secretes esterases into the medium. In contrast, the ovarioles hydrolyze the hormone by means of cell-associated enzyme. The relationship of these phenomena to insect development is discussed. This work supported by NSF Grant PCM 76-02229 and University of Kansas Biomedical Sciences Grant RR-07037.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

Nonspecific regulatory mechanism of contact sensitivity: nonspecific suppressor factor (NSF)-treated intermediate cells produce a second nonspecific suppressor factor (NSFint).

Nonspecific suppressor factor (NSF), which inhibits the passive transfer of contact sensitivity (CS), is produced spontaneously from macrophage-like suppressor cells induced by intravenous administration of oxazolone (Ox)-conjugated spleen cells. NSF binds selectively to Ia-positive, cyclophosphamide (CY)-sensitive, and plastic-adherent cells (named intermediate cells) present in the normal spleen. NSF-treated intermediate cells acquire the ability to suppress the passive transfer of CS nonspecifically. In this study, NSF-treated intermediate cells were found to release a second nonspecific suppressor factor (NSFint) during a 2-hr culture, while retaining their suppressor activity. Investigation of the relationship between these two factors showed that both NSF and NSFint were trypsin-sensitive, nondialyzable proteins. However, gel chromatography revealed that NSF was about 43 kDa, while NSFint was about 20 kDa. NSF was released from macrophage-like suppressor cells after RNA-dependent protein synthesis. In contrast, production of NSFint was energy dependent but did not require protein synthesis. Intermediate cells pretreated with lysosomotropic agents, such as ammonium chloride or chloroquine, did not acquire suppressor activity nor release suppressor factors due to NSF treatment. These observations suggest that NSFint is an altered form of NSF released by the intermediate after having undergone some modification; the biochemical mechanism is not known. This study showed that the intermediate cells play an active role in the suppressor cascade of NSF.     

Chemical synthesis of a 7 kDa insect gonadotropic neurohormone

An original insect neurohormone of 65 residues was synthesized by the solid-phase methodology using t-Boc strategy and Boc-Val-PAM–resin. The purification, conducted by several steps of liquid chromatography having mass, polarity or charge as separative criteria, yielded the product with the correct molecular weight of 6922 Da determined by mass spectrometry. The synthetic peptide had both the same affinity for the antinative neurohormone serum and the same biological activity as the native neurohormone.     

From bioassays to Drosophila genetics: strategies for characterizing an essential insect neurohormone, bursicon

We describe the molecular analysis and cellular expression of the insect peptide neurohormone, bursicon. Bursicon triggers the sclerotization of the soft insect cuticle after ecdysis. Using protein elution analyses from SDS gels, we determined the molecular weight of bursicon from different insects to be approximately 30 kDa. Four partial peptide sequences of Periplaneta americana bursicon were obtained from purified nerve cord homogenates separated on two-dimensional gels. Antibodies produced against one of the sequences identified the cellular location of bursicon in different insects and showed that bursicon is co-produced with crustacean cardioactive peptide (CCAP) in the same neurons in all insects tested so far. Additionally, using the partial peptide sequences, we successfully searched the Drosophila genome project for the gene encoding bursicon. With Drosophila as a tool, we can now verify the function of the sequence using transgenic flies. Sequence comparisons also allowed us to verify that bursicon is conserved, corroborating the older data from bioassays and immunohistochemical analyses. The sequence of bursicon will enable further analysis of its function, release, and evolution.     

Sites of action of a peptide neurohormone that controls hindgut muscle activity in the cockroach Leucophaea maderae

A peptide neurohormone from the brain and nervous system of the Madeira cockroach Leucophaea maderae has stimulating effects on both the mechanical and electrical events of hindgut visceral muscle. The peptide initiated action potentials at silent recording sites in the circular muscles of the rectum after prior treatment with tetrodotoxin (10⁻⁶ g/ml). The neurohormone also caused an increase in the amplitude and frequency of spontaneous postsynaptic potentials. However, the isolated hindgut failed to respond to the neurohormone after depolarization in high potassium saline solutions. Both the potassium contracture and the action of the neurohormone were calcium dependent.Although some hindguts were responsive to the neurohormone in a Ca free medium, such preparations failed to respond in 0·5 mM EGTA. Moreover, 1 mM Mn blocked the action of the peptide. The sodium ion was also essential for effective hormone action. These results suggest the presence of a loosely bound source of Ca at the surface of muscle membranes that in some way interacts with the neurohormone to change muscle excitability.     

Octopamine-Mediated Neuromodulation of Insect Senses

Octopamine functions as a neuromodulator, neurotransmitter, and neurohormone in insect nervous systems. Octopamine has a prominent role in influencing multiple physiological events: (a) as a neuromodulator, it regulates desensitization of sensory inputs, arousal, initiation, and maintenance of various rhythmic behaviors and complex behaviors such as learning and memory; (b) as a neurotransmitter, it regulates endocrine gland activity; and (c) as a neurohormone, it induces mobilization of lipids and carbohydrates. Octopamine exerts its effects by binding to specific proteins that belong to the superfamily of G protein-coupled receptors and share the structural motif of seven transmembrane domains. The activation of octopamine receptors is coupled with different second messenger pathways depending on species, tissue source, receptor type and cell line used for the expression of cloned receptor. The second messengers include adenosine 3′,5′-cyclic monophosphate (cAMP), calcium, diacylglycerol (DAG), and inositol 1,4,5-trisphosphate (IP₃). The cAMP activates protein kinase A, calcium and DAG activate protein kinase C, and IP₃ mobilizes calcium from intracellular stores. Octopamine-mediated generation of these second messengers is associated with changes in cellular response affecting insect behaviors. The main objective of this review is to discuss significance of octopamine-mediated neuromodulation in insect sensory systems.