Release of identified adipokinetic hormones during flight and following neural stimulation in Locusta migratoria

Fractionation of methanol extracts of perfusate and haemolymph on thin-layer chromatography was used to separate hormones associated with haemolymph lipid regulation in Locusta. Electrical stimulation of the nervi corporis cardiaci II (NCC II) of isolated corpora cardiaca resulted in the release of three hormones into the perfusate; hypolipaemic hormone and two adipokinetic hormones. The two adipokinetic hormones co-migrated with synthetic adipokinetic hormone (adipokinetic hormone I) and with the R_F value similar to Carlsen's peptide (adipokinetic hormone II).These two adipokinetic hormones were also present in small amounts in the haemolymph of unflown Locusta, and shown to be released during a 30-min flight. The adipokinetic hormone II fraction from the NCC II-stimulated perfusate and haemolymph also possessed hyperglycaemic activity when assayed in ligated locusts.It is concluded that NCC II controls the release of adipokinetic hormones during flight and that two adipokinetic hormones are released during flight. One of these hormones adipokinetic hormone II also acts as a hyperglycaemic hormone illustrating that a hyperglycaemic hormone is released, during flight.     

Effects of adrenalectomy on photoperiod-induced changes in release of luteinizing hormone and prolactin in ovariectomized ewes

Finnish Landrace x Southdown ewes were ovariectomized (OVX) and subjected to daily photoperiods of 16L:8D (Group I) or 8L:16D (Group II) for 84 days. Ewes were then either adrenalectomized (ADX) (N = 5 for Group I; N = 4 for Group II) or sham ADX (N = 6 for Groups I + II). After surgery, ewes in Group I were subjected to 8L:16D for 91 days and 16L:8D for 91 days whereas ewes in Group II were exposed to 16L:8D for 91 days and 8L:16D for 91 days. Oestradiol implants were inserted into all ewes on Day 148. Sequential blood samples were taken at 28, 56, 91, 119, 147 and 168 days after surgery to determine secretory profiles of LH and prolactin. Photoperiod did not influence LH release in Group I in the absence of oestradiol. Although photoperiod influenced frequency and amplitude of LH pulses in Group II before oestradiol treatment, adrenalectomy did not prevent these changes in patterns of LH release. However, in Group II the increase in LH pulse amplitude during exposure to long days was greater (P less than 0.01) in adrenalectomized ewes than in sham-operated ewes. Mean concentrations of LH increased in ADX ewes on Days 91 (P = 0.07) and 119 (P less than 0.05). Adrenalectomy failed to influence photoperiod-induced changes in mean concentrations of LH, amplitude of LH pulses and frequency of LH pulses in the presence of oestradiol. Concentrations of prolactin were influenced by photoperiod. In Groups I and II concentrations of prolactin increased (P less than 0.01) after adrenalectomy, but the magnitude of this effect decreased over time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurosecretion

Summary Protrusions of bounding membranes of neurosecretory granules, comparable to those demonstrated by Castel (1977) in the mammalian neurohypophysis, were observed under various experimental conditions in the corpora cardiaca of the insects Leucophaea maderae and Periplaneta americana. Electrical stimulation in vitro of the nervus corporis cardiaci I, which elicited a marked rise in the amount of neurohormone discharged, as determined by bioassay, also yielded a significantly larger number of membrane protrusions than were observed in unstimulated controls. However, no comparable response was obtained in glands subjected to stimulation of hormone release by exposure to serotonin or high potassium concentrations. On the other hand, membrane protrusions were numerous under certain conditions not expected to stimulate neurohormone release, i.e., in tissue exposed to a zinc iodide mixture without prior fixation. The present results support the conclusion drawn by Castel that these configurations appear to be related to the process by which neurosecretory material is discharged. Too transient to be much in evidence under physiological conditions, they become more prominent not only after appropriate acceleration of the rate of release, but also when such membrane arrangements are frozen by procedures that interfere with the regular milieu.Dedicated to Professor Pierre Drach in recognition of his many contributions to arthropod biology. — Supported by research grants NB-05219, NB-00840, and 5 PO1-NS-07512 from the U.S.P.H.S. and by N.S.F. grant BMS74-12456     

Release of a neurosecretory protein from the corpora cardiaca of Locusta migratoria induced by high potassium saline and compound action potentials

Electrical stimulation of nervus corpus cardiacum I (NCCI) resulted in the propagation of a compound action potential into the storage and glandular lobes of the corpus cardiacum (CC) of Locusta migratoria. The compound action potential was abolished in the presence of both sodium-free saline and tetrodotoxin (TTX). Calcium-free saline had variable effects.The release of a neurosecretory protein (estimated following precipitation with an antiserum directed against neurosectetory protein) was examined after treatment with high potassium saline and electrical stimulation of NCC I. Release was induced by elevated potassium salines and by the propagation of a compound action potential along NCC I. The release was calcium-dependent. TTX and sodium-free saline abolished the electrically-induced release of protein. Concomitant with the release of protein was the release of a factor with diuretic activity, illustrating that hormones are also being released along with the neurosecretory protein. The release of this protein was dependent upon the frequency of electrical stimulation (up to approx. 5 Hz) and the patterning of electrical stimulation. This neurosecretory protein which has previously been shown to be very similar in both size and amino acid composition to the pituitary neurophysins, now also shares the characteristic of being released along with hormone.     

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.     

Adrenaline induces mitochondrial biogenesis in rat liver

We studied the effects of adrenaline administration and depletion (induced by reserpine) on rat liver oxidative metabolism. We showed that adrenaline increases, and reserpine decreases aerobic capacity (inferred by cytochrome oxidase activity) in tissue modifying the hepatic content of mitochondrial proteins without changing mitochondrial aerobic capacity. The changes in tissue cytochrome oxidase activity, which agreed with the expression levels of factors involved in mitochondrial biogenesis, such as PGC-1, NRF-1, and NRF-2, were associated with similar changes in tissue and mitochondrial State 3 respiration. Adrenaline and reserpine induced extensive lipid and protein oxidative damage in tissue and mitochondria. The increase in H₂O₂ release by respiring mitochondria and the decrease in the activities of the antioxidant enzymes glutathione peroxidase and reductase contributed to the reserpine effect on oxidative damage. The adrenaline effect is more difficult to explain, since the hormone increased the antioxidant enzyme activities but, in respiring mitochondria, increased ROS release rate in the presence of succinate and decreased it in the presence of pyruvate/malate. These opposite changes were due to the increased content of the autoxidizable electron carrier located at complex III and decreased content of that located at complex I. Our data suggest that adrenaline can be involved in the mitochondrial population adaptation which verify in conditions in which an increased body energy expenditure verify such as cold exposure.     

Evidence for a Critical Role of Catecholamines for Cardiomyocyte Lineage Commitment in Murine Embryonic Stem Cells

Catecholamine release is known to modulate cardiac output by increasing heart rate. Although much is known about catecholamine function and regulation in adults, little is known about the presence and role of catecholamines during heart development. The present study aimed therefore to evaluate the effects of different catecholamines on early heart development in an in vitro setting using embryonic stem (ES) cell-derived cardiomyocytes. Effects of catecholamine depletion induced by reserpine were examined in murine ES cells (line D3, αPIG44) during differentiation. Cardiac differentiation was assessed by immunocytochemistry, qRT-PCR, quantification of beating clusters, flow cytometry and pharmacological approaches. Proliferation was analyzed by EB cross-section measurements, while functionality of cardiomyocytes was studied by extracellular field potential (FP) measurements using microelectrode arrays (MEAs). To further differentiate between substance-specific effects of reserpine and catecholamine action via α- and β-receptors we proved the involvement of adrenergic receptors by application of unspecific α- and β-receptor antagonists. Reserpine treatment led to remarkable down-regulation of cardiac-specific genes, proteins and mesodermal marker genes. In more detail, the average ratio of ∼40% spontaneously beating control clusters was significantly reduced by 100%, 91.1% and 20.0% on days 10, 12, and 14, respectively. Flow cytometry revealed a significant reduction (by 71.6%, n = 11) of eGFP positive CMs after reserpine treatment. By contrast, reserpine did not reduce EB growth while number of neuronal cells in reserpine-treated EBs was significantly increased. MEA measurements of reserpine-treated EBs showed lower FP frequencies and weak responsiveness to adrenergic and muscarinic stimulation. Interestingly we found that developmental inhibition after α- and β-adrenergic blocker application mimicked developmental changes with reserpine. Using several methodological approaches our data suggest that reserpine inhibits cardiac differentiation. Thus catecholamines play a critical role during development.     

Synthesis, transport, and release of a neurohormone by cultured neuroendocrine glands from the cockroach, Leucophaea maderae

A hindgut-stimulating neurohormone synthesized in vitro by the neurosecretory cells of cultured brains of Leucophaea maderae passes through the nervi corporis cardiaci I into the corpora cardiaca and is released into the culture medium. As much as 90 per cent of the hormone breaks down in the medium during a 3-day incubation period, and the amount recovered represents only a small fraction of the amount actually released.     

Hormone-stimulated polyphosphoinositide breakdown in rat liver plasma membranes. Roles of guanine nucleotides and calcium

Calcium-sensitive inositide release in a purified rat liver plasma membrane preparation is increased by calcium-mobilizing hormones in the presence of guanine nucleotides. Vasopressin-stimulated inositide release is evident in the presence of GTP or its nonhydrolyzable analogs guanyl-5'-yl imidodiphosphate and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The stimulation of inositide release by (-)-epinephrine (alpha 1), angiotensin II, or vasopressin in the presence of either 1 microM or 10 microM GTP gamma S correlates with the number of receptors present for each hormone. The guanine nucleotide and hormonal stimulation is evident on both inositol trisphosphate production and phosphatidylinositol bisphosphate degradation. Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (1 mM) completely abolishes stimulation by guanine nucleotides and hormone. Prior treatment of plasma membranes with cholera toxin or islet activating protein or prior injection of animals with islet activating protein does not affect stimulation of inositide release by GTP gamma S or GTP gamma S plus vasopressin. Stimulation by GTP gamma S is dependent upon magnesium and is inhibitable by guanosine 5'-(2-O-thio) diphosphate. Inositide release from the plasma membrane exhibits half-maximal stimulation by calcium at approximately 100 nM free calcium in the presence of 1.5 mM MgCl2 and at approximately 10 microM free calcium in the presence of 10 mM MgCl2. Addition of guanine nucleotides decreases the requirement for calcium and also increases the activity at saturating calcium. The results presented suggest that calcium-mobilizing hormones stimulate polyphosphoinositide breakdown in rat liver plasma membranes through a novel guanine nucleotide binding protein.     

Release from the brain and acquisition by the corpus cardiacum of a neurohormone in vitro

Hindgut stimulating neurohormone (HSN) was synthesized and stored by cultured brains of the cockroach, Leucophaea maderae. The presence of either HSN or cultured corpora cardiaca in the medium caused the brains to release their accumulated HSN. The corpora cardiaca were able to sequester HSN from the medium when the concentration was above a threshold level. Thus, the corpus cardiacum may provide an homeostatic mechanism for maintaining physiological levels of neurohormone.     

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.     

Neurosecretion

Summary Ultrastructural changes were examined in corpora cardiaca ofPeriplaneta americana in which acute electrical stimulationin vitro had brought about hormone release.Tissue specimens from which a substantial amount of cardioaccelerator substance had been discharged into the incubation medium showed morphological indications of an increased rate of release of neurosecretory substance. These consisted in the presence, after stimulation, of a greater number of synaptoid configurations whose properties and distribution implicate them as sites of neurohormone release. This interpretation, originally arrived at by an analysis of corpora cardiaca not stimulated by experimental procedures, and fixedin situ, is now strengthened by the cytophysiological response observed as a consequence of the stimulus.According to the type of neurosecretory axon in which the formation of synaptoid structures occurs, apparently on demand, the cardioaccelerator tested for in the presentin vitro experiments, seems to be derived from large neurosecretory granules stored within the corpus cardiacum. On the basis of morphological and physiological information, their site of origin may be the perikarya of intrinsic or extrinsic neurosecretory neurons or possibly both.Supported by grants AM-3984, NB-00840, NB-04989, and NB-05219 from the U.S.P.H.S., and N.S.F. GB-4847. — We are indebted to Mrs.Sarah Wurzelmann for her excellent technical assistance.Grantee of an NDEA Title IV Predoctoral Fellowship.     

Specificity Profiling of Dual Specificity Phosphatase Vaccinia VH1-related (VHR) Reveals Two Distinct Substrate Binding Modes

Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/φ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/φ)(T/S)(D/E)pY(G/A/S/Q) (where φ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X_1–6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp¹⁶⁴, which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo.     

Effect of juvenile hormone on acid phosphatases in the degenerating flight muscles of the Douglas-fir beetle, Dendroctonus pseudotsugae

Changes in acid phosphatase activity were used to estimate flight muscle degeneration in Dendroctonus pseudotsugae. Acid phosphatase positive sites increased numerically as well as in size in flight muscles of beetles attacking the host logs and those maintained on host bark chips. Topical treatment of beetles with a juvenile hormone analogue further increased the phosphatase activity. Juvenile hormone produced similar effects when applied to insects injected with eserine. An increase in the activity of acid phosphatases was also observed after incubation in culture medium with added juvenile hormone. The rôle of nerves and hormones in the degenerating muscles of D. pseudotsugae is compared with that in the permanently degenerating muscles during metamorphosis of some moths.     

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.     

Oxidation-reduction potentials of the hemes in cytochrome C3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy.

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.     

The surface receptor is involved in annexin I-stimulated insulin secretion in MIN6N8a cells

This study investigated the effect of extracellular annexin I on regulating insulin secretion in MIN6N8a (an insulin secreting cell line) cells. The properties of annexin I receptor in MIN6N8a cells were also determined. Annexin I stimulated insulin release in MIN6N8a cells, regardless of the presence or absence of extracellular Ca(2+). Confocal microscopy revealed that annexin I bound to the surface of MIN6N8a cells. In addition, FACs analysis showed that annexin I bound to the surface of MIN6N8a cells in a dose-dependent manner. However, the annexin I-stimulated insulin secretion and the annexin I binding were abolished in MIN6N8a cells treated with proteases. Annexin I receptors were regenerated time-dependently. Furthermore, annexin I-stimulated insulin secretion was inhibited by cycloheximide but not by actinomycin D. These results showed that annexin I binds to the surface receptor in order to regulate the stimulation of insulin release in MIN6N8a cells.     

The role of octopamine and cyclic AMP in regulating hormone release from corpora cardiaca of the American cockroach

Incubation of corpora cardiaca from adult male Periplaneta americana in the presence of octopamine results in elevated tissue levels of cyclic AMP. The octopamine-induced elevation of cyclic AMP is partially blocked by phentolamine, gramine and cyproheptadine but not by propranolol. Dopamine and 5-hydroxytryptamine also increase cyclic AMP levels in the corpus cardiacum and additivity studies indicate that separate octopamine- and dopamine-binding sites are present within the tissue. Cyclic AMP levels in the corpus cardiacum also increase in response to electrical stimulation of nervi corporis cardiaci II (NCC II) and the electrically induced effect is eliminated in the presence of phentolamine.A factor, which causes elevated haemolymph trehalose levels when injected into adult cockroaches, is released from corpora cardiaca incubated in the presence of octopamine. The active factor is denatured by incubation in the presence of pronase. The hypertrehalosemic factor is also released when corpora cardiaca are incubated in the presence of dibutyryl cyclic AMP or 40 mM potassium chloride; however dopamine and 5-hydroxytryptamine fail to effect a marked release of the hypertrehalosemic factor.The results are discussed in light of the proposal that the release of hypertrehalosemic hormone from corpora cardiaca is regulated by octopaminergic neurones contained within NCC II.     

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.     

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.