Melatonin-induced neuropeptide release from isolated locust corpora cardiaca

A method, based on a combination of mass spectrometry and liquid chromatography, was developed to investigate the release of neuropeptides from isolated locust corpora cardiaca. Melatonin, octopamine, trehalose and forskolin were administered to the perifused glands. The neuropeptides present in the releasates (spontaneous versus induced) were visualized by either conventional or capillary HPLC. Identification was achieved by means of MALDI-TOF MS and/or nanoflow-LC-Q-TOF MS. The observed effects of these chemicals regarding AKH release were in line with previous studies and validate the method. The most important finding of this study was that administration of melatonin stimulated the release of adipokinetic hormone precursor related peptides (APRP 1 and APRP 2), neuroparsins (NP A1, NP A2 and NP B) and diuretic peptide.     

Locust corpora cardiaca contain an inactive adipokinetic hormone

A neuropeptide from the migratory locust, Locusta migratoria, has been identified as a novel member of the family of adipokinetic hormones (AKHs). The peptide is probably synthesised in the brain because it is the first AKH found in the storage lobe, whilst the three 'classic' Locusta AKHs are present in the glandular lobe of the corpora cardiaca. In locusts, the peptide has no biological activity usually associated with AKHs. There is only 36-56% sequence identity with the three Lom-AKHs, but 78% identity with the Drosophila melanogaster AKH, Drm-HrTH. The new peptide is active in the American cockroach, Periplaneta americana, and was provisionally named 'L. migratoria hypertrehalosaemic hormone', Lom-HrTH; its biological role in locusts remains to be established. The high degree of identity with Drm-HrTH suggests that Lom-HrTH is an ancient molecule.     

Preliminary characterization of glycogen phosphorylase activating hormone and adipokinetic hormone from Manduca sexta corpora cardiaca

ABSTRACT. An attempt was made to separate glycogen phosphorylase activating hormone (GPAH) and adipokinetic hormone (AKH) from the corpora cardiaca (CC) of the moth Manduca sexta (Lepidoptera: Sphingidae) by separating extracts of CC on various chromotographic media, but it was not possible to conclude whether GPAH and AKH are activities of one or of two different peptides. Both activities elute together from glass beads, from Sephadex G-25 and from Sephadex LH-20 columns. In the separation experiments with glass beads and G-25 the activities eluted as a single peak, but using LH-20 we found two peaks exhibiting both activities. The major peak eluted at 1.25 × V_t, which is very similar to locust AKH, while the smaller second peak eluted at O.74 × V _t. Cross injections of CC extracts from M. sexta into Locusta migratoria and CC extracts from L. migratoria into M. sexta suggest that GPAH and the AKH from M. sexta are not identical with the decapeptide AKH from locusts.     

Quantitative studies on the release of locust adipokinetic hormone

Fractionation of methanolic extracts of haemolymph on Sephadex LH-20 made possible the measurement of the titre of adipokinetic hormone in the haemolymph of locusts. Experimentally produced high concentrations of haemolymph carbohydrate caused a delay in the mobilization of lipid during flight, and very low titres of the hormone were present in the haemolymph of locusts injected with trehalose immediately before a 25 min flight. In these locusts flight speed was higher than saline-injected controls. Although delayed lipid mobilization during flight was also seen in locusts injected with sucrose, sucrose is not utilized for flight metabolism and flight speed was not increased by the injection. Tentative estimates of the release rate (c. 1000pg/20min flight) and half life (c. 20 min) of adipokinetic hormone during flight are made. The results described suggest that during flight the rate at which trehalose disappears from the haemolymph does not play a major role in the initiation of the release of adipokinetic hormone.     

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.     

Anti-juvenile effect of neuroparsin A,a neuroprotein isolated from locust corpora cardiaca

Neuroparsin A, a sulfur-containing protein synthesized by the medial part of the brain of Locusta and transported to the corpora cardiaca (CC) via the nervi corporis cardiaci I (Girardie et al., 1987), was satisfactorily isolated using electro-elution. A specific immune serum against electro-eluted neuroparsin A was generated. On serial histological sections of the brain treated with the immune serum, only the median neurosecretory cells [stained in blue following the double staining Victoria blue-paraldehyde fuchsin (Al type)] were revealed using peroxidase-antiperoxidase technique. Inactivation of neuroparsin A by antigen-antibody complex formation following injections of immune serum induced green pigmentation, intermediary forms and precocious sexual maturation. These symptoms also follow juvenile hormone (JH) injections. Injections of immune serum antineuroparsin A or the electro-eluted neuroparsin A produced opposite effects on oocyte growth but had no effect on the rate of JH biosynthesis evaluated by radiochemical assay. The neurohormone neuroparsin A could be the median humoral inhibiting factor of the JH system which was previously demonstrated (Girardie, 1966, 1967) in the central area of the pars intercerebralis.     

A comparative study on the isolation of adipokinetic and hypertrehalosaemic factors from insect corpora cardiaca

ABSTRACT. Extracts of corpora cardiaca from two cockroaches, Nauphoeta cinerea Olivier and Leucophaea maderae F., from a cricket, Gryllus bimaculatus De Geer, from the Colorado potato beetle, Leptinotarsa decemlineata Say, and from the sphinx moth, Sphinx ligustri L. were assayed for adipokinetic and hypertrehalosaemic activity, in acceptor locusts ( Locusta migratoria L.) and cockroaches ( Periplaneta americana L.) respectively. Both bioassays give positive results with all corpus cardiacum material tested except that from the sphinx moth; in this insect haemolymph lipid concentrations (but not those of the total carbohydrate) are, however, increased after injection of an extract of corpora cardiaca from the same species. A similar result is obtained when specimens of G. bimaculatus are injected with an extract of corpora cardiaca from G. bimaculatus. Biological activities of corpus cardiacum extracts from all species investigated can be resolved on reversed-phase high-performance liquid chromatography. Gland extracts from the two cockroach species each show a single absorbance peak which has hypertrehalosaemic activity, but with a (common) retention time distinct from all previously described arthropod neuropeptides. The corpora cardiaca of G. bimaculatus contain also a novel adipokinetic factor with a retention time distinct from previously characterized arthropod hormones, as well as from the new cockroach factor described in this study. The two hypertrehalosaemic factors from the corpora cardiaca of the potato beetle coelute with the hypertrehalosaemic hormones I and II of the American cockroach. The active (adipokinetic) compound from glands of S. ligustri appears to coelute with locust adipokinetic hormone I.     

Phylogenetic differentiation of glandular cells in corpora cardiaca as studied immunocytochemically with region-specific antisera to adipokinetic hormone

Immunocytochemical reactions with region-specific antibodies to adipokinetic hormone were used to study the stainability and structural organization of the intrinsic glandular cells of the corpora cardiaca of 19 insect species belonging to 10 orders. There was considerable variation in the nature of the glandular product as well as in morphology of the glandular cells, and in their association with the storage compartment of the corpus cardiacum. The apterygote Folsomia only contains substances reacting with the C-terminal specific antibody No. 241, whereas Thermobia has glandular cells which stain either with this serum or with the N-terminal specific antiserum No. 433. Glandular cells in Locusta may either react with only the N-terminal specific antiserum or with both types of antiserum. All other species studied contain only glandular cells reacting with the N-terminal specific antiserum 433, except for the dipteran species which were negative.It was attempted to relate the immunological staining with the chemical properties of the substances so far isolated from the corpora cardiaca. The glandular cells are variable in the number, shape, and the degree of clustering and association with the storage zones. In some species they are entirely separated, in others they are embedded in the storage zone. Cell processes may be very long or absent.Neurones in the brain were generally left unstained by serum 433, indicating that the substance revealed earlier by antiserum 241 differs from adipokinetic hormone.     

Structure elucidation and biological activity of an unusual adipokinetic hormone from corpora cardiaca of the butterfly, Vanessa cardui.

A structurally unusual member of the adipokinetic hormone/red pigment-concentrating hormone peptide family was isolated from corpora cardiaca of the painted lady butterfly, Vanessa cardui. Its primary structure was assigned by Edman degradation and nano-electrospray-time-of-flight mass spectrometry as pQLTFTSSWGGK (Vac-AKH). Vac-AKH represents the first 11mer and the first nonamidated peptide in this family. The peptide shows significant adipokinetic activity in adult specimens of V. cardui. Injection of 10 pmol of synthetic Vac-AKH into 4-day-old decapitated males resulted in an approximately 150% increase of hemolymph lipids after 90 min. Half maximal adipokinetic activity was achieved with about 0. 1 pmol of Vac-AKH. During a 2-h incubation of corpora cardiaca/corpora allata complexes in medium containing 50 mM KCl, significant amounts of Vac-AKH were released from the glands.     

The hyperglycaemic activity of locust adipokinetic hormone

ABSTRACT. Pure adipokinetic hormone (AKH) extracted from the glandular lobes of locusts induces hyperglycaemia in cockroaches. Larger doses (20–200 pmol) of AKH are required to induce hyperglycaemia in the cockroach than are required to produce hyperlipaemia in locusts (1–20 pmol).     

The inhibition of lipid synthesis in vitro in the locust, Schistocerca gregaria, by factors from the corpora cardiaca

ABSTRACT. The rate of lipid synthesis from [¹⁴C]acetate in fat body from Schistocerca americana gregaria has been studied in vitro. Maximum incorporation is found on days 6–10 in adults and day 4 of the fifth stadium. The label appeared in the fatty acid components of triacyl-glycerol, diacylglycerol and phospholipid.
Lipid synthesis in vitro was inhibited by extracts of corpora cardiaca, and such inhibition was most marked (up to 85%) in fat bodies from insects at stages where fatty acid synthesis was greatest. HPLC separation of corpora cardiaca extracts gave several active fractions of which the most active was adipokinetic hormone 1 (AKH-1).     

Studies on the purification of locust adipokinetic hormone

$\text{Locusta}$ adipokinetic hormone (AKH) has been purified from glandular lobes of corpora cardiaca by a series of chromatographic steps that have allowed a 58 to 77% recovery of activity. Methanolic extracts of the glandular lobes were fractionated on Sephadex LH-20 and then on thin-layer plates. The average recovery of AKH activity after separation on Sephadex LH-20 approached 100%, whereas recoveries of between 58 to 77% were obtained after TLC on cellulose plates. In practice, the highest recoveries were obtained with separations of large amounts of the hormone on cellulose plates. In the course of this investigation it was found that AKH activity can be extracted efficiently from aqueous solutions using Florisil. The hormone activity was recovered from the Florisil by eluting with 80% methanol. The purified hormone was found to be homogeneous in composition when subjected to rechromatography on Sephadex LH-20 and to amino acid analysis of the acid hydrolysate. From the amino acid analysis we estimate that one pair of glandular lobes contains on average 188 pmoles of AKH.     

Separation and characteristics of antidiuretic factors from the corpora cardiaca of the migratory locust

Summary

Corpora cardiaca (CC) of the migratory locust contain two antidiuretic (AD) factors increasing the fluid reabsorption of isolated recta over a 5-h period.

They are contained in storage lobes (SL) and glandular lobes (GL) of the CC. They differ in size and can be separated from each other in different ways (extraction, dialysis, electrophoresis).

The AD factor contained in GL of the CC is stable at 4°C and quickly destroyed at 100°C. It stimulates the fluid reabsorption in a dose-dependent manner. Its release into the haemolymph seems to be controlled by octopamine, which is probably synthesized in the lateral neurosecretory areas of the brain.     

Possible involvement of monoamines in the release of adipokinetic hormone in the locust Schistocerca gregaria.

1. The adipokinetic hormone release, which can be induced by anticholinesterases, is reduced by depleting the content of monoamines in the nervous system. 2. The participation of monoamines in the pathway of release of adipokinetic hormone is studied in vivo and in vitro. 3. A possible mechanism for anticholinesterase-induced release of this hormone involving cholinergic and aminergic transmission is postulated.     

Remodeling of the corpora cardiaca and the corpora allata during adult metamorphosis in <Emphasis Type=Italic>Bombyx mori</Emphasis>: identification of invisible corpora cardiaca by the expression of adipokinetic hormone

It is problematic to identify the corpora cardiaca (CC) of pupal and adult stages of Bombyx mori because of the presence of tissues similar in appearance near the associated corpora allata (CA). To find the CC, we performed in situ hybridization experiments using the adipokinetic hormone II (AKH-II) gene as a probe, as AKH-II expression occurs only in the CC. Shortly after pupation, the CC and CA began to move toward the esophagus and had attached there tightly by 4 days; during this period, the distance between the CC and CA increased from 65 to 507 μm. The CC became flattened during the remodeling period, although the CA stayed globular until adult ecdysis. In situ hybridization analyses, after the CC had been correctly identified during adult development, showed that the mRNA expression of all the mevalonate enzymes and juvenile hormone acid O-methyltransferase involved in JH synthesis occurred only in CA cells, indicating that the fluctuations of enzyme mRNA amounts in CC-CA complexes (Kinjoh et al. in Insect Biochem Mol Biol 37:808–818, 2007) were derived solely from those in the CA.     

Synthesis of adipokinetic hormone (AKH-I) in the locust brain

Methanol extracts of vitellogenic female locust brains contain two factors that inhibit protein synthesis in fat body tissue excised from such individuals. One of these factors (BI) elicits lipid mobilization when injected into adult male locusts. The retention times of BI on an RP-18 column and on an RP-4 column are identical to those of synthetic locust adipokinetic hormone (AKH-I) on each of these columns. Half maximal inhibition of protein synthesis in excised adult locust fat bodies is exerted by 0.05 brain extract equivalents of BI, which is equivalent to activity elicited by 1.5 pmol of AKH-I, as previously determined by AKH-radioimmunoassay. Enzymatic hydrolysis of the N-terminal pyroglutamate, followed by amino acid sequence analysis, indicates that the structure of BI is similar to that of the decapeptide AKH-I synthesized in the glandular lobe of the locust corpora cardiaca (CC). Incorporation of [5-³H]tryptophan into BI of locust brains incubated in vitro indicates that the AKH-I present in the brain is synthesized in situ and is not transported from the CC. Similar incorporation of radiolabel into AKH-I is obtained when excised CC are incubated in vitro.     

Identification of gonadotropic and antigonadotropic factors from the nervous part of the corpora cardiaca in the African locust

Summary

The nervous part of the locust corpora cardiaca (NCC) was extracted using 70% methanol and subjected to fast protein liquid chromatography (FPLC). Twenty fractions were collected and injected into females from the end of the IIIrd larval instar to day-12 of the adult life to screen the effects on metamorphosis, body pigmentation, and oocyte growth. The rate of juvenile hormone (JH) biosynthesis by the corpora allata of injected animals was evaluated in vitro. One fraction was found to stimulate ovarian maturation, and 3 to delay oocyte growth. None of them disturbed (i) either metamorphosis and body color (which are JH dependent), or (ii) rate of JH biosynthesis. Of the 3 fractions which inhibited ovarian maturation, 2 of them were identified as the neuroparsins A and B. The results clearly illustrate that antagonist factors from the brain are involved in the regulation of ovarian maturation in the locust.     

In vivo studies on the release of hormones from the corpora cardiaca of locusts

Summary Sectioning of the afferent nerves (NCCl and NCCll) to the locust corpus cardiacum prevents thein vivo release of adipokinetic hormone from the glandular lobes. This failure to release the hormone during flight and the consequent lack of lipid mobilisation brings about an impairment of flight performance which can be corrected by injections of corpus cardiacum extracts. Sectioning of the NCCl and NCCll reduces markedly the activity of the corpora allata. However, the poor flight performance of allatectomised locusts is not related to an inability to mobilise lipid since injections of corpus cardiacum extract which will mobilise fat body lipid in these locusts have no effect on flight performance. The results of individual sectioning of the NCCl and NCCll suggest that a double innervation of the glandular lobes functionsin vivo to control adipokinetic hormone release but that the NCCl alone may control the release of the diuretic hormone.     

Further characterization of locust low density lipophorin induced by adipokinetic hormone

This study was designed to resolve basic questions concerning the nature of low density lipophorin (LDLp) which is induced by adipokinetic hormone (AKH). For this purpose, lipophorin was fractionated by density gradient ultracentrifugation and each fraction containing lipophorin was analyzed for diacylglycerol and associated apolipophorin-III (apoLp-III). The diacylglycerol content of LDLp fractions increased significantly as the density of the fraction decreased (116 micrograms/100 micrograms protein at a high density to 209 micrograms/100 micrograms protein at a lower density). On the other hand, the content of diacylglycerol in each fraction of HDLp remained almost constant (33 micrograms/100 micrograms protein). It was also found that the number of apoLp-III molecules associated with LDLp increased as the density decreased (from 6.9 mol/mol LDLp to 13.2 mol/mol LDLp). However, electron microscopic observation showed that LDLp particles in each of the fractions were extremely heterogeneous in size with diameters of 29.4 +/- 6.8 nm, 27.1 +/- 5.5 nm, and 26.3 +/- 5.7 nm for low, medium, and high density fraction, respectively. HDLp particles were very homogeneous in size irrespective of the fraction (15.9 +/- 1.5 nm, 15.6 +/- 1.5 nm, and 15.6 +/- 1.3 nm for the respective fractions). A theoretical analysis based on all the experimental data strongly supports the hypothesis that the heterogeneity in the size of LDLp particles does not reflect different densities, but rather, heterogeneity is the result of intermolecular fusion between LDLp particles of the same density.