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.     

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.     

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.     

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.     

Heart-accelerating peptides from cockroach corpora cardiaca

Four heart-accelerating factors were separated by means of a column of Sephadex G-15 in 0·1 M acetic acid from extracts of Periplaneta americana corpora cardiaca (1a, 1b, 2a, and 2b) (first purification step). Peak 2b also shows hyperglycaemic activity. The heart-accelerating activity recovered in the four peaks accounts for 40 per cent of the activity of the whole extract.The peptidic nature of the four factors is demonstrated by experiments with proteolytic enzymes. Factors 1a and 1b are inactivated by trypsin (TPCK), whereas factors 2a and 2b are inactivated by chymotrypsin but not by trypsin.Factors 2a and 2b are better preserved if the extraction from the tissue is performed by heating, whereas factors 1a and 1b are relatively heat-labile. In order to extract factors 2a and 2b the former condition was therefore chosen. Further purification steps involved: (II) a column of Sephadex LH-20 operated with a mixture of butanol and water, (III) a small column of Sephadex G-15 in 0·1 M acetic acid. After these passages factors 2a and 2b were both inactive on the trehalose content of the haemolymph. Both had absorption and fluorescence spectra coinciding with those of tryptophan; at least one tryptophan residue is therefore probably present in each of the two molecules. An amino acid analysis of the hydrolysates revealed the presence in both peptides of the following amino acids: lysine, aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, leucine, and in 2a also of threonine and phenylalanine. The two peptides show considerable similarities. According to one of two alternative interpretations of the data, they may contain 17 (2a) and 12 (2b) amino acid residues.     

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.     

Adipokinetic hormone content of the corpora cardiaca in gregarious and solitary migratory locusts

Abstract. The adipokinetic hormone (AKH-I and AKH-II) content of the corpora cardiaca from adult males of crowded (gregarious) and isolated (solitary) Locusta migratoria migratorioides (Reiche & Fairmaire) was quantified by reverse-phase high-performance liquid chromatography.Significantly less total hormone was found in the corpora cardiaca of crowded locusts than in those glands of isolated locusts at the age of 12–19 days after fledging.The ratio of AKH-I/AKH-II was higher in crowded than in isolated locusts at this age.From the age of 12–19 days to that of 25–30 days, AKH content increased significantly in the corpora cardiaca of crowded locusts, but no such increase was found in the glands of isolated locusts, and at 25–30 days there were no significant differences in the AKH content of the glands from crowded and isolated locusts.     

Intercellular junctions in the corpora cardiaca of locusts

Summary The intercellular junctions in the corpora cardiaca of the locusts Schistocerca gregaria and Locusta migratoria were investigated by transmission electron microscopy. In the glandular lobes, complexes consisting of scalariform junctions and associated mitochondria, comparable to those previously observed in ion transporting epithelia, are formed between gland cells, and more rarely between gland cells and the neurons innervating them. Their structure and abundance are apparently unaffected by the stage of development or by the various experimental conditions employed. In the neural lobe, scalariform junctions form between glial cells and show close association with the endoplasmic reticulum. Gap junctions are present among glandular, neural and glial elements, and are formed between cells of the same type and of different types. Contacts resembling punctate tight junctions are widely distributed in the gland, but would be unlikely to form a barrier to diffusion. Septate junctions are formed exclusively between glial cells.     

The role of the glandular lobes of the corpora cardiaca during flight in Locusta

ABSTRACT. Flight performance in Locusta is reduced following severance of the major afferent nerves to the corpora cardiaca or removal of the glandular lobes of the corpora cardiaca. These operations prevent the release of adipokinetic hormone and the consequent mobilization of stored lipid. However, locusts deprived of about 90% of their glandular lobe tissue, while flying poorly, did mobilize lipid. It is suggested that the remaining glandular parenchyma cells are capable of secreting enough hormone to stimulate lipid mobilization, but that the concentration may be inadequate to encourage lipid utilization. After removal of all the glandular lobe parenchyma, the blood carbohydrate concentration was temporarily depressed. Nevertheless flight performance was equally poor, both when haemolymph carbohydrate levels were low and when they had returned to normal. After the injection of trehalose into operated control locusts and locusts deprived of their glandular lobes, flight was still markedly poorer in the operated insects, even though the injection of trehalose prevented adipokinetic hormone release in the intact locust. It seems that the poor flight performance of locusts deprived of their glandular lobes cannot be fully explained by the simple absence of adipokinetic hormone.     

Primary sequence analysis by fast atom bombardment mass spectrometry of a peptide with adipokinetic activity from the corpora cardiaca of the cricket Gryllus bimaculatus

The octapeptide AKH-G, isolated from the cricket Gryllus bimaculatus, stimulates lipid mobilization in both the cricket itself and in migratory locusts. The structure of AKH-G has been assigned by fast atom bombardment mass spectrometry (FABMS) as pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH2, and it is most closely related to AKH II-S from Schistocerca species, which has a Leu2 unit. Synthetic AKH-G, prepared by solid phase techniques, had the same bioactivity as the natural substance. FABMS measurements, including high-resolution and metastable studies, were made employing 400-900 pmole of sample.     

A possible site of action for adipokinetic hormone on the flight muscle of locusts

In mitochondrial preparations the oxidation of palmitate and of palmitoyl carnitine is stimulated by dilute extracts of the glandular lobes of the corpora cardiaca. Extracts of the storage lobes of the corpora cardiaca or of corpora allata are without effect. In a working single muscle preparation the entry of diglyceride into the muscle is also stimulated by tissue extracts containing adipokinetic hormone. This stimulation is, however, prevented by the addition of 2-bromostearic acid to the perfusion fluid. It is suggested that adipokinetic hormone may have a single site of action in the flight muscle and this may be in stimulating the entry of acyl groups into the mitochondria; perhaps by acting on the inner acyl carnitine transferase.