Isolation and amino acid composition of the adipokinetic neuropeptide from the corpus cardiacum of the cricket, Gryllus bimaculatus

ABSTRACT. The corpora cardiaca of the cricket, Gryllus bimaculatus De Geer, contain peptidic material which elicits hyperlipaemia in migratory locusts and in crickets. Appreciable amounts of this adipokinetic factor are found only in the corpora cardiaca of the cricket, and not in other parts of the nervous system. The factor is heat-stable and retains its activity after incubation with the exopeptidases leucine aminopeptidase and carboxypeptidase A. Fractionation of a methanolic extract of corpora cardiaca from Gryllus bimaculatus by RP-HPLC shows that the adipokinetic activity is confined to a single absorbance peak. The amino acid composition of the purified adipokinetic neuropeptide was determined after acid hydrolysis with HCI and with methanesulfonic acid. The factor is an octapeptide containing the following amino acid residues in equimolar amounts: Asx, Thr, Ser, Glx, Gly, Val, Phe and Trp. This composition closely resembles the AKHI II molecule from Schistocerca species , and the Gryllus peptide can be assigned to the AKH/RPCH family of neuropeptides.     

Regulation of glycolytic intermediates in cockroach fat body by the corpus cardiacum

Glycolytic intermediates and related metabolites were measured in the fat body of the American cockroach (Periplaneta americana) to locate the rate-limiting reactions that regulate glycolysis during the action of the corpus cardiacum (CC) in vitro.

The metabolic neuropeptides of the corpus cardiacum from the potato beetle and the American cockroach are identical

Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high performance liquid chromatography from the corpus cardiacum of the Colorado potato beetle, Leptinotarsa decemlineata. The sequences of both peptides, designated Led-CC-I and Led-CC-II, were determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. The C-terminal of both peptides were blocked and neither molecule was cleaved by carboxypeptidase. Both peptides were found to be octapeptides; Led-CC-I has the primary structure pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂, and Led-CC-II has the primary sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂. These structures are identical to the two hypertrehalosaemic hormones from the American cockroach. Preliminary experiments show that the synthetic peptides are apparently involved in the control of amino acid metabolism during flight of the potato beetle.     

Activation of fat body glycogen phosphorylase in Locusta migratoria by corpus cardiacum extract and synthetic adipokinetic hormone

Between 10 and 20 per cent of the total glycogen phosphorylase in the fat body of mature Locusta migratoria of both sexes is in the active form. Injection of an aqueous corpus cardiacum (CC) extract results in a rapid activation: within 2 min the level of active phosphorylase is significantly increased and full activation is reached within 10 to 20 min. As little as 0.002 CC gland equivalents stimulate fat body glycogen phosphorylase significantly and maximum activation is obtained with 0.05 CC gland equivalents. From experiments with known quantities of injected synthetic adipokinetic hormone (SAKH), it appears that this hormone cannot account for all the activation. This is supported by results obtained when extracts of carefully isolated storage lobes are injected; at the dose used here these have no adipokinetic activity, but activate fat body phosphorylase. Furthermore, when locusts are ‘stressed’ by rotation, although no adipokinetic hormone is released, an activation of phosphorylase occurs. Starvation causes also an increase in the active form of the enzyme. The fat body receptor sites of the locust recognise also the crustacean red pigment concentrating hormone (RPCH), whose structure closely resembles that of the locust adipokinetic hormone, leading to activation of the phosphorylase. However, RPCH is about 2.5–5 times less potent than SAKH. Crude CC extracts of a stick insect (Carausius morosus), a cockroach (Periplaneta americana) and the tobacco hornworm (Manduca sexta) activate locust fat body phosphorylase, although this last extract has no effect on lipid elevation. On the other hand, CC extracts of the death's head hawk moth (Acherontia atropos) and purified crustacean hyperglycaemic hormone from a crayfish (Orconectes limosus) have no effect.     

Isolation and structure elucidation of a novel adipokinetic hormone (Lom-AKH-III) from the glandular lobes of the corpus cardiacum of the migratory locust, Locusta migratoria

A new adipokinetic hormone (named Lom-AKH-III) was isolated from the glandular lobes of the corpora cardiaca of Locusta migratoria. At the N-terminus it is blocked by a 5-oxoproline (pyroglutamic acid) residue (less than Glu). After enzymatic deblocking, the amino acid sequence of the N-terminus was partly established by automatic Edman degradation to be [less than Glu]-Leu-Asn-Phe-Thr-Pro-. Fast-atom-bombardment spectrometry (FAB-MS) revealed that the new hormone is an octapeptide, which is amidated at the C-terminus, and has a relative molecular mass of 1072. Based on the FAB-MS data the complete sequence is less than Glu-Leu-Asn-Phe-Thr-Pro-Trp-Trp-NH2, which was confirmed by chemical synthesis. All characteristics from HPLC, FAB-MS and biological activity of the natural hormone and the synthetic peptide appeared to be identical. Although the structure of this new hormone resembles that of Lom-AKH-I (less than Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2), its amino acid sequence points to a completely different route for its biosynthesis, involving a third prohormone. High-[K+]-containing media can cause release of all three adipokinetic hormones in vitro. Interestingly, the new hormone is absent in another locust species. Schistocerca gregaria. Based on in vitro biosynthesis experiments the turnover for this hormone is very high, suggesting an important physiological function. Locusta migratoria is the first insect species in which three different adipokinetic hormones have been demonstrated.     

The hormonal content of the locust corpus cardiacum

Summary The amounts of adipokinetic and diuretic hormone in the separate storage and glandular lobes of the locust corpora cardiaca during the imaginal moult and up to the onset of sexual maturation have been measured. The levels of the hormones are high prior to the imaginal moult, fall at emergence and increase during the somatic growth period. The effects of surgical interference with the neuroendocrine system upon the hormonal content of the corpora cardiaca have been investigated. Cautery of the brain neurosecretory cells or allatectomy in mature locusts has no effect on the content of adipokinetic hormone. Diuretic hormone is absent from the storage lobes of locusts deprived of their cerebral neurosecretory cells but normal levels are present in the corpora cardiaca of allatectomised animals. Severance of the nervus corporis cardiacum I and II reduces the level of diuretic hormone in the storage lobes of the corpora cardiaca but is without effect on the levels of adipokinetic hormone in the glandular lobes. This work is supported by grants from the Science Research Council and the Royal Society.     

Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus

Summary The ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160–350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160–350 nm in diameter. Synaptoid configurations with 20–50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.This work was done while JTB was supported by USPHS Training Grant HD-0266 from NICHDI wish to express my thanks to Dr. Richard A. Cloney for sharing his expertise in electron microscopy     

Presence of an adipokinetic peptide in the corpus cardiacum of Dermaptera but not in the neurohaemal aorta, and chemical and functional identification of the peptide

Corpora cardiaca of the earwigs Labidura riparia and Forficula auricularia contain a substance that causes hyperlipaemia in migratory locusts and hypertrehalosaemia in the American cockroaches. A conspecific bioassay in L. riparia revealed that this factor is lipid-mobilizing. Isolation of the neuropeptide was achieved by single-step RP-HPLC. The primary structure of the earwig adipokinetic peptide was elucidated by automated Edman degradation in combination with matrix-assisted laser desorption/ionization mass spectrometry. It is a blocked octapeptide, pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH₂, previously denoted as Grb-AKH and first identified in Gryllus bimaculatus ( Gäde & Rinehart 1987 ). The synthetic peptide co-chromatographed under various conditions with the native peptide and, in biological assays, resulted in lipid-mobilization in L. riparia when injected in low concentration.     

Identification of a secretomotor centre in the brain of Locusta migratoria,controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum

Summary After retrograde filling of axons terminating in the glandular lobe of the corpus cardiacum (CC) of Locusta migratoria with cobalt chloride, a paired group of about 15 cobalt containing cells was demonstrated in the lateral area of the protocerebrum. The axons of these cells run via the NCC II into the glandular lobe of the CC. These small neurons have the characteristics of secretory cells; they contain secretory granules of about 1000 Å in diameter. The axon terminals in the glandular lobe, making synaptic contacts with the glandular cells, contain secretory granules of the same size. It is therefore concluded that the cell groups in the protocerebrum control the activity of the glandular cells which produce an adipokinetic hormone. Arborizations of fibers of the lateral secretomotor cells are present in the dorsal neuropile of the protocerebrum, ventral of the mushroom bodies and along the tracts of the NCC I within the brain. It is proposed that these arborizations are sites of synaptic input. It is discussed that the axons of these cells might receive additional synaptic input in the storage lobe of the CC.The localization of cell bodies, the axons of which enter the storage part of the CC is described. The course of the axon tracts of the various cell groups in the protocerebrum and their connections with the NCC I and NCC II are demonstrated.Supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). The electron microscopical investigations were performed at the EM-unit of the Faculty of Biology, State University of Utrecht (Director: Prof. Dr. J.C. van de Kamer)The author is greatly indebted to Dr. A.M.Th. Beenakkers and Dr. H.H. Boer for their active interest and helpful advise. Thanks are also due to Mr. H. van Kooten and his staff for making the macro- and microphotographs, to Mr. L.W. van Veenendaal for preparing the electron micrographs and final assistance in the preparation of the photo pages and to Mr. D. Smit, who made the drawings     

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.     

Co-localization of the adipokinetic hormones I and II in the same glandular cells and in the same secretory granules of corpus cardiacum of Locusta migratoria and Schistocerca gregaria

Summary The immunocytochemical reactivity of the glandular cells of the corpus cardiacum (CCG-cells) of Locusta migratoria and Schistocerca gregaria was investigated at the electron-microscopic level, using the protein A-gold method, with three antisera against fragments of the adipokinetic hormones AKH I and AKH II. This combination of antisera permitted discrimination between anti-AKH I and anti-AKH II immunoreactivity. Fixation in a mixture of 2% glutaraldehyde and 2% formaldehyde, in combination with low-temperature embedding in Lowicryl K₄M, produced the highest and most consistent selective immunogold labelling of the secretory and ergastoplasmic granules. All secretory granules in all CCG-cells investigated possessed a distinct anti-AKH I-immunopositive reaction, whereas most secretory granules showed a weaker anti-AKH II immunoreaction. Ergastoplasmic granules reacted similar to the secretory granules. The average immunolabelling of the secretory granules was higher in the processes than in the cell bodies of the CCG-cells. The results in Schistocerca gregaria were essentially similar to those in Locusta migratoria. It is concluded that (i) the individual CCG-cells synthesize AKH I as well as AKH II; (ii) these hormones coexist in the same ergastoplasmic and secretory granules; and (iii) these granules contain a higher content of AKH I than AKH II.     

Pharmacology of octopamine-, dopamine-, and 5-hydroxytryptamine-stimulated cyclic amp accumulation in the corpus cardiacum of the american cockroach,Periplaneta americana L

A variety of potential receptor agonists were incubated with isolated corpora cardiaca from the American cockroach Periplaneta americana to determine their effects on cyclic AMP production in this gland. Octopamine, dopamine, and 5-hydroxytryptamine elevated cyclic AMP levels in a dose-dependent manner with estimated K_a values of 15.8, 1.7, and 1.1 m?M, respectively, and their stimulation was found to be additive. Several vertebrate receptor antagonists were tested against the three amines and a preliminary pharmacological profile developed.     

Morphology of neurones in the storage part of the corpus cardiacum of Locusta migratoria: no evidence for their involvement in the regulation of adipokinetic cell activity

The storage part of the corpus cardiacum of Locusta migratoria consists of two compartments: a neural part on the haemocoelic side containing neuronal cell bodies that are protected by a blood-brain barrier, and a neurohaemal part adjacent to the aorta. Intracellular filling of the neurones in the neural part with Lucifer yellow followed by confocal laser scanning microscopy has revealed that these neurones can be divided into several classes. None of the neurones has processes extending into the glandular part of the corpus cardiacum. They are, therefore, not directly involved in the regulation of adipokinetic cell activity.     

Immunoreactive material resembling vertebrate neuropeptides in the corpus cardiacum and corpus allatum of the insect Leucophaea moderae

Summary The presence and differential distribution of substances antigenically related to known vertebrate neuropeptides demonstrated within the corpus cardiacum of the insect Leucophaea are as follows: Of ten mammalian antisera tested, six yielded substantial immunoreactive deposits resembling oxytocin, somatostatin, Substance P, met-enkephalin, bombesin, and neurotensin, respectively. In the remaining four, the reaction was moderate (vasopressin, -endorphin) or marginal (LH-RF, calcitonin). With regard to their regional distribution, these biochemically distinct reaction products seem to fall into two groups: (1) Materials resembling oxytocin, vasopressin, met-enkephalin, -endorphin (and presumably also neurotensin and LH-RF) predominate in the central release area of the organ and are considered to be of extrinsic (cerebral) origin. (2) Substances localized primarily in areas rich in intrinsic glandular cells of the corpus cardiacum, and revealed by antisera raised against somatostatin, Substance P, and bombesin, are judged to be synthesized and stored within this organ. In peptidergic fibers entering the adjacent corpora allata, thus far Substance P-, -endorphin-, and LH-RF-like immunoreactivities have been demonstrated. Some of these new neuropeptides may be contained in classical neurosecretory neurons, formerly identified by less specific methods, others must be assigned to additional peptidergic neurons heretofore unknown.Supported by NSF grant BMS 74-12456 (B.S.). The excellent technical assistance of Mrs. Sarah Wurzelmann is gratefully acknowledged     

Methylxanthine inhibition of neurosecretion in the brain and corpus cardiacum of Cecropia

The fine-structure of the median neurosecretory cells and corpora cardiaca of the Cecropia silkmoth during the first 7 days after transfer from cold conditions to room temperature was compared to that of similar animals whose development was arrested with aminophylline. The major difference observed was the failure of the intrinsic secretory cells of the corpus cardiacum to degenerate in the arrested animals. This failure to degenerate coincides with the expected period of brain hormone release. After long periods of arrest, the medial neurosecretory cells and their axons became distended with neurosecretory granules. The significance of these observations in the initiation of adult development is discussed.     

Proctolin: A possible releasing factor in the corpus cardiacum/corpus allatum of the locust

The corpus cardiacum (CC) and corpus allatum (CA) of the locust, Locusta migratoria, contain intense proctolin-like immunoreactivity (PLI) within processes and varicosities. In contrast, in the cockroach, Diploptera punctata, although a similar staining pattern occurs within the CC, PLI appears absent within the CA. The possible role of proctolin as a releasing factor for adipokinetic hormone (AKH) and juvenile hormone (JH) was investigated in the locust. Proctolin caused a dose-dependent increase in AKH I release (determined by RP-HPLC) from the locust CC over a range of doses with threshold above 10(-8)M and maximal release at about 10(-7)M proctolin. Isolated glandular lobes of the CC released greater amounts of AKH I following treatment with proctolin and in these studies AKH II was also released. Confirmation of AKH I release was obtained by injecting perfusate from incubated CCs into locusts and measuring hemolymph lipid concentration. Perfusate from CC incubated in proctolin contained material with similar biological activity to AKH. Proctolin was also found to significantly increase the synthesis and release of JH from locust CA, with the increase being greatest from CAs that had a relatively low basal rate of JH biosynthesis (<35 pmol h(-1) per CA). In contrast, proctolin did not alter the synthesis and release of JH from the cockroach CA. These results suggest that proctolin may act as a releasing factor for AKHs and JH in the locust but does not act as a releasing factor for JH in the cockroach.     

Serotonin-immunoreactive neurones in the brain of Locusta migratoria innervating the corpus cardiacum

Summary The serotoninergic innervation of the corpus cardiacum (CC) of Locusta migratoria was investigated using two antisera against serotonin. A dense network of immunoreactive nerve fibres was present in the storage lobe of the CC. Immunopositive fibres only sporadically crossed the border between the storage lobe and the glandular lobe of the CC. Immunopositive fibres entered the storage lobe of the CC via the nervus corporis cardiaci I (NCCI); NCCII was immunonegative. Unilateral retrograde fillings of the NCCI with the fluorescent tracer Lucifer yellow, followed by antiserotonin immunocytochemistry, revealed about 20 double-labelled neurones in the anterior part of the pars intercerebralis. The double-labelled neurones were scattered between fluorescent non-immunoreactive neurones. Additionally, 5–7 neurones labelled only with Lucifer yellow were found at the ventrolateral side of the tritocerebrum. No immunopositive neurones were observed in the hypocerebral ganglion. Immunopositive fibres from neurones in the frontal ganglion ran via the recurrent nerve and the neuropile of the hypocerebral ganglion into the paired oesophageal nerve. At most, a few immunopositive nerve fibres occurred in the cardiostomatogastric nerves II, which connect the storage lobe of the CC with the paired oesophageal nerve at the caudal end of the hypocerebral ganglion.     

Effect of the brain and corpus cardiacum on egg production in Rhodnius prolixus

Using total egg production corrected for size of blood meal as an index of the activity of the corpus allatum (CA), the effects of various surgical manipulations of the neuroendocrine system have been examined. Isolation of the CA from its nervous connections increases egg production well beyond that of a normal insect, thus confirming that the CA is at least partly controlled by inhibitory nerves from the brain. Removal of the corpora cardiaca (CC) reduces the level of this increased egg production, and decapitation anterior to the CC results in a level of egg production that is greater than that found in females decapitated between the CA and CC. Implanting a CC together with a CA into a decapitated female results in a higher egg production than implanting a CA alone. These results demonstrate that an allatotropic influence is exerted by the CC. Experiments designed to examine the role of the brain were inconclusive and did not eliminate the possibility that the allatotropin from the CC originated in the brain.     

Effect of corpus cardiacum extracts on the ATPase activity of locust rectum

The Mg²⁺ dependent and Na⁺K⁺-activated ATPase activities of microsomal preparations from the rectum of Locusta migratoria were both stimulated, to varying extents, by crude extracts of the corpora cardiaca of this species. Mg²⁺ ATPase activity increased by approximately 549% whereas the hormonal stimulation of Na⁺K⁺-activated ATPase depended upon the concentration of sodium and potassium ions. At 100 mM Na⁺ and 20 mM K⁺, conditions which approximate to optimum for this enzyme system, Na⁺K⁺-activated ATPase activity increased by about 14%. At sub-optimum concentrations of these ions, i.e. 50 and 5 mM Na⁺ and K⁺ respectively, the increase in Na⁺K⁺-activated ATPase activity was about 205%. Ouabain at a concentration of 10^?3 M completely abolished this stimulated activity and was consistently effective in partially reducing the stimulation of Mg²⁺ ATPase activity by corpora cardiaca extracts.