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Endocrinology Vol. 138, No. 1 138-142
Copyright © 1997 by The Endocrine Society

ARTICLES

Isolation and Characterization of an Adipokinetic Hormone Release-Inducing Factor in Locusts: The Crustacean Cardioactive Peptide

D. Veelaert, P. Passier, B. Devreese, J. Vanden Broeck, J. Van Beeumen, H. G. B. Vullings, J. H. B. Diederen, L. Schoofs and A. De Loof

Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute K. U. Leuven (D.V., J.V.B., L.S., A.D.L.), Leuven, Belgium; and the Department of Experimental Zoology, University of Utrecht (P.P., H.G.B.V., J.H.B.D.), Utrecht, The Netherlands; and the Department of Biochemistry, Physiology and Microbiology, Universiteit Gent (B.D., J.V.B.), Gent, Belgium

Address all correspondence and requests for reprints to: Dr. J. Vanden Broeck, Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute K. U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium. E-mail: Jozef.VandenBroeck{at}bio.kuleuven.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A methanolic extract of 7000 desert locust (Schistocerca gregaria) brains contains several factors that stimulate the in vitro release of adipokinetic hormone (AKH) by glandular cells of locust (Locusta migratoria and Schistocerca gregaria) corpora cardiaca. The most potent one has now been fully identified. Matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis revealed a mass of 954.6 Da. The primary structure of the peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, appeared identical to that of a previously identified crustacean cardioactive peptide. This myotropin was first isolated from the shore crab, Carcinus maenas, and later from several insect species, but was never reported in the context of AKH release.

The present study shows that synthetic crustacean cardioactive peptide induces the release of AKH from corpora cardiaca in a dose-dependent manner when tested in concentrations ranging from 10-5-10-9 M. This is the first demonstration in invertebrates of a peptide neurohormone controlling the release of a second peptide hormone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING PROLONGED flight, the African migratory locust, Locusta migratoria, releases adipokinetic hormones (AKH) from endocrine cells in the glandular part of the corpus cardiacum (CC), glandular part (CCg), into the hemolymph. These hormones are involved in mobilization from the fat body of lipids and carbohydrates that serve as energy substrates for the flight muscles. Up until now, three different AKHs (1, 2, 3) have been isolated from the CCg of Locusta migratoria. They are members of a large family of structurally related, but functionally diverse, peptides (4).

The CC is connected to the brain via two paired nerves, the nervi corporis cardiaci I and II (NCC I and II). NCC II fibers are directly involved in AKH release; the secretomotor neurons from the lateral part of the protocerebrum project via the NCC II to the adipokinetic cells in the CCg (5), and electrical stimulation of the NCC II evokes AKH release (6). Stimulation of NCC I fibers enhances the effect of NCC II activity (6, 7). These NCC I fibers contain dopamine (8) and serotonin (9). In vitro, both substances potentiate AKH release induced by cAMP-activating agents (10).

Recently, it has been demonstrated immunocytochemically that some NCC II fibers contain locustatachykinins (Lom-TKs) (11). Until now, five Lom-TK analogs have been isolated from L. migratoria brain-CC-corpora allata (CA) complexes (12, 13, 14). To date, Lom-TK I is the only neuroactive substance known to initiate the release of AKH from corpora cardiaca without simultaneous addition of substances that stimulate the accumulation of cAMP [e.g. 3-isobutyl-1-methylxanthine (IBMX)] (11). Recently, all five known Lom-TK analogs were isolated from the CCg (Passier, P., et al., unpublished results). Some NCC II fibers contain RFamide-related peptides (FaRPs). (These peptides have an Arg-Phe-amide C-terminus in common.) In vitro, both FMRFamide and Schisto-FLRFamide inhibited the release of AKH that was induced by IBMX (15).

In addition to neurotransmitters/modulators (NCC II nerve terminals), some humoral factors can play a role in the release of AKH. It is known that resting levels of trehalose (80 mM) in the hemolymph prevent the release of AKH, whereas flight levels (40 mM) have no effect (16, 17). Although Orchard and co-workers (18, 19, 20) suggested that octopamine might induce AKH release by acting as a neurotransmitter, the amine could not be detected either immunocytochemically or electrochemically in the CCg (10, 21). Moreover, in the absence of IBMX, octopamine is not able to elicit a significant effect on AKH release. In the presence of IBMX, however, it clearly enhances the IBMX-induced release of AKH (10). During the first minutes of flight, the octopamine titer in the hemolymph increases to a level 3 times higher than that during rest (22). These data strongly suggest a neurohormonal role of octopamine in the regulation of AKH release.

In this study, we present the purification and identification of a potent AKH-releasing factor from the locust brain-CC-CA complex. To monitor biological activity after subsequent chromatographic separation steps, a method to measure the release of AKH from isolated corpora cardiaca using HPLC was employed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, tissue extraction, and purification
Schistocerca gregaria was raised at 32 C and fed cabbage leaves and oat flakes. Brain-CC-CA complexes (n = 7000) were dissected from adults, 12–14 days after their final ecdysis, and immediately placed in ice-cold methanol-water-acetic acid (90:9:1, vol/vol/vol) solution. The complexes were sonicated and centrifuged for 30 min (10,000 x g; 4 C). The methanol was evaporated, and the remaining aqueous residue was reextracted with ethylacetate and n-hexane to remove the bulk of the lipids. The organic solvent layer was decanted, and the aqueous solution was dried in siliconized round bottom flasks. Subsequently, it was redissolved in aqueous 0.1% trifluoroacetic acid (TFA) and prepurified on Megabond Elute C18 cartridges (Varian, Harbor City, CA) that had been activated with acetonitrile CH3CN-H2O-TFA (80:19.9:0.1, vol/vol/vol) and afterward rinsed with aqueous 0.1% TFA. The cartridges were eluted with 25 ml 50% and 80% CH3CN in 0.1% aqueous TFA. Columns and operation conditions for HPLC on a Gilson (Gilson Medical Electronics, Villiers le Bel, France) HPLC system with variable wavelength detector (set at 214 nm) were 1) Deltapak C18 column (25 x 100 mm; Waters Associates, Milford, MA); solvent A, 0.1% TFA in water; solvent B, 50% CH3CN in 0.1% aqueous TFA; column conditions: 100% solvent A for 8 min, followed by a linear gradient to 100% solvent B in 150 min; flow rate, 6 ml/min; detector range, 1 absorption unit full scale (AUFS); 2) Vydac C4 column (4.6 x 250 mm; solvent A, 0.1% TFA in water; solvent B, 50% CH3CN in 0.1% aqueous TFA; column conditions: 100% solvent A for 20 min, followed by a linear gradient to 100% solvent B in 50 min; flow rate, 1 ml/min; detector range, 0.5 AUFS; 3) phenyl Spheri-5 column (4.6 x 250 mm; Brownlee, Applied Biosystems, Foster City, CA); solvent A, 15% CH3CN in 0.1% aqueous TFA; solvent B, 65% CH3CN in 0.1% aqueous TFA; column conditions: 100% solvent A for 20 min, followed by a linear gradient to 100% solvent B in 50 min; flow rate, 1.5 ml/min; detector range, 0.5 AUFS; and 4) Vydac C18 (4.6 x 250 mm); solvent A, 10% CH3CN in 0.1% aqueous TFA; solvent B, 40% CH3CN in 0.1% aqueous TFA; column conditions: 100% solvent A for 10 min, followed by a linear gradient to 100% solvent B in 60 min; flow rate, 1.5 ml/min; detector range, 0.5 AUFS. Peaks were collected manually. After each run, a volume containing about 10 brain-subesophageal ganglion (SOG)-CC-CA complex equivalents were taken from each fraction, dried, and assayed in duplicate.

Mass and structure determination
A sample containing 0.5–1.0 pmol purified peptide was subjected to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis (23). One microliter was mixed with 1 µl of a 50 mM solution of {alpha}-cyano-4-hydroxycinnamic acid in H2O-CH3CN-TFA (60:39.9:0.1, vol/vol/vol) and applied to the multisample target. This mixture was air-dried, and the target was then introduced in the instrument, a VG Tofspec (Fisons Instruments, Analytical MS, Wythenshawe, UK) equipped with a N2 laser (337 nm). The sample was measured either in the linear mode (acceleration voltage, 25 kV) or in the reflectron mode (acceleration voltage, 25 kV; reflectron voltage, 28.5 kV). In both cases, the laser energy was reduced until an optimal resolution and signal/noise ratio was obtained. The results of 10–20 shots were averaged to obtain the final spectrum. Amino acid sequencing was performed on an AVI476A protein sequencer (Applied Biosystems) according to the method of Hewick et al. (24).

Retention time of synthetic crustacean cardioactive peptide (CCAP)
Ten nanograms of the synthetic peptide mixed with one tenth of the purified amount of native peptide were injected together on a Gilson HPLC system with variable wavelength detector (214 nm) on a Microsorb-MV C18 column (4.6 x 250 mm; Rainin Instruments Co., Woburn, MA; solvent A, 15% CH3CN in 0.1% aqueous TFA; solvent B, 30% CH3CN in 0.1% aqueous TFA). Column conditions were 100% solvent A, followed by a linear gradient to 100% solvent B in 60 min (flow rate, 1 ml/min; detector range, 0.2 AUFS).

AKH release bioassay
Adult male African migratory locusts, Locusta migratoria were used, 14 days after their final moult. The animals were reared under controlled conditions as described earlier (10). Corpora cardiaca (CC) were excised and collected on watchglasses in pools of five in 100 µl insect saline buffer (150 mM NaCl, 10 mM KCl, 4 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, pH 7.0). After rinsing three times with insect saline buffer, the pooled glands were incubated in 100 µl insect saline buffer under continuous and moderate shaking in a moist chamber for 30 min at 30 C. Incubation media were collected in Eppendorf tubes and diluted 1:1 with 2.5 M acetic acid to prevent sticking of AKH to the wall of the tubes (recovery 95–98%). The watchglasses and the pipette tips used were siliconized with 1% diethylchlorosilane diluted in chloroform. The CC were rinsed with insect saline buffer and incubated for another 30 min under the conditions as described above in 100 µl insect saline buffer, but now provided with the dried HPLC-fractions. For dose response experiments, increasing amounts of synthetic CCAP (Saxon Biochemicals GmBH (Bachem), Hannover, Germany) were added to the second incubation medium resulting in final CCAP concentrations of 10-4 M to 10-10 M. The incubation media of the second incubation period were also collected and diluted 1:1 with 2.5 M acetic acid. The amounts of AKH I released into the incubation media were quantified via HPLC (Pharmacia LKB, Uppsala, Sweden) using a Spherisorb C18 column (4 x 250 mm) (Pharmacia LKB, Uppsala, Sweden). Solvent A contained 15% CH3CN, solvent B 90% CH3CN. The column conditions were: 100% A followed by a linear gradient to 17% B in 6 min, and then by a linear gradient to 22% B in 10 min, flow rate 0.9 ml/min AUFS). Fluorescence was detected with a spectrophotometric detector (Shimadzu RF10A (Shimadzu Corp., Kyoto, Japan; extension, 276 nm; emission, 340 nm). Peaks were integrated, and the mean ratio between the amounts of AKH I released during the second and the first incubation period was used as a parameter for the effect on the release of AKH I of the fraction to be tested.

Similar conditions were used to determine AKH release of synthetic CCAP by CC of Schistocerca gregaria. The experimental data are given as the mean ± SEM of n experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Two Megabond Elute columns were used to prepurify the extract of 7000 brain/SOG/CC/CA equivalents. All AKH-releasing activity was present in the fraction eluting with 50% CH3CN in 0.1% aqueous TFA. This fraction was then separated on a Delta Pak C18 column. The fraction eluting from this column at 66–74 min stimulated AKH release by 250%. This fraction was further purified. It had a retention time of 41–42 min on the second column, 38.8 min on the third column, and 34.16 min on the fourth column (Fig. 1Go). After the fourth column purification step, the peptide showed apparent homogeneity. MALDI-TOF-mass spectrometry of ±1 pmol revealed a mass of 954.5 Da (Fig. 2Go). Automated Edman sequence analysis of the isolated peptide designated the primary structure of the peptide as Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2. The primary structure of the peptide was identical to that of the CCAP (25). This CCAP has been shown to contain a disulfide bridge. Assuming the presence of this disulfide bridge, the calculated monoisotopic mass of 954.6 Da was in good agreement with the mass analysis data.



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  		Figure 1. Final HPLC purification of CCAP using a Vydac C18 column. The peak eluting at 37 min contained the biologically active (black area) Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2 peptide.

 


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  		Figure 2. MALDI-TOF-mass spectrometry of CCAP. Three different ions are observed: CCAP and H+ (955.6), CCAP and Na+ (977.8), and CCAP and K+ (993.6).

 
To test the validity of the mass spectroscopy and the amino acid sequence data, we compared the elution characteristics of the isolated peptide with those of the synthetic peptide (Saxon Biochemicals, Hannover, Germany). Both peptides coeluted perfectly synchronously under the conditions used.

Synthetic peptide was used for establishing a dose-response curve. As L. migratoria was used as a model in other studies on AKH release and functioning (see introduction), a full dose-response curve of CCAP on the release of AKH was performed for this species. Increasing concentrations of CCAP were tested for their ability to release AKH from pools of isolated corpora cardiaca. The effect ranged between 10 µM and 10 nM. A concentration of 1 nM had no effect compared to the control, whereas 10 nM doubled the amount of AKH released into the incubation medium (Fig. 3Go). The dose-response curve in L. migratoria and the effect of 10 µM CCAP on the release of AKH in S. gregaria are shown in Fig. 3Go (n = 5).



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  		Figure 3. Dose-response curve for stimulation of AKH release by CCAP on CC of S. gregaria and L. migratoria. The rates of AKH release by treated CC were expressed as a percentage of those during the first incubation period (n = 5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The AKH release-inducing factor isolated and identified from Schistocerca gregaria is identical to CCAP. This peptide was first isolated and sequenced from the shore crab, Carcinus maenas (25). Since then, CCAP was isolated from the lobster Homarus americanus (26), the African locust, Locusta migratoria (27), and the tobacco hornworm, Manduca sexta (28) and now from the desert locust Schistocerca gregaria. The structure of CCAP has been highly conserved during evolution, providing support for the speculation that this peptide must have had an early evolutionary origin. Therefore, this peptide is a good candidate for future endocrinological investigations in other animal phyla.

In the shore crab, C. maenas, CCAP strongly accelerates the heart beat in a semiisolated preparation (25). In insects, this peptide also elicits myotropic responses. In M. sexta, one of the cardioactive peptides (CAPs) has been identified as CCAP (CAP2a) (28). From physiological studies, it was concluded that CAPs stimulated the heartbeat, while acting as neurohormones. During flight episodes, CAPs enhance the hemolymph circulation between the abdomen and the thorax of M. sexta (29). According to Tublitz (29), the increased hemolymph circulation probably facilitates the transport of energy substrates from the fat body to the flight muscles during flight. The specificity for the CAP2a target is dependent on the developmental stage. In early developmental stages, CAP2a acts predominantly on hindgut contractility. During metamorphosis, the sensitivity of the heart for CAP2a increases, whereas the hindgut becomes less sensitive.

In locusts, immunolocalization studies support the suggested role of this peptide in the control of heartbeat (29) and abdominal ventilatory and visceral muscle (e.g. the hindgut) (27) activity. As the central body in the brain contains strong immunopositive staining, CCAP is probably also involved in the integration of visual information and the control of locomotion, walking, and flying (30, 31).

In the present study, CCAP purification was based on its effect on the release of AKH I from isolated corpora cardiaca of L. migratoria. The original abbreviation remarkably covers this novel function, since CCAP appears to be a "corpus cardiacum-activating peptide" in locusts. In the brain extract of S. gregaria, this peptide was the only substance that had such a strong stimulatory effect on the in vitro release of AKH I. This result suggests an in vivo role for CCAP in the release of AKH I. Detailed morphological studies of the nervous system of L. migratoria, however, could not demonstrate the existence of a direct connection of CCAP-immunopositive nerve fibers coming from the brain with the glandular cells in the corpus cardiacum (31). Therefore, CCAP is not likely to act as a neurotransmitter because it is not present in NCC II axon terminals on AKH-producing cells. Occasionally, very few weakly immunopositive fibers were encountered in the CCg, but the origin of these fibers remains unknown. In the neurohemal part of the CC, some immunopositive fibers were found. These originate from cell bodies present in the SOG and project to the CCs via the NCC III (31, 32). In C. maenas, CCAP acts as a neurohormone released from the pericardial organs in the direct vicinity of the heart (25, 27). Dircksen and Homberg (31) suggest that in the locust, CCAP is released by neurosecretory cells of the SOG and, thus, is also a neurohormone. Indeed, CCAP meets the requirements for a neurohormonal status, in that it is active at low concentrations. As in insects still other functions are described for CCAP, it is likely to be a pleiotropic messenger molecule, just like many other insect neuropeptides that have been identified (14, 33). In insects, the circulation system is an open one, and factors released from neurohemal sites can reach the CCg, as well as many other organs, some of which may be targeted by this factor as well. Most of the humoral effects that were reported for CCAP appear to be related to a physiological situation that corresponds with periods of increased activity and higher energy metabolism. Other humoral factors that are usually involved in the induction of such conditions are octopamine and AKH. The effects elicited by CCAP appear to be perfectly complementary to the effects of these two hormones. In addition to neurohormones, such as CCAP and octopamine, neurotransmitters/modulators present in NCC II terminals (e.g. locustatachykinins and FaRPs) may also be involved in controlling AKH release (11, 15). The precise spatio-temporal relationships among these factors certainly have to be determined in future physiological investigations.

Although other factors may have an influence on AKH release, the effect of CCAP reported in the present paper provides the first evidence for the existence in invertebrates of neuropeptides that act as hormone-releasing factors (CCAP) for other peptide hormones (AKH). The mechanism, however, differs from that in vertebrates, in which hypothalamic releasing factors are released in a specialized portal vein system in which the blood circulation is directed toward the pituitary gland. Nevertheless, the basic principles encountered in vertebrate endocrinology are analogous to those encountered in many studies on insect hormones.

Received April 23, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals