Identification of a calcitonin-like diuretic hormone that functions as an intrinsic modulator of the American lobster, Homarus americanus, cardiac neuromuscular system.

In insects, a family of peptides with sequence homology to the vertebrate calcitonins has been implicated in the control of diuresis, a process that includes mixing of the hemolymph. Here, we show that a member of the insect calcitonin-like diuretic hormone (CLDH) family is present in the American lobster, Homarus americanus, serving, at least in part, as a powerful modulator of cardiac output. Specifically, during an ongoing EST project, a transcript encoding a putative H. americanus CLDH precursor was identified; a full-length cDNA was subsequently cloned. In silico analyses of the deduced prepro-hormone predicted the mature structure of the encoded CLDH to be GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide (Homam-CLDH), which is identical to a known Tribolium castaneum peptide. RT-PCR tissue profiling suggests that Homam-CLDH is broadly distributed within the lobster nervous system, including the cardiac ganglion (CG), which controls the movement of the neurogenic heart. RT-PCR analysis conducted on pacemaker neuron- and motor neuron-specific cDNAs suggests that the motor neurons are the source of the CLDH message in the CG. Perfusion of Homam-CLDH through the isolated lobster heart produced dose-dependent increases in both contraction frequency and amplitude and a dose-dependent decrease in contraction duration, with threshold concentrations for all parameters in the range 10(-11) to 10(-10) mol l(-1) or less, among the lowest for any peptide on this system. This report is the first documentation of a decapod CLDH, the first demonstration of CLDH bioactivity outside the Insecta, and the first detection of an intrinsic neuropeptide transcript in the crustacean CG.

Presence and activity of a Dippu-DH31-like peptide in the blood-feeding bug, Rhodnius prolixus.

The blood-feeding bug, Rhodnius prolixus, ingests large blood meals, then undergoes a period of rapid diuresis which is under neurohormonal control. In both cockroach (Diploptera punctata) and fruit fly (Drosophila melanogaster) a calcitonin-like DH31 neuropeptide has been identified [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74] and demonstrated to be active on Malpighian tubule secretion [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74]. Using an antibody raised against D. punctata (Dippu) DH31, we demonstrate the presence of Dippu-DH31-like immunoreactivity in the CNS, salivary glands, hindgut and neurohemal sites of 5th instar Rhodnius. Double-label immunohistochemistry for Dippu-DH31-like and serotonin-like immunoreactivity demonstrates some co-localization of these factors in cells of the mesothoracic ganglionic mass (MTGM) and in neurohemal sites on the abdominal nerves. When tested on Rhodnius 5th instar Malpighian tubules, Dippu-DH31 stimulated minor increases in rate of secretion. Dippu-DH31 tested in combination with serotonin resulted in increases in the rate of secretion which were at least additive.

A study of signal transduction for the two diuretic peptides of Diploptera punctata.

We investigated second messengers involved in the action of the CRF-related peptide Dippu-DH46 and the calcitonin-like peptide Dippu-DH31 in Diploptera punctata. Dippu-DH46 causes a dose-dependent increase in intracellular cAMP levels, its diuretic activity is mimicked by cAMP agonists, but is attenuated by Rp-cAMPS. Dippu-DH46 acts synergistically with kinins and thapsigargin; both mobilize intracellular Ca2+. Dippu-DH46 also acts synergistically with cAMP agonists, and its effect is inhibited by a PKC inhibitor, suggesting it also activates intracellular Ca2+. Dippu-DH31 has no effect on cAMP levels and its activity is not blocked by cAMP agonists. Neither peptide stimulated cGMP levels in a dose-dependent manner, nor does cGMP have any effect on fluid secretion.

The biological activity of diuretic factors in Rhodnius prolixus.

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones. In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards. Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.

Antagonistic control of fluid secretion by the Malpighian tubules of Tenebrio molitor: effects of diuretic and antidiuretic peptides and their second messengers.

Fluid secretion by insect Malpighian tubules is controlled by haemolymph-borne factors. The mealworm Tenebrio molitor provides the first known example of antagonistic interactions between endogenous neuropeptides acting on Malpighian tubules. The two corticotropin-releasing-factor (CRF)-related diuretic peptides previously isolated from Tenebrio molitor, Tenmo-DH(37) and Tenmo-DH(47), were found to stimulate Tenebrio molitor tubules in vitro in a dose-dependent manner with EC(50) values of 0.12 nmol l(-1) and 26 nmol l(-1) respectively. However, no synergistic or additive effect was observed when these two peptides were tested simultaneously. We then investigated antagonism between second messengers: dose-response curves were constructed for stimulation of Tenebrio molitor tubules by cyclic AMP and their inhibition by cyclic GMP. When both cyclic nucleotides were included in the bathing Ringer, the stimulatory effect of cyclic AMP was neutralised by cyclic GMP. Similarly, the stimulatory effect of Tenmo-DH(37) was reversed on addition of an antidiuretic peptide (Tenmo-ADF), which was recently isolated from Tenebrio molitor and acts via cyclic GMP. The cardioacceleratory peptide CAP(2b), originally isolated from Manduca sexta, also increases intracellular cyclic GMP levels and inhibited fluid secretion by Tenebrio molitor tubules, with an EC(50) value of 85 nmol l(-1). This inhibitory effect was reversed by Tenmo-DH(37). Endogenous diuretic and antidiuretic peptides, effective at low concentrations and acting via antagonistic second messengers, have the potential for fine control of secretion rates in the Malpighian tubules of Tenebrio molitor.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.

Isolation and identification of a diuretic hormone from Zootermopsis nevadensis.

A diuretic hormone (DH) was isolated from extracts of heads of Zootermopsis nevadensis, a dampwood termite. The peptide has 46 residues, M(r) = 5,328.2 Da, with the sequence TGAVPSLSIVNPLDVLRQRLLLEIARRRMRQSQDQIQANREMLQTI-NH(2,) showing it to be a CRF-related DH. This peptide increases cyclic AMP production in Malpighian tubules of Manduca sexta. We detected another factor in the head extracts which behaved as a more basic peptide on ion exchange chromatography. The latter factor also stimulated cyclic AMP production in the bioassay, but two large scale attempts to isolate this peptide were unsuccessful. We believe the second peptide is acid labile.

The distribution of a kinin-like peptide and its co-localization with a CRF-like peptide in the blood-feeding bug, Rhodnius prolixus.

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay. In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s). The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.

Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects.

Insect diuretic hormones are crucial for control of water balance. We isolated from the cockroach Diploptera punctata two diuretic hormones (DH), Dippu-DH(31) and Dippu-DH(46), which increase cAMP production and fluid secretion in Malpighian tubules of several insect species. Dippu-DH(31) and -DH(46) contain 31 and 46 amino acids, respectively. Dippu-DH(46) belongs to the corticotropin-releasing factor (CRF)-like insect DH family, whereas Dippu-DH(31) has little sequence similarity to the CRF-like DH, but is similar to the calcitonin family. Dippu-DH(46) and -DH(31) have synergistic effects in D. punctata but have only additive effects in Locusta migratoria. Dippu-DH(31) represents a distinct type of insect DH with actions that differ from those of previously identified insect peptides with diuretic activity.

Isolation and characterization of CRF-related diuretic hormones from the whitelined sphinx moth Hyles lineata.

We have isolated and characterized two diuretic hormones (DH), Hylli-DH41 and Hylli-DH30, from extracts of whole heads of the lepidopteran Hyles lineata. We monitored the isolation by measuring the ability of fractions to affect levels of cyclic AMP production by Malpighian tubules of Manduca sexta maintained in vitro. These DH are related to a family of vertebrate neuropeptides which includes sauvagine, corticotropin-releasing factor (CRF), and urotensin I. Both Hylli-DH41 (RMPSLSIDLPMSVLRQKLSLE KERKVQALRAAANRNFLNDI-NH2) and Hylli-DH30 (SFSVNPAVEILQHRYMEKVAQNNRNFLNRV-NH2) show extremely high similarity with two DH from the tobacco hornworm M. sexta. This is not surprising because both H. lineata and M. sexta are sphingid moths. The discovery of these DH provides a third example of two CRF-related DH occurring in one insect species.

Antidiuretic effects of a factor in brain/corpora cardiaca/corpora allata extract on fluid reabsorption across the cryptonephric complex of Manduca sexta.

Extracts of the brain/corpora cardiaca/corpora allata (Br/CC/CA) complex of Manduca sexta larvae elicit an antidiuretic effect, measured by an increase in fluid reabsorption across the cryptonephric complex of larval M. sexta. Separation of the extract by reversed-phase liquid chromatography gave two fractions with antidiuretic effects. The more potent of these two factors was further characterized for its effects on the cryptonephric complex. Its antidiuretic effect is not inhibited by bumetanide, a drug that inhibits M. sexta diuretic hormone (Mas-DH)-stimulated fluid reabsorption. These data indicate that the mechanism of the antidiuretic effect of the factor is different from that of Mas-DH on the cryptonephric complex. The basal reabsorption of the cryptonephric complex is blocked when treated on the lumen side with bafilomycin A(1), an inhibitor of the H(+)-ATPase, or with amiloride, an inhibitor of the H(+)/K(+) antiporter. However, the antidiuretic-factor-stimulated fluid reabsorption is not affected by either bafilomycin A(1) or amiloride. The increase in reabsorption triggered by the semi-purified factor can be inhibited by Cl(-) channel blockers or by removing Cl(-) from the lumen side of the cryptonephric complex. It appears that this factor activates a Cl(-) pump associated with the cryptonephric complex. Forskolin mimics the effect of this factor on fluid reabsorption, and the effect of forskolin is not inhibited by bumetanide. A selective and potent inhibitor of protein kinase A, H-89, also inhibits antidiuretic-factor-stimulated fluid reabsorption. Addition of the factor to cryptonephric complexes maintained in vitro caused a significant increase in cyclic AMP levels extracted from these tissues compared with values for controls. These data suggest that the antidiuretic effect of the factor in Br/CC/CA extract is mediated by cyclic AMP.

The distribution of a CRF-like diuretic peptide in the blood-feeding bug Rhodnius prolixus.

The blood-feeding bug Rhodnius prolixus ingests a large blood meal, and this is followed by a rapid diuresis to eliminate excess water and salt. Previous studies have demonstrated that serotonin and an unidentified peptide act as diuretic factors. In other insects, members of the corticotropin-releasing factor (CRF)-related peptide family have been shown to play a role in post-feeding diuresis. Using fluorescence immunohistochemistry and immunogold labelling with antibodies to the Locusta CRF-like diuretic hormone (Locusta-DH) and serotonin, we have mapped the distribution of neurones displaying these phenotypes in R. prolixus. Strong Locusta-DH-like immunoreactivity was found in numerous neurones of the central nervous system (CNS) and, in particular, in medial neurosecretory cells of the brain and in posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM). Positively stained neurohaemal areas were found associated with the corpus cardiacum (CC) and on abdominal nerves 1 and 2. In addition, Locusta-DH-like immunoreactive nerve processes were found over the posterior midgut and hindgut. Double-labelling studies for Locusta-DH-like and serotonin-like immunoreactivity demonstrated some co-localisation in the CNS; however, no co-localisation was found in the medial neurosecretory cells of the brain, the posterior lateral neurosecretory cells of the MTGM or neurohaemal areas. To confirm the presence of a diuretic factor in the CC and abdominal nerves, extracts were tested in Malpighian tubule secretion assays and cyclic AMP assays. Extracts of the CC and abdominal nerves caused an increase in the rate of secretion and an increase in the level of cyclic AMP in the Malpighian tubules of fifth-instar R. prolixus. The presence of the peptide in neurohaemal terminals of the CC and abdominal nerves that are distinct from serotonin-containing terminals indicates that the peptide is capable of being released into the haemolymph and that this release can be independent of the release of serotonin.

A cardioactive peptide from the southern armyworm, Spodoptera eridania.

A cardioactive peptide was isolated from extracts of whole heads of the southern armyworm, Spodoptera eridania. This peptide has the sequence ENFAVGCTPGYQRTADGRCKPTF (Mr = 2516.8), determined from both Edman sequencing and tandem mass spectrometry in combination with off-line micropreparative capillary liquid chromatography. This peptide, termed Spoer-CAP23, has excitatory effects on a semi-isolated heart from larval Manduca sexta, causing an inotropic effect at low concentrations of peptide and chronotropic and inotropic effects at high doses. The threshold concentration for stimulatory effects of the synthetic peptide on the semi-isolated heart was about 1 nM, suggesting a physiological role as a neuropeptide.

Isolation and identification of a second diuretic hormone from Tenebrio molitor.

A diuretic hormone (DH) of unusual structure was isolated from extracts of heads of Tenebrio molitor. The hormone is a 47 amino acid peptide, Mr = 5,029.9, with the sequence AGALGESGASLSIVNSLDVLRNRLLLEIARKKAKEGANRNRQILLSL. This peptide increases cyclic AMP production in Malpighian tubules of T. molitor. We recently identified a smaller DH from T. molitor with 37 amino acids; these peptides have only 15 identical amino acids when aligned to maximize similarity to other members of the insect DH family. This family has sequence similarity to the corticotropin-releasing factor superfamily of vertebrate peptides.

Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway.

A system is described for the heterologous expression of peptides in Saccharomyces cerevisiae. A synthetic gene encoding a precursor of the 41 amino acid Manduca sexta diuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide. A precursor of a mutant peptide of Mas-DH, Mas-DH[K22Q] was also expressed. The peptides were purified, then treated with peptidylglycine alpha-amidating enzyme to generate the alpha-amidated, mature, form of Mas-DH or Mas-DH[K22Q], which were biologically active. Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain. In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors. Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases. This protease-deficient strain may prove of general utility for expression of peptides. Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites. Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys downward arrow-Glu-Arg site. Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys. This paper is the first report on the in vivo activity and specificity of Yap3 and Mkc7 expressed at physiological levels.

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry.

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor-urotensin-sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of approximately 1 microM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29-R30 and R30-A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

Isolation and identification of a diuretic hormone from the mealworm Tenebrio molitor.

A diuretic hormone of unusual structure was isolated from extracts of whole heads of the mealworm Tenebrio molitor. The hormone is a 37-aa peptide of 4371 Da, with the sequence SPTISITAPIDVLRKTWEQERARKQMVKNREFLNSLN. This peptide increases cAMP production in Malpighian tubules of T. molitor. The amino acid sequence reveals that this peptide is a member of the family of sauvagine/corticotropin-releasing factor/urotensin I-related insect diuretic hormones. The C-terminal sequence of this peptide is quite different from other members of this family, which have a hydrophobic C terminus (isoleucinamide or valinamide). When aligned comparably, T. molitor diuretic hormone has a more hydrophilic C terminus, leucylasparagine (free acid). In contrast to all other known diuretic hormones of this family, this peptide has exceptionally low stimulatory activity on cAMP production in Malpighian tubules of Manduca sexta. However, at nanomolar concentrations it stimulates cAMP production in Malpighian tubules of T. molitor. Diuretic hormones of this family have been isolated previously from Lepidoptera, Orthoptera, Dictyoptera, and Diptera. This appears to be the first diuretic hormone isolated from a coleopteran insect.

A comparative catabolism study of isoleucine by insect and mammalian tissues.

The metabolism of [U-14C]isoleucine was examined in different tissues of five species of lepidopteran and four species of non-lepidopteran insects. Slices of fat body, epidermis, Malpighian tubule, gut, and muscle were incubated in a culture medium containing [U-14C]isoleucine; the medium was analyzed by ion-exclusion LC to quantify labeled metabolites. Tissues of lepidopteran insects secrete high levels of metabolites including 2-keto-4-methylvalerate, 2-methylbutyrate, propionate, and acetate. Tissues of non-lepidopteran insects secrete low amounts of these acids. Analysis of isoleucine transaminase activity in selected tissues of non-lepidopteran insects indicated that those tested contain significant activity. These results demonstrate that tissues of lepidopteran insects have a unique ability to secrete short chain acids, derived from isoleucine, into the medium. The secretion of propionate correlates with the ability to synthesize ethyl-branched juvenile hormones and indicates the presence of an efficient transport system for short chain acids. We also monitored the secretion of acidic metabolites of isoleucine by different tissues of the rat. Muscle was most active in secreting keto acid whereas heart secreted high levels of 2-methylbutyrate. Negligible quantities of metabolites of isoleucine were secreted by the liver.

Isolation and identification of a cardioactive peptide from Tenebrio molitor and Spodoptera eridania.

We isolated several cardioactive peptides from extracts of whole heads of the mealworm, Tenebrio molitor, and the southern armyworm, Spodoptera eridania, using a semi-isolated heart of Manduca sexta for bioassay. We have now isolated from each species the peptide with the strongest effect on rate of contraction of the heart. The peptides were identified using micro Edman sequencing and mass spectrometric methods. This cardioactive peptide has the same primary structure from both species: Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, a cyclic nonapeptide which is identical to crustacean cardioactive peptide (CCAP) originally isolated from the shore crab, Carcinus maenas, and subsequently isolated from Locusta migratoria and Manduca sexta. This is additional evidence that CCAP has widespread occurrence in arthropoda.

Complete structure of eclosion hormone of Manduca sexta. Assignment of disulfide bond location.

The locations of the three disulfide bonds of eclosion hormone (EH) isolated from Manduca sexta were assigned by sequence analysis of thermolysin fragments and by comparison of a key heterodimeric fragment to regiospecifically synthesized parallel and antiparallel isomers. We elucidated the complete structure of Manduca EH as a 62-residue peptide which has three disulfide bonds between Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49.