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.

Cyclopropanecarboxylic acid: chain elongation to omega-cyclopropyl fatty acids by mammals and plants.

Rats dosed orally which [carboxyl-14C]cyclopropanecarboxylic acid (or its hexadecyl ester) retain radioactivity in tissue as novel triacylglycerols. The most abundant 14C-labeled metabolites were identified by gas-liquid chromatography-mass spectrometry as 13-cyclopropyltridecanoic and 15-cyclopropylpentadecanoic acids. Similar omega-cyclopropyl fatty acids are produced by beagle dogs and a lactating cow, as well as by apple and orange trees.

JH Zero: New Naturally Occurring Insect Juvenile Hormone from Developing Embryos of the Tobacco Hornworm.

A new insect juvenile hormone was isolated from developing embryos of the tobacco hornworm moth, Manduca sexta. The new hormone was found with juvenile hormone I and is a 1-carbon homolog of this substance. The assigned structure is methyl (2E,6E,10-cis)-10,11-epoxy-3,7-diethyl-11-methyl-2,6-tridecadienoate, which constitutes a trishomosesquiterpenoid skeleton. This is the first chemical idetification of any juvenile hormone from insect eggs.

Identification of an allatotropin from adult manduca sexta.

A peptide that strongly stimulates the secretion of juvenile hormone from corpora allata in vitro (allatotropin) has been purified from extracts of heads of pharate adult Manduca sexta. The primary structure of this 13-residue peptide has been determined: H-Gly-Phe-Lys-Asn-Val-Glu-Met-Met-Thr-Ala-Arg-Gly-Phe-NH(2). This neurohormone has no sequence similarity with any known neuropeptide from other organisms. Synthetic allatotropin, as well as truncation fragments, including one with the five amino terminal residues deleted, showed in vitro activity indistinguishable from that of native allatotropin.

Identification of a juvenile hormone-like compound in a crustacean.

Juvenile hormone (JH) has central roles in the regulation of insect development and reproduction but has not previously been identified in other arthropod classes. The hemolymph of a crustacean, Libinia emarginata (Leach), has now been analyzed for JH-like compounds. Samples contained 0.003 to 0.030 nanogram of JH III per milliliter and 10 to 50 nanograms of methyl farnesoate per milliliter; methyl farnesoate is a compound structurally related to JH III that has JH bioactivity. Several tissues were examined for synthesis and secretion of JH-like compounds. Of these tissues, only the mandibular organs produced and secreted JH III and methyl farnesoate. However, microchemical analysis revealed that this JH III was racemic, and thus likely an artifactual oxidation product of methyl farnesoate. Secretion of methyl farnesoate was related to reproduction in females, with the highest rates observed in Libinia near the end of the ovarian cycle when oocyte growth and vitellogenesis are greatest. These results indicate that JH-like compounds such as methyl farnesoate have regulatory roles in crustaceans.

Biosynthesis of 3-hydroxy-3-methylglutaryl-CoA, 3-hydroxy-3-ethylglutaryl-CoA, mevalonate and homomevalonate by insect corpus allatum and mammalian hepatic tissues.

Both radioactively labeled 3-hydroxy-3-methyglutarylcoenzyme A and its homolog 3-hydroxy-3-ethylglutarylcoenzyme A are produced by a cytosolic fraction obtained from corpora allata-corpora cardiaca complexes of the moth Manduca sexta, incubated with [1-14C]acetylcoenzyme A plus unlabeled propionylcoenzyme A. A particulate fraction isolated from the same tissue was able to reduce [Me-3H]hydroxymethylglutaryl-CoA and [3-14C]hydroxyethylglutaryl-CoA to mevalonate and homomevalonate, respectively, when NADPH was used as the electron donor. These reactions are in keeping with the proposed homoisoprenoid biosynthetic pathway for certain of the insect juvenile hormones. Measurement of products produced in picomole levels was accomplished by their conversion to ultraviolet absorbing derivatives which were subsequently analyzed by thin-layer chromatography and liquid chromatography followed by collection and liquid scintillation counting of appropriate fractions. Similar assays conducted with cell-free preparations from corpora allata of the grasshopper Schistocerca nitens, the mealworm beetle Tenebrio molitor, and from rat liver, species in which homoisoprenoid compounds have not been detected, also resulted in formation of the normal and homologous products (although no hydroxymethylglutaryl-CoA/hydroxyethylglutaryl-CoA reductase activity could be demonstrated in T. molitor). The latter results indicate a rather loose substrate specificity of the enzymes leading to mevalonate formation.

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.

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.

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.

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.

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.

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.

Metabolism of proctolin, a pentapeptide neurotransmitter in insects.

The in vitro metabolism of [tyrosyl-3, 5-3H]proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) was studied in the following tissues from the American cockroach, Periplaneta americana: proctodeum, midgut, hemolymph, brain, terminal ganglion, and coxal depressor muscles. In all tissues assayed, the Tyr-Leu bond is the primary cleavage site, but scission of the Arg-Tyr bond is also significant. Greater than 90% of the degradative activity is found in the 100,000 X g supernatant from homogenates. In vivo studies with the tobacco hornworm, Manduca sexta, show that topically applied proctolin does not penetrate larval cuticle; proctolin is readily degraded to constituent amino acids (at least to Tyr) upon ingestion.

Detection of only JH III in several life-stages of Nauphoeta cinerea and Thermobia domestica.

We have conducted a reinvestigation into both the identification and quantification of juvenile hormone (JH) from several developmental stages of the cockroach, Nauphoeta cinerea, and the firebrat, Thermobia domestica, using a gas chromatography-mass spectrometric (GC/MS) method. We detected only JH III in these animals in contrast to prior studies in which JH I, II, and/or III had been reported using a different scheme relying on HPLC purification and subsequent GC/MS analysis under chemical ionization (CI) conditions. Very high levels (approximately 800 ng/g) of JH III were found in N. cinerea embryos at stages after dorsal closure whereas first stadium nymphs and female penultimate stadium nymphs contained only low levels (approximately 1 ng/g and approximately 7 ng/ml respectively); in adult females at the stage of rapid oocyte growth approximately 150 ng JH III per ml of hemolymph was measured. T. domestica nymphs and egg laying adults contained only low levels (approximately 1 ng/g) of JH III. The results emphasize the caution which must be used in interpreting results of procedures for analysis of JH at parts-per-billion levels, and also enforce prior observations that the higher JH homologs are not present except in the Lepidoptera.

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.

A survey of the chromatographic analysis of natural insect juvenile hormones and the insect growth regulator, altosid.

A brief survey of gas and liquid chromatographic methods for analysis, purification and quantitation of natural insect juvenile hormones and one commercially used analoh, altosid (methoprene) insect growth regulator, includes references to the most recent literature.