Ion Transport Peptide Research Articles

Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells.

Ion transport peptide (ITP) stimulates Cl(-) transport (measured as short-circuit current, I(sc)) and fluid reabsorption in Schistocerca gregaria ilea. We report that Drosophila Kc1 cells transfected with preproITP cDNA secrete a peptide (KcITP(75)) that, while cleaved correctly at the N-terminus, had reduced (10-fold) stimulatory activity on ileal I(sc) compared to both native ITP (ScgITP) and synthetic ITP (synITP). We provide evidence that the reduced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP(75)) to L-amide. In support of this, in vitro amidation of glycine extended ITP (i.e., KcITP(73) ending in LG) but not KcITP(75) (ending in LGKK) significantly increased specific activity in the bioassay. Further evidence for C-terminus involvement includes complete loss of stimulation by truncated mutants (e.g., KcITP(71) which lacks LGKK) and a mutant in which alanine is substituted for the terminal glycine in KcITP(73). Moreover a natural homologue (KcITP-L, which differs only in the C-terminal sequence) expressed by Kc1 cells does not stimulate ileal I(sc). Rather KcITP-L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). KcITP(70) has no antagonistic effect. A short synthetic peptide fragment of the C-terminus (VEIL-amide) does not stimulate ileal I(sc), indicating that other regions of ITP are also essential to biological activity. Arch.

Mutational analysis of the C‐terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells

Mutational analysis of the C‐terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells

Isolation of a cDNA encoding a CHH-family peptide from the silkworm Bombyx mori

The crustacean hyperglycemic hormone (CHH) peptide family includes four types of neuropeptide in decapod and isopod crustaceans, and the ion-transport peptide in orthopteran insects. To identify a new member of this family in Insecta, a PCR-based search for cDNAs encoding CHH-family peptides was carried out in the silkworm Bombyx mori. A cDNA, named BmCHHL ( B ombyx m ori CHH - l ike protein), with an open reading frame of 110 amino acids was isolated. Sequence analyses suggested that the conceptual protein was a precursor of a peptide of 72 amino acids which was amidated at the carboxy terminus. The BmCHHL sequence exhibited significant similarities to members of the CHH family including the orthopteran ion-transport peptide. BmCHHL expression was detected in five or six cells (per hemisphere) in the frontal area of the brain in day 4 fifth instar larvae.

Analysis of an insect neuropeptide, Schistocerca gregaria ion transport peptide (ITP), expressed in insect cell systems.

We have produced an active form of Schistocerca gregaria ion transport peptide (ITP) in an insect cell expression system. Transformed Drosophila Kc1 cells secreted a form of ITP into the cell culture medium that was proteolytically cleaved correctly at the amino (N)-terminus. Concentrated culture supernatant from transformed Kc1 and Hi5 cells had high biological activity when tested on isolated locust ilea. Conversely, ITP expressed by baculovirus-infected Sf9 cells was larger in size and had decreased specific activity compared to ITP produced by Kc1 cells due to incorrect cleavage of the peptide at the N-terminus in the baculovirus system. This demonstrates how processing of the secreted foreign protein (ITP) expressed under the late polyhedrin promoter is compromised in a baculovirus-infected cell. Transient transformation of Kc1 cells results in supernatants containing two forms of ITP; one form (A) co-elutes with synthetic ITP and the other form (B) has reduced electrophoretic mobility. In contrast, in stably transformed Kc1 cell supernatant, ITP is expressed in a single form, which has the same electrophoretic mobility and specific biological activity as form A produced by transiently transformed Kc1 cells. Arch.

Target organ specificity of major neuropeptide stimulants in locust excretory systems

The major stimulant of ileal fluid reabsorption in Locusta migratoria and Schistocerca gregaria corpora cardiaca, ion-transport peptide (ITP), had no stimulatory action on fluid secretion by isolated Malpighian tubules of S. gregaria, nor did it have a synergistic or antagonistic effect in combination with locustakinin (Lom-K) or Locusta-diuretic hormone (Locusta-DH). Stimulants of locust Malpighian tubules (Lom-K and Locusta-DH) had no action on either active transport of Cl(-) (measured as short-circuit current, I(sc)) or the rate of fluid reabsorption across S. gregaria ilea and recta in vitro. Thus, hormonal control of these major organs of the excretory system appears to be clearly separated. Lom-K and Locusta-DH acted synergistically to stimulate secretion by S. gregaria Malpighian tubules, and the diuretic response was more rapid than the response of the ileum and rectum to hindgut stimulants. Taken together, these data suggest that, in the initial phase of post-prandial diuresis, urine flow will exceed fluid uptake in the hindgut, thereby allowing excess water to be eliminated.

Recent Advances in Arthropod Endocrinology

Part I. Moulting, Metamorphosis and Reproduction: 1. Structures, functions and occurence of insect allatostatic peptides R. J. Weaver, J. P. Edwards, W. G. Bendena, and S. S. Tobe 2. Neuropeptides inhibiting growth and reproduction of crustaceans S. G. Webster 3. Molecular, cytological and physiological aspects of the crustacean hyperglycemic hormone family F. Van Herp 4. Endocrine effectors in insect vitellogenesis X. Belles 5. Endocrine regulation of development and reproduction in Acarines L. O. Lomas, and H. H. Rees 6. Ecdysteroid synthesis in the crustacean Y-organ: role of cyclic nucleotides and Ca 2+ D. Sedlmeier, and A. Seinsche 7. Regulation of steroidogenesis: role of transaldolase in crab moulting glands F. Lachaise, and G. Somme Part II. Control of Intermediary Metabolism, Ion and Water Balance: 8. New perspectives on the structures, assays and actions of locust adipokinetic hormones M. J. Lee, and G. J. Goldsworthy 9. Signal transduction of adipokinetic hormone W. J. A. Van Marrewijk, and D. J. Van der Horst 10. The regulation of primary urine production in insects G. M. Coast 11. Locust ion transport peptide (ITP): function, structure, DNA and expression J. E. Phillips, J. Meredith, N. Audsley, M. Ring, A. Macins, H. Brock, D. Theilmann, and D. Littleford Part III. Myotropic and Myoininhibitory Arthropod Neuropeptides: Structures and Functions?: 12. The dipteran Leu-callatostatins: structural and functional diversity in an insect neuroendocrine peptide family H. Duve, A. Thorpe, A. H. Johnsen, J-L. Maestro, A. G. Scott, and P. D. East 13. An insect peptide family in search of functions the tachykinin-related peptides D. R. Nassel, C. T. Lundquist, J. E. Muren, and A. S. A. Winther 14. The distribution, biological activity, and pharmacology of SchistoFLRFamide and related peptides in insects I. Orchard, and A. B. Lange 15. Ontogenetic, phylogenetic and physiological aspects of the conserved crustacean cardioactive peptide (CCAP) neural networks in arthropods H. Dirksen 16. Control of the insect oviduct: the role of the neuropeptide CCAP in the tobacco hornworm, Manduca sexta A. K. Marshall, and S. E. Reynolds Part IV. Peptidases, Peptide and Pseudopeptide Mimetics: Toward New Strategies of Insect Pest Control?: 17. Insect angiotensim-converting enzyme: comparative biochemistry and evolution R. E. Isaaac, D. Coates, T. A. Williams, and L. Schoofs 18. Mimetic analogues of the myotropic diuretic insect kinin neuropeptide family R. J. Nachman, G. M. Holman, and G. M. Coast.

Biological actions of synthetic locust ion transport peptide (ITP)

Locust Ion Transport Peptide (ITP) a member of the arthropod neuropeptide family which includes hyperglycemic, vitellogenesis-inhibiting, and moult-inhibiting hormones (CHH, VIH, MIH, respectively) was synthesized as proposed by Meredith et al. (1996)with terminal amidation of amino acid residue 72 and with 3 disulphide bridges. This is the first member of this family to be synthesized. Biological activities of synthetic ITP (synITP) were very similar to those previously reported for ITP purified from Schistocerca corpora cardiaca (ScgITP) and partially sequenced by Audsley et al. (1992a, b). Dose–response curves for both synITP and ScgITP on ileal transport of Cl − (measured as increased short-circuit current, Δ I sc), were similar with a EC 50 of 1–2 nM. The I sc time course and maximum Δ I sc across ileal epithelia at different dosages of synITP and ScgITP had similar patterns as did changes in transepithelial open-circuit potential ( V t) and resistance ( R t), reflecting changes in salt transport which drives fluid absorption. Disulphide bridges were shown to be required for biological activity of synITP, which caused the same 4-fold increase in ileal fluid transport rate ( J v) as previously reported for ScgITP. Both synITP and ScgITP caused only partial stimulation of rectal I sc and had no significant effect on rectal J v. These results indicate that the structure of ITP predicted earlier from cDNA is correct.

Locust Ion Transport Peptide (ITP): A Putative Hormone Controlling Water and Ionic Balance in Terrestrial Insects

SYNOPSIS. The anterior (ileum) and posterior (rectum) segments of the locust hindgut constitute the reabsorptive regions of the excretory system, which conserves or eliminates body water and solutes as required for osmotic homeostasis. Hindgut transport mechanisms in the desert locust have previously been well described but the neuropeptide hormones that may naturally control these processes are only now being identified. Ion Transport Peptide (ITP) has been isolated from locust corpus cardiacum (CC) and its full sequence of 72 amino acids deduced from its cDNA. Native ITP has the same actions as crude CC extracts in stimulating Cl , Na + , K + , and fluid absorption and inhibiting H + secretion (i.e., influencing pH regulation). The deduced amino acid sequence of ITP was confirmed by showing biological activity of expressed and synthetic forms of this peptide. ITP has high sequence homology with a large family of crustacean hormones that include hyperglycaemic (CHH) and moult-inhibiting hormones (MIH) and is the first member reported outside crustaceans.

Expression of Schistocerca gregaria ion transport peptide (ITP) and its homologue (ITP-l) in a baculovirus/insect cell system

We expressed an N-terminally extended Schistocerca gregaria ion transport peptide (ScgITP) and its homologue (ion transport peptide-like; ITP-L) in insect Sf9 cells using baculovirus expression vectors. Antibodies raised against peptide fragments of ITP and ITP-L were used to detect and characterize the baculovirus expressed peptides (bacITP, bacITP-L). Biological activity of the expressed peptides was assayed using the highly specific bioassay for native ITP, namely the increase in ileal short-circuit current which is a measure of active Cl − transport. BacITP and bacITP-L expression was optimal in Sf9 cells infected at a multiplicity of infection of 1, grown in Grace's medium, and harvested 2–3 days after infection. Western blots showed that bacITP was 2 kDa larger than native or synthetic ITP. This difference was not due to glycosylation and could in part be attributed to post-translational cleavage of the ITP propeptide at a site 11 amino acids upstream of the cleavage site used by S. gregaria to produce native ITP. BacITP stimulated ileal short-circuit current but is significantly less active (270–fold) than synthetic ITP (synITP) possibly as a result of the N-terminal extension. Production of bacITP-L permitted us to show that it is not stimulatory in the bioassay but reduces the synITP response in vitro and thus may have some potential for enhancing the effectiveness of biological control agents such as baculoviruses.

Actions of Ion-Transport Peptide From Locust Corpus Cardiacum on Several Hindgut Transport Processes

ABSTRACT Schistocerca gregaria ion-transport peptide (Scg-ITP), a neuropeptide isolated from locust corpora cardiaca, stimulates ileal Cl − transport (Isc) in a dose-dependent manner and causes increases in Na+, K+ (IK) and fluid reabsorption (Jv) as previously observed with crude extracts of corpus cardiacum and with cyclic AMP. Unlike cyclic AMP, Scg-ITP does not stimulate ileal NH4+ secretion. H+ secretion (JH) in the ileum, which is not affected by cyclic AMP, is almost completely abolished by Scg-ITP. Although ITP may act via cyclic AMP as second messenger to stimulate NaCl, KC1 and fluid reabsorption, it apparently acts through a different intracellular pathway to influence xJH. Scg-ITP is unlikely to be the chloride transport stimulating hormone previously reported to act on the rectum, because it did not produce a maximum rectal Isc response and had no effect on either rectal Jv (which is Cl− -depcndent) or IK.

Isolation of a neuropeptide from locust corpus cardiacum which influences ileal transport.

1. Schistocerca gregaria ion-transport peptide (Scg-ITP) was isolated from aqueous extracts of the corpus cardiacum by a four-step procedure, utilizing reverse-phase high-performance liquid chromatography for separation and stimulation of a Cl(-)-dependent short-circuit current (Isc) across locust ilea as the bioassay. 2. Scg-ITP has an unblocked N terminus and an apparent relative molecular mass of 7700. Thirty-one residues (of an estimated 65) were identified by sequence analysis. 3. Scg-ITP is structurally related to a crustacean family of neuropeptides which includes the crustacean hyperglycaemic hormones from the shore crab Carcinus maenas and the crayfish Orconectes limosus and moult-inhibiting hormone and vitellogenesis-inhibiting hormone from the lobster Homarus americanus. 4. Scg-ITP has no sequence homology with neuroparsins (Nps). Nps are the only other neuropeptides isolated to date that might regulate reabsorption in an insect hindgut (rectum).