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Purification of a neuropeptide from the corpus cardiacum of the desert locust which influences ileal transport

University of British Columbia

Previous studies on the regulation of salt and water reabsorption in the insect excretory system have concentrated on the rectum, while regulation of the ileum has received little attention. Cl⁻ transport is the predominant ion transport process in both the ileum and rectum of the desert locust and drives fluid absorption. The central nervous system (CNS) was surveyed for factors which stimulate Cl⁻-dependent short-circuit current (I[formula omitted]) using in vitro flat sheet preparations of locust ileum as a bioassay. All ganglia extracts tested (except the corpora allata) caused significant increases in ileal I[formula omitted]. Extracts of muscle tissue, used as a control, had no effect on ileal I[formula omitted] indicating that stimulants were not general metabolites present in locust tissue. Crude extracts of the corpus cardiacum (CC) and fifth ventral ganglion (VG5) stimulated ileal I[formula omitted] in a dose-dependent manner and both caused an increase in K⁺ and Na⁺ absorption as previously observed with cAMP. CC and VG5 had no effect on ileal NH₄⁺ secretion but both inhibited ileal H⁺ secretion. Most of the stimulatory effects of CC and VG were largely abolished by treatment with trypsin and chymotrypsin, suggesting that the stimulants were peptides. CC and VG5 factors were apparently separate compounds because they differed in the time courses of ileal I[formula omitted] response, thermal stability, and extraction properties. Reversed-phase high performance liquid chromatography (RP-HPLC) of water extracts of CC identified two distinct factors (fractions D and F) which stimulated ileal I[formula omitted] and a third factor (fraction G) which had little effect on I[formula omitted], but which caused a five-fold increase in ileal fluid transport (J[formula omitted]). None of these fractions increased rectal J[formula omitted]; moreover, fraction D stimulated rectal I[formula omitted] at higher doses. These results provided the first indication that separate stimulants act on locust rectum and ileum. The most potent factor in CC acting on ileal I[formula omitted] was isolated using a four-step purification procedure, utilizing C₈ and phenyl RP-columns for separation. Amino acid analysis of this purified peptide indicated a molecular weight of 7700 daltons and a near complete amino acid sequence (50 out of 65) was determined. The purified factor (S. gregaria ion transport peptide; ScgITP) was assayed on all ileal ion transport processes influenced by crude CC extracts. ScgITP caused quantitatively the same range of effects as crude CC extracts, in that it stimulated Cl⁻, K⁺, and Na⁺ reabsorption and inhibited H⁺ secretion. High doses of ScgITP (5 CC equiv.ml⁻¹) caused the same maximum response on all these systems as crude CC extracts (0.25 CC equiv.ml⁻¹). ScglTP is unlikely to be chloride transport stimulating hormone, previously reported to act on the rectum, because a maximum rectal I[formula omitted] response was not achieved and there was no effect on rectal J[formula omitted], which is Cl⁻-dependent. It appears that ScgITP acts through cAMP as the second messenger to stimulate reabsorptive processes because this cyclic nucleotide mimicked the actions of ScgITP and crude CC extracts. In support of this view, ileal I[formula omitted] was also stimulated to maximum levels by 5mM theophylline and 50μM forskolin. The inhibition of H⁺ secretion by ScgITP must occur through a different intracellular pathway because this action was not mimicked by cAMP.

Text

2011-01-25

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http://hdl.handle.net/2429/30818

Zoology

University of British Columbia

Graduate