Isolation of two arginine vasopressin‐like factors from ganglia of Locusta migratoria

Abstract

Tissues of Locusta migratoria are known to contain a material which crossreacts with an antibody against arginine vasopressin (AVP), and this factor has been correlated with the diuretic hormone of this species. In this paper, we report the isolation of two AVP-like factors from suboesophageal ganglia and thoracic ganglia of Locusta migratoria. The less abundant, more hydrophobic of these AVP-like factors shows diuretic activity in an assay where excretion of amaranth dye from Locusta migratoria hemolymph is used as the scoring criterion. After extracting a total of -51,000 ganglia with an acidic solvent, the crude extract was prepurified by batch adsorption/ elution from disposable reversed-phase cartridges. The prepurified extract was then sequentially purified by reversed-phase liquid chromatography using solvent programs of substantially differing selectivity. The more abundant factor was isolated to apparent homogeneity in three steps, while the less abundant factor required four or five steps. Use of a C, reversedphase column minimized losses of the minor, more hydrophobic factor. Key words: diuretic hormone, reversed-phase liquid chromatography, migratory locust INTRODUCTIONA number of researchers have reported the existence of a material in tissues of several insect species, including Locusfa rnigratoria, which crossreacts with antibodies that recognize AW*. Proux and Rougon-Rapuzzi have *Abbreviations: AVP = arginine vasopressin; BSA = bovine serum albumin; F1 = AVP-like factor 1; F2 = AVP-like factor 2; HFBA = heptafluorobutyric acid; LC = liquid chromatography; I-PrOH = I-propanol; RIA = radioimmunoassay; SOG = suboesophogeal ganglion; TFA = trifluoroacetic acid; TG = thoracic ganglion.Acknowledgments: We thank Dr. Richard R. Randall for preliminary studies on the separation of F1 and F2 using the DuPont PEP R P l l column, Bernadette Proux for her patience and understanding while one of us 0.P.P.) spent 5% months in California, the members of the CNRS Neuroendocrinology Laboratory who helped collect the 51,000 ganglia, Prof. John E. Phillips (Univ. of British Columbia) for supplying specimens of Schistocerca gregaria, and Prof. M. Delaage (Immunotech, Marseille, France) who provided the AVP antibodies.

Malpighian tubules of larvalAedes aegypti are hormonally stimulated by 5-hydroxytryptamine in response to increased salinity

“…An important aspect of this homeostasis is the regulation of rates of primary urine formation by the Malpighian tubules. There is an extensive literature concerning factors which affect fluid secretion rates of insect Malpighian tubules in vitro, including peptides (e.g., Blackburn et al, 1991;Hayes et al, 1989;Holman et al, 1987;Kataoka et al, 1989;Kay et al, 1991;Lehmberg et al, 1991;Schooley et al, 1987), 5-hydroxytryptamine (5-HT*) (Maddrell et al, 1991), and ionic concentrations within the hemolymph (Maddrell, 1969).…”

Isolation and identification of a diuretic hormone from the tobacco hornworm, Manduca sexta

“…To date, isolation of two insect DHs, both from Locusta migratoria, has been reported (13,14 Several species of butterflies exhibit significant diuresis soon after adult eclosion. This phenomenon allowed development of a facile in vivo DH assay (16).…”

“…By using in vitro Mt assays, substances with diuretic activity have been found in ganglia from the head, thorax, and abdomen, and in a glandular tissue, the corpora cardiaca (CC) (4-8). Partial purifications of DH from several species indicate that there may be several DHs which differ in size and possibly in modes of action (3,5,(8)(9)(10)(11)(12).To date, isolation of two insect DHs, both from Locusta migratoria, has been reported (13,14 Several species of butterflies exhibit significant diuresis soon after adult eclosion. This phenomenon allowed development of a facile in vivo DH assay (16).…”

“…To date, isolation of two insect DHs, both from Locusta migratoria, has been reported (13,14). One of these was isolated from subesophageal and thoracic ganglia, using its immunological cross-reactivity with an antibody to [Arg8]-vasopressin (AVP) as an assay (14).…”

“…One of these was isolated from subesophageal and thoracic ganglia, using its immunological cross-reactivity with an antibody to [Arg8]-vasopressin (AVP) as an assay (14). This DH was identified as an antiparallel dimer of [Leu2,Thr4,Arg8]vasotocin (15), and it occurs in tissue with the corresponding monomer, which has no diuretic activity (15).…”