Abstract

In this study, for the first time, both neuropeptides isotocin (IT) and arginine vasotocin (AVT) have been identified and measured in urophysis, the neurohaemal organ of the caudal neurosecretory system of teleost fish. So far, AVT, but not IT, was quantified by radioimmunoassay (RIA) in urophysis of several fish species. We have used high-performance liquid chromatographic assay with fluorescence detection (HPLC-FL) preceded by solid-phase extraction (SPE) and liquid chromatography-electrospray ionization triple-quadrupole tandem mass spectrometry (LC-ESI MS/MS) technique to determine both neuropeptides in urophysis of three fish species. The efficiency of peptide’s SPE extraction was 79–85 %. In HPLC-FL method, the limits of detection (LOD) and quantification (LOQ) were estimated as 1.0 and 3.4 pmol/mL for IT and 0.25 and 2.20 pmol/mL for AVT. In LC–MS/MS method, LOD and LOQ were estimated as 0.4 and 1.2 pmol/mL for IT and 0.06 and 0.2 pmol/mL for AVT. The chromatographic methods are good alternative for RIA, because enable to measure both nonapeptides simultaneously in one sample. In round goby (Neogobius melanostomus), three-spined stickleback (Gasterosteus aculeatus) and sea bream (Sparus aurata), urophysial IT concentrations ranged between 0.056 and 0.678 pmol/mg tissue and AVT concentrations ranged between 0.0008 (or even below detection threshold) and 0.084 pmol/mg tissue.

Highlights

  • In teleostean fish, neurones localized in the parvocellular and magnocellular nuclei of the preoptic area, synthesize nonapeptides, isotocin (IT) and arginine vasotocin (AVT)

  • We have used high-performance liquid chromatographic assay with fluorescence detection (HPLC-FL) preceded by solid-phase extraction (SPE) and liquid chromatography-electrospray ionization triple-quadrupole tandem mass spectrometry (LC-ESI MS/MS) technique to determine both neuropeptides in urophysis of three fish species

  • It is well known that urophysis is a site of storage and release of peptides, that is, urotensin I (UI), urotensin II (UII), corticotropin-releasing factor (CRF) and parathyroid hormone-related protein (PTHrP) (Pearson et al 1980; Lederis et al 1982; Ingleton et al 2002), which are synthesized by Dahlgren cells

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Summary

Introduction

Neurones localized in the parvocellular and magnocellular nuclei of the preoptic area, synthesize nonapeptides, isotocin (IT) and arginine vasotocin (AVT). Neurones project both to the neurohypophysis, where neuropeptides are released to the circulation, and to multiple regions of the brain (Holmqvist and Ekstrom 1995; Saito et al 2004). The CNSS consists of large magnocellular neurons—Dahlgren cells—located in the terminal vertebral segments of the spinal cord, which project axons to the urophysis, a neurohaemal organ in teleost fishes (Fridberg and Bern 1968). The benefit of using the chromatographic methods is that both IT and AVT are quantified simultaneously in the same sample

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