Studies on rat gastrointestinal neuropeptide Y
Creators: name: Dadgar, Anoushiravan
Publisher: name: University of British Columbia
Year: 2010

description: A sensitive and specific radioimmunoassay (RIA) for Neuropeptide Y (NPY) was developed and quantitation, characterization and release studies were performed. The development of the RIA required the purification of the NPY tracer due to both multiple iodinated products resulting from the five tyrosine residues in its amino acid sequence, and the presence of unlabelled NPY. Ion-exchange and reverse phase high performance chromatography (HPLC) purification of ¹²⁵I-NPY were performed. Optimal purification of ¹²⁵I-NPY was achieved using a HPLC with a μBondapak C₈ column and a 45-50% acetonitrile concentration gradient. A polyethylene glycol separation technique was used in conjunction with the HPLC purified tracer to improve assay conditions. Although many studies aimed at elucidating the actions of NPY have been performed, little information is available on the distribution of gastrointestinal (GI) NPY in the rat. Therefore the NPY-immunoreactivity (IR) in extracts of the various regions of the GI tract were determined using the developed RIA. The tissue content of NPY was found to be highest in the various segments of the rat stomach, with a decreasing trend in NPY-IR down the GI tract until the level of the ascending colon where an increase was detected. Characterization studies on the tissue extracts were performed using gel filtration chromatography and HPLC. One immunoreactive species was detected in the corpus and ileum extracts using gel filtration chromatography, and in the corpus and colon extracts using HPLC. This immunoreactive species eluted in a position similar to synthetic porcine NPY and later than peptide YY (PYY). In the physiological investigation of the the role of neuropeptides in the regulation of GI functions, release studies are crucial. The presence of high levels of NPY-IR in the stomach allowed the investigation of the release mechanisms of NPY in the perfused isolated rat stomach. However, due to the low basal secreted levels of NPY in comparison with other gastric peptides, as well as the enzymatic degradation and/or peptide uptake that occurs in the stomach vasculature, certain steps had to be taken to allow for the detection of the endogenously released peptide. Sep Pak extraction was found to be required to concentrate the endogenously released peptide. Proteolytic inhibitors were also added to the perfusate to reduce enzymatic degradation. A low basal level of NPY was detected which ranged from 98 to 147 fmole/min. Neuropeptide Y was found to be released into the gastric vasculature in response to high potassium depolarization. A few studies have been performed on cholinergic effects on NPY release, however no direct release studies have been performed on NPY-containing neurones innervating the stomach. Therefore, the actions of cholinergic agonists and antagonists on NPY secretion in the isolated perfused rat stomach were investigated. Acetylcholine and the nicotinic ganglionic agonist dimethyl-phenyl-piperazinium (DMPP) stimulated NPY secretion. The acetylcholine-stimulated secretion was not blocked by the cholinergic muscarinic antagonist atropine and was partially blocked by the ganglionic cholinergic antagonist hexamethonium. The effects of α- and β-adrenergic agonists and antagonists on NPY secretion into the stomach vasculature were investigated in order to elucidate possible adrenergic release mechanisms. There were conflicting results on the α-adrenergic release of NPY. Both the α-adrenergic antagonist phentolamine and the agonist phenylephrine had a stimulatory effect on NPY secretion in the stomach. The β -adrenergic agonist isoproterenol had a stimulatory effect on NPY release. The β -adrenergic antagonist propranolol caused an initial small increase in mean NPY levels followed by a decrease, but this was not found to be statistically significant. These studies demonstrated the presence of NPY in the GI tract of the rat with the highest content being in the rat stomach. There are cholinergic stimulatory mechanisms involved in the secretion of NPY which are partially ganglionically mediated. The results did not conclusively demonstrate as to whether there is an α-adrenergic stimulatory or inhibitory action on NPY-containing neurones in the gut, however preliminary release studies suggest there is a β-adrenergic stimulatory mechanism involved in NPY secretion.