Jc Yeats

Radioimmunoassay of neuropeptide Y

The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences.

Intramural distribution of regulatory peptides in the sigmoid-recto-anal-region of the human gut

The distribution of regulatory peptides was studied in the separated mucosa, submucosa and muscularis externa taken at 10 sampling sites encompassing the whole human sigmoid colon (five sites), rectum (two sites), and anal canal (three sites). Consistently high concentrations of VIP were measured in the muscle layer at most sites (proximal sigmoid: 286 (16) pmol/g, upper rectum: 269 (17), a moderate decrease being found in the distal smooth sphincter (151 (30) pmol/g). Values are expressed as mean (SE). Conversely, substance P concentrations showed an obvious decline in the recto-anal muscle (mid sigmoid: 19 (2.0) pmol/g, distal rectum: 7.1 (1.3), upper anal canal: 1.6 (0.6)). Somatostatin was mainly present in the sigmoid mucosa and submucosa (37 (9.3) and 15 (3.5) pmol/g, respectively) and showed low, but consistent concentrations in the muscle (mid sigmoid: 2.2 (0.7) pmol/g, upper anal canal: 1.5 (0.8]. Starting in the distal sigmoid colon, a distinct peak of tissue NPY was revealed, which was most striking in the muscle (of mid sigmoid: 16 (3.9) pmol/g, upper rectum: 47 (7.8), anal sphincter: 58 (14)). Peptide YY was confined to the mucosa and showed an earlier peak (upper sigmoid: 709 (186) pmol/g, mid-distal sigmoid: 1965 (484)). A clear differential distribution of regulatory peptides was thus shown in the region studied. A possible role is suggested for NPY and VIP containing nerves in the effector control of the human internal anal sphincter.

Effect of reserpine, phenoxybenzamine and cold stress on the neuropeptide Y content of the rat peripheral nervous system

The effect of reserpine treatment on the neuropeptide Y content of the rat adrenal gland, heart, kidney and vasculature was studied using a specific radioimmunoassay. One hour after reserpine administration (5 mg/kg) the neuropeptide Y concentration in the adrenal gland was significantly reduced and after 4 h a similar reduction was seen in the heart and kidney. After 48 h, neuropeptide Y concentrations were reduced in all tissues. The greatest reduction occurred in the cardiac septum (77%) and the least in the inferior vena cava (25%). Phenoxybenzamine (2 mg/kg) also caused a reduction in neuropeptide Y concentrations which was less marked than after reserpine, except in the adrenal gland where it was similar. Cold stress caused no change in neuropeptide Y concentrations. The neuropeptide Y depletion induced by reserpine was compared to that following 6-hydroxydopamine. In the heart and pial arteries both drugs caused a similar neuropeptide Y depletion whilst in the pineal gland and renal artery 6-hydroxydopamine had more effect than reserpine. The implications of these results on NPY storage sites are discussed.

Effect of 6-hydroxydopamine on neuropeptides in the rat female genitourinary tract

The occurrence and distribution of neuropeptide Y has been determined in the rat female genitourinary tract by radioimmunoassay and chromatographic analysis. Within the bladder, higher concentrations of neuropeptide Y were found in the trigone (48.8 +/- 5.2 pmol/g) than in the dome (36.0 +/- 2.1 pmol/g). In the genital tract, highest concentrations were identified in the vagina (41.4 +/- 2.1 pmol/g). Treatment of rats with 6-hydroxydopamine resulted in significant depletion of neuropeptide Y concentrations in both parts of the bladder, together with vagina, uterine horn and fallopian tube. No change was observed in the cervix, uterine body and ovary. Concentrations of vasoactive intestinal polypeptide were unaffected by treatment with 6-hydroxydopamine except in the area of the cervix where concentrations rose from 64.1 +/- 5.7 pmol/g to 133.6 +/- 15.1 pmol/g (p less than 0.05). There was a generalised, but statistically insignificant rise in substance P concentrations.

Neuropeptide Y in neuroblastoma × glioma hybrid cells: Response to dexamethasone and nerve growth factor

High concentrations of a newly-identified biologically potent peptide, neuropeptide Y, have been demonstrated in 3 related mouse neuroblastoma-derived clonal cell lines, N18TG2 0.35 pmol/mg protein, NG108-15 0.44 pmol/mg protein and NCB-20 0.39 pmol/mg protein. The NG108-15 cell line was chosen for further evaluation. Dexamethasone (10 microM) and nerve growth factor (10 ng/ml) resulted in a 2-fold increase in cellular neuropeptide Y concentrations. The response to dexamethasone was demonstrated to be dose-dependent. Exposure to both agents in combination resulted in a more than additive effect, indicating synergism.High concentrations of a newly‐identified biologically potent peptide, neuropeptide Y, have been demonstrated in 3 related mouse neuroblastoma‐derived clonal cell lines, N18TG2 0.35 pmol/mg protein, NG108‐15 0.44 pmol/mg protein and NCB‐20 0.39 pmol/mg protein. The NG108‐15 cell line was chosen for further evaluation. Dexamethasone (10 μM) and nerve growth factor (10 ng/ml) resulted in a 2‐fold increase in cellular neuropeptide Y concentrations. The response to dexamethasone was demonstrated to be dose‐dependent. Exposure to both agents in combination resulted in a more than additive effect, indicating synergism.