Neuropeptide Y (3-36) (human, rat)

Recently, using molecular cloning approaches, three new neuropeptide Y (NPY)/peptide YY (PYY) receptors have been described in rodent brain, with pharmacological profiles that differ from the three previously described Y1, Y2 and Y3 NPY receptors and the Y4 pancreatic polypeptide- (PP-) preferring receptor. Two of these new receptors are spice variants and are called Y5 receptors, whilst a third receptor has been called Y6 and has been suggested to be expressed only in the mouse. In the absence of a totally selective Y5 and/or Y6 radioligands, we have examined [125I]PYY(3-36) binding, which binds Y2 and Y5/Y6 receptors, using homogenate assays and quantitative receptor autoradiography to study the distribution of the three newly discovered Y5/Y6 receptors by masking binding to Y1 receptors with high concentrations of the non-peptidergic selective Y1 antagonist, BIBP3226, and using either [Leu31,Pro34]NPY or human PP to mask binding to Y5 and Y6 receptors, leaving binding to Y2 receptors. Using this approach, [125I]PYY(3-36) labels a small population of Y1 receptors and a larger population of binding sites that are insensitive to BIBP3226, human PP and [Leu31,Pro34]NPY, presumed to be Y2 receptors. There was also [125I]PYY(3-36) binding to sites sensitive to NPY, human PP and [Leu31,Pro34]NPY, but insensitive to BIBP3226, located in the hypothalamus, amygdala, hippocampus and thalamus. As one of the recently cloned Y5 receptors is synthesized in these regions, as shown by in-situ hybridization techniques, we suggest that the small population of [125I]PYY(3-36) binding sites which are sensitive to human PP and [Leu31,Pro34]NPY, but insensitive to BIBP3226, may represent binding to Y5 receptors. We have been unable, however, to visualize a smaller population of Y6 receptors which are labelled by [125I]PYY3-36 and sensitive to [Leu31,Pro34]NPY, but not to BIBP3226 and human PP, confirming that the murine Y6 receptor does not appear to be expressed in rat brain.

Leptin may act as a negative feedback signal to the brain in the control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine- and amphetamine-regulated transcript (CART), a new anorectic peptide. We aimed at studying whether leptin, NPY, and CART have related effects on the hypothalamic control of the pituitary-gonadal system and the developmental changes in NPY and CART effects. Using retrochiasmatic hypothalamic explants from prepubertal 15-day-old male rats, the GnRH interpulse interval (mean +/- SD: 62 +/- 5 min) was significantly reduced by 10(-7) M of leptin (46 +/- 3.3 min) as well as 10(-7) M of NPY (47 +/- 4.4 min) and 10(-6) M of CART (46 +/- 2.7 min), whereas the GnRH pulse amplitude was not affected. The stimulatory effects of different NPY receptor agonists [human PYY 3-36, porcine NPY 13-36, human (D-Trp 32) NPY, porcine (Leu 31 Pro 34) NPY, human pancreatic polypeptide (PP)], as well as the absent effects of rat PP were consistent with the involvement of the Y5-receptor subtype in mediation of NPY effects. Incubation with 10(-7) M of a Y5-receptor selective antagonist prevented the effect of NPY (61 +/- 4 vs. 46 +/- 2 min), whereas leptin and CART effects were not (47 +/- 3 vs. 46 +/- 3 min and 46 +/- 3 vs. 46 +/- 2 min, respectively), suggesting that NPY was not involved in leptin and CART effects. Using an anti-CART antiserum (1:1000), the reduction of GnRH interpulse interval caused by leptin was partially prevented (56.2 +/- 4 vs. 47.9 +/- 3.8 min), whereas the reduction of GnRH interval caused by NPY was not affected (45.9 +/-2.5 vs. 47.8 +/- 3.7). The GnRH interpulse interval was decreased by 10(-7) M of NPY at 5 days (72 +/- 3.8 vs. 91.9 +/- 3.5) as well as at 15 days, whereas such an effect was not observed anymore at 25 and 50 days. Similar effects were observed using 10(-6) M of CART-peptide. Using 10(-6) M of the Y5-receptor antagonist, the GnRH interpulse interval was significantly increased at 15 days (66.6 +/- 2.7 min), 25 days (56.5 +/- 39.9 min), and 50 days (52.5 vs. 38.2 min), whereas no change was observed at 5 days. Using the anti-CART antiserum, a significant increase of GnRH interpulse interval was observed at 25 days only. In conclusion, the stimulatory effects of leptin and NPY on the frequency of pulsatile GnRH secretion before puberty involve two distinct mechanisms. NPY causes acceleration of GnRH pulsatility via the Y5-receptor subtype, which is not involved in leptin effects while the CART is involved in leptin effects on GnRH secretion but not in NPY effects. The reduction of pulsatility by the Y5 antagonist provides evidence of endogenous NPY involvement in the control of GnRH secretion from the time of onset of puberty.

Implants of diethylstilbestrol inducing anterior pituitary prolactinomas in female Fischer-344 rats produced a considerable elevation of high-affinity binding of either rat or human pancreatic polypeptide (hPP). No comparable upregulation of high-affinity binding sites was noted for the ligand [125I](Leu31,Pro34)hPYY (LP-PYY) (masked by 2 nM hPP to force selectivity for the Y1 sites), or of the Y2-selective ligand [125I]hPYY(3-36). The Y5-like sites displayed the rank order of potency of hPP = rPP, hNPY, LP-PYY > pPYY(1-36) > hNPY(2-36) > hPYY(3-36) >> aPP, similar to the previously described rabbit kidney or hypothalamic Y5-like receptors. The PP binding in the anterior pituitary was not sensitive to the Y1-selective non-peptidic antagonist BIBP-3226. The PP binding was highly sensitive to alkali metal cations, and to a N5-substituted amiloride antagonist of sodium transport, but not to a C2-guanido substituted amiloride antagonist of sodium channels. The binding was also sensitive to phospholipase C antagonist U-73122, and to alkylating alpha-adrenergic agonist chloroethylclonidine. Lactotrophs isolated in Percoll gradients after enzymic dispersion of the anterior pituitary gland retained the high-affinity PP binding. Following removal of estrogen implants, the hPP binding sites decreased to very low levels within 3 days, in parallel to the decrease of plasma prolactin.