[Leu31,Pro34]-Neuropeptide Y (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.

Quantitative receptor autoradiography was performed on sections of rat and human brain using [125I]peptide YY ([125I]PYY) to measure the anatomical distribution of neuropeptide Y (NPY) receptors. Masking Y1- and Y2-NPY subtypes with the agonists (Leu31,Pro34]NPY and NPY13-36, respectively demonstrated a differential distribution of Y1 and Y2 receptors between human and rat brain. In human brain, the highest density of [125I]PYY binding was found in pyramidal layers (CA4-CA1) of hippocampus, head and tail regions of caudate nucleus, locus coeruleus and substantia nigra. There was moderate [125I]peptide YY binding to NPY receptors in the molecular layers of the hippocampus, frontal and temporal cerebral cortex, especially in superficial layers, anterior amygdala, central grey and inferior colliculus. Low levels of binding were observed in white matter. The selective Y1 receptor agonist, [Leu31,Pro34]NPY did not effectively reduce [125I]PYY binding to any human brain region examined except for approximately 20-40% of the binding sites in the molecular layer of the dentate gyrus, layer IV of the frontal cortex and the radiatum and oriens layers of the hippocampal complex. In contrast, the Y2 agonist, NPY13-36 was effective at reducing [125I]PYY binding in all human brain regions examined. In rat brain, high densities of [125I]PYY binding was measured in cerebral cortex, thalamus and inferior colliculus which was sensitive to [Leu31,Pro34]NPY. In contrast, high densities of the NPY13-36 sensitive binding was found in the hippocampus, striatum and nucleus accumbens. Medium to low densities of NPY13-36 sensitive binding was found in medulla and pons. This data suggests that human brain contains primarily Y2-type NPY receptors with only a few regions expressing Y1-type receptors. No human brain region examined contained solely Y1-type receptors. In contrast to human brain, rat brain contains regions which express only Y1 receptors as well as regions containing only Y2 receptors and regions containing both Y1 and Y2 receptors.

Two types of binding sites have previously been described for 36-amino acid neuropeptide Y (NPY), called Y1 and Y2 receptors. Y2 receptors can bind long C-terminal fragments of NPY-e.g., NPY-(13-36)-peptide. In contrast, Y1 receptors have until now only been characterized as NPY receptors that do not bind such fragments. In the present study an NPY analog is presented, [Leu31, Pro34]NPY, which in a series of human neuroblastoma cell lines and on rat PC-12 cells can displace radiolabeled NPY only from cells that express Y1 receptors and not from those expressing Y2 receptors. The radiolabeled analog, [125I-Tyr36] monoiodo-[Leu31, Pro34]NPY, also binds specifically only to cells with Y1 receptors. The binding of this analog to Y1 receptors on human neuroblastoma cells is associated with a transient increase in cytoplasmic free calcium concentrations similar to the response observed with NPY. [Leu31, Pro34]NPY is also active in vivo as it is even more potent than NPY in increasing blood pressure in anesthetized rats. It is concluded that [Leu31, Pro34]NPY is a specific Y1 receptor agonist and that the analog or variants of it can be useful in delineating the physiological importance of Y1 receptors.