Stimulation of neuropeptide Y (NPY) Y2 receptors induced an intracellular free Ca2+ ([Ca2+]i) increase in a human neuroblastoma cell line, CHP-234. When NPY in a Ca(2+)-free solution was applied, this increase was abolished. Depolarization with high KCl evoked no response, suggesting that the responses were not mediated by voltage-gated Ca2+ channels. There was no evidence that the NPY response consisted of a capacitative Ca2+ entry sensitive to internal Ca2+ store levels. The [Ca2+]i elevation was diminished by Ni2+, a blocker of Ca2+ entry. Mn2+ induced a quench of the fura-2 fluorescence, which ceased promptly upon the removal of NPY, indicating that Ca2+ entry was linked tightly to receptor activation. Although thapsigargin- and ryanodine-sensitive Ca2+ stores were present, NPY-induced responses were not impaired by pretreatment with either drug. Furthermore, NPY had no effect on the thapsigargin-sensitive store. Pertussis toxin did not affect the NPY-stimulated [Ca2+]i increase, although it abolished the NPY-dependent inhibition of cAMP production. It is concluded that the Y2 receptors couple directly to receptor-operated Ca2+ channels without the involvement of intracellular Ca2+ stores. The results also indicate that Y2 receptors can activate both pertussis toxin-sensitive and -insensitive mechanisms in the same cell.

Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is widely distributed in the central nervous system and periphery. Pharmacological studies have suggested that there are at least three receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been reported previously. Here we report the isolation by expression cloning of a cDNA encoding a human NPY receptor displaying a pharmacology typical of a Y2 receptor. COS-7 cells transfected with the cDNA express high affinity binding sites for NPY, peptide YY, and NPY13-36, whereas [Leu31,Pro34]NPY binds with lower affinity. The receptor is 381 amino acids in length and has seven putative transmembrane regions typical of G-protein-coupled receptors. Comparison of the amino acid sequence of this Y2 receptor to that of the human Y1 receptor indicates that the two receptors are 31% identical at the amino acid level. Northern blot analyses reveal a single 4-kilobase mRNA species and indicate that the messenger RNA is present in many areas of the central nervous system. NPY induced calcium mobilization and inhibited forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells that stably express the Y2 receptor cDNA, indicating that the recombinant Y2 receptor is functionally coupled to second messenger systems.

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiological activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system. Two major subtypes of NPY receptor (Y1 and Y2) have been defined by pharmacological criteria. We report here the molecular cloning of a cDNA sequence encoding a human NPY receptor and the corrected sequence for a rat homologue. Analysis of this sequence confirms that the receptor is a member of the G protein-coupled receptor superfamily. When expressed in Chinese hamster ovary (CHO) or human embryonic kidney (293) cells, the receptor exhibits the characteristic ligand specificity of a Y1 type of NPY receptor. In the 293 cell line, the receptor is coupled to a pertussis toxin-sensitive G protein that mediates the inhibition of cyclic AMP accumulation. In the CHO cell line, the receptor is coupled not to the inhibition of adenylate cyclase but rather to the elevation of intracellular calcium. These results demonstrate that second messenger coupling of the NPY-Y1 receptor is cell type specific, depending on the specific repertoire of G proteins and effector systems present in any cell type.

Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is widely distributed in the central nervous system and periphery. Pharmacological studies have suggested that there are at least three receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been reported previously. Here we report the isolation by expression cloning of a cDNA encoding a human NPY receptor displaying a pharmacology typical of a Y2 receptor. COS-7 cells transfected with the cDNA express high affinity binding sites for NPY, peptide YY, and NPY13-36, whereas [Leu31,Pro34]NPY binds with lower affinity. The receptor is 381 amino acids in length and has seven putative transmembrane regions typical of G-protein-coupled receptors. Comparison of the amino acid sequence of this Y2 receptor to that of the human Y1 receptor indicates that the two receptors are 31% identical at the amino acid level. Northern blot analyses reveal a single 4-kilobase mRNA species and indicate that the messenger RNA is present in many areas of the central nervous system. NPY induced calcium mobilization and inhibited forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells that stably express the Y2 receptor cDNA, indicating that the recombinant Y2 receptor is functionally coupled to second messenger systems.

OBJECTIVES: To identify genes contributing to variation in echocardiographic left ventricular mass and related traits using linkage and linkage disequilibrium analysis in sibships ascertained on hypertension. METHODS: The Hypertension Genetic Epidemiology Network (HyperGEN) Study of left ventricular hypertrophy characterized left ventricular mass, relative wall thickness (RWT), and aortic root diameter (ARD) with echocardiograms collected using a standardized protocol at four HyperGEN field centers. A high-throughput scanning fluorescence detector system genotyped 387 polymorphisms distributed throughout the genome. Linkage analyses were conducted once genotyping results became available for 885 siblings from 382 sibships. RESULTS: Although single logarithm of the odds (LOD) score peaks of 1.2 or more were found on chromosomes 1, 4, 5, 6, 7, 8, 9, 10, 12, 14, 17, and 21, we observed a broad band of peaks in both ethnic groups (white and black) on chromosome 4 and selected candidate genes (NPY1R, NPY2R, NPY5R, SFRP2, CPE, IL15, and EDNRA) from this region. Using cases and controls from extremes of the left ventricular mass index, RWT, and ARD distributions, we assessed associations with these phenotypes and haplotype-tagging single-nucleotide polymorphisms (SNPs) in the candidates. Among blacks, SNPs in IL15, NPY2R, and NPY5R showed strong evidence for association (P < 0.005); all candidates except EDNRA showed suggestive association (P < 0.05). In whites, NPY2R, NPY5R, and SFRP2 SNPs offered suggestive evidence of association with one or more traits (P < 0.05). CONCLUSION: Genetic variation in NPY1R, NPY2R, NPY5R, CPE, IL15, and SFRP2, detected using linkage analysis in hypertensive siblings, was associated with left ventricular phenotypes in blacks and/or whites.

The neuropeptide Y Y2 receptor is one of six receptor subtypes mediating the multiform physiological actions of neuropeptide Y. The Y2 receptor has been demonstrated to be the most predominant receptor subtype in the human brain and appears to be involved in many neuropeptide Y actions, such as the regulation of locomotor activity, cardiovascular functions, memory processing, circadian rhythms and release of other neurotransmitters. We have recently demonstrated the widespread and abundant distribution of neuropeptide Y Y1 receptor messenger RNA in the human cerebral cortex (different laminar patterns within distinct cortical regions), hippocampal dentate gyrus and striatum. To assess a possible differential distribution of Y1 and Y2 receptor messenger RNAs, the regional expression of neuropeptide Y Y2 messenger RNA-containing cells in the human brain was analysed, in particular within the cerebral cortex and striatum. In situ hybridization experiments revealed the localization of the Y2 messenger RNA signal throughout all cortical regions, with the highest intensity per cell apparent in lamina IV, with the exception of the striate cortex, which showed an intense labelling primarily in layer VI. The striatum expressed low to undetectable levels of the Y2 receptor messenger RNA. The dentate gyrus and the CA2 region presented the highest hybridization signals, while a very weak Y2 messenger RNA expression was found in the CA1 region and subiculum. Positive Y2 messenger RNA hybridization signals were also detected in the lateral geniculate nucleus, amygdala, substantia nigra, hypothalamus, cerebellum and choroid plexus. These results demonstrate the widespread distribution of neuropeptide Y Y2 receptor messenger RNA in the human brain, with a pattern of expression distinct from the Y1 subtype, suggesting that these two receptor subtypes may mediate different neuropeptide Y functions in the human brain, mainly through actions on different neuronal systems.

OBJECTIVES: To identify genes contributing to variation in echocardiographic left ventricular mass and related traits using linkage and linkage disequilibrium analysis in sibships ascertained on hypertension. METHODS: The Hypertension Genetic Epidemiology Network (HyperGEN) Study of left ventricular hypertrophy characterized left ventricular mass, relative wall thickness (RWT), and aortic root diameter (ARD) with echocardiograms collected using a standardized protocol at four HyperGEN field centers. A high-throughput scanning fluorescence detector system genotyped 387 polymorphisms distributed throughout the genome. Linkage analyses were conducted once genotyping results became available for 885 siblings from 382 sibships. RESULTS: Although single logarithm of the odds (LOD) score peaks of 1.2 or more were found on chromosomes 1, 4, 5, 6, 7, 8, 9, 10, 12, 14, 17, and 21, we observed a broad band of peaks in both ethnic groups (white and black) on chromosome 4 and selected candidate genes (NPY1R, NPY2R, NPY5R, SFRP2, CPE, IL15, and EDNRA) from this region. Using cases and controls from extremes of the left ventricular mass index, RWT, and ARD distributions, we assessed associations with these phenotypes and haplotype-tagging single-nucleotide polymorphisms (SNPs) in the candidates. Among blacks, SNPs in IL15, NPY2R, and NPY5R showed strong evidence for association (P < 0.005); all candidates except EDNRA showed suggestive association (P < 0.05). In whites, NPY2R, NPY5R, and SFRP2 SNPs offered suggestive evidence of association with one or more traits (P < 0.05). CONCLUSION: Genetic variation in NPY1R, NPY2R, NPY5R, CPE, IL15, and SFRP2, detected using linkage analysis in hypertensive siblings, was associated with left ventricular phenotypes in blacks and/or whites.