Identification of a novel neuromedin U receptor subtype expressed in the central nervous system.

Neuromedin U is a neuropeptide prominently expressed in the upper gastrointestinal tract and central nervous system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific receptor for neuromedin U. A BLAST search of the GenBank(TM) genomic database using the NmU-R1 cDNA sequence revealed a human genomic fragment encoding a G protein-coupled receptor that we designated NmU-R2 based on its homology to NmU-R1. The full-length NmU-R2 cDNA was subsequently cloned, stably expressed in 293 cells, and shown to mobilize intracellular calcium in response to neuromedin U. This response was dose-dependent (EC(50) = 5 nm) and specific in that other neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to be most highly expressed in central nervous system tissues. Based on these data, we conclude that NmU-R2 is a novel neuromedin U receptor subtype that is likely to mediate central nervous system-specific neuromedin U effects.

Expression of a novel neuropeptide Y receptor subtype involved in food intake: an in situ hybridization study of Y5 mRNA distribution in rat brain.

Our group has reported on the cloning of a novel rat neuropeptide Y (NPY) receptor involved in NPY-induced food intake, the Y5 receptor. The distribution in rat brain of the mRNA encoding this receptor has been determined by in situ hybridization histochemistry, using radiolabeled oligonucleotide probes. Control experiments were carried out in cell lines transfected with either rat Y1 or rat Y5 cDNAs. With the exception of the cerebellum, only the antisense probes yielded hybridization signal in rat brain tissue sections. A number of brain regions contained hybridization signals indicative of Y5 mRNA localization. Chief among these were various hypothalamic nuclei, including the medial preoptic nucleus, the supraoptic nucleus, the paraventricular nucleus, and the lateral hypothalamus. Other regions with substantial hybridization signals included the midline thalamus, parts of the amygdala and hippocampus, and some midbrain and brain-stem nuclei. In general a low density of Y5 mRNA was observed in most cortical structures, with the exception of the cingulate and retrosplenial cortices, each of which contained a moderate abundance of Y5 hybridization signal. The distribution of this receptor mRNA is consistent with a role for the Y5 receptor in food intake and also suggests involvement in other processes mediated by NPY.

A receptor subtype involved in neuropeptide-Y-induced food intake.

Neuropeptide Y (NPY) is a powerful stimulant of food intake and is proposed to activate a hypothalamic 'feeding' receptor distinct from previously cloned Y-type receptors. This receptor was first suggested to explain a feeding response to NPY and related peptides, including NPY2-36, that differed from their activities at the Y1 receptor. Here we report the expression cloning of a novel Y-type receptor from rat hypothalamus, which we name Y5. The complementary DNA encodes a 456-amino-acid protein with less than 35% overall identity to known Y-type receptors. The messenger RNA is found primarily in the central nervous system, including the paraventricular nucleus of the hypothalamus. The extent to which selected peptides can inhibit adenylate cyclase through the Y5 receptor and stimulate food intake in rats correspond well. Our data support the idea that the Y5 receptor is the postulated 'feeding' receptor, and may provide a new method for the study and treatment of obesity and eating disorders.

Distribution of phosphatase inhibitor-1-immunoreactive neurons in the suprachiasmatic nucleus of the Syrian hamster.

The protein phosphatase inhibitor-1 (I-1) is phosphorylated by a cyclic AMP-dependent protein kinase, and is itself involved in the regulation of phosphorylation of other proteins. The enzyme has been shown to be present in skeletal muscles and in distinct neuronal systems of the brain. The suprachiasmatic nucleus is essential in generation of circadian rhythms, but the cellular mechanisms by which the oscillator is entrained are not understood. Since cyclic AMP is known to phase shift the rhythm of electrical activity in SCN neurons in vitro, we aimed by an avidin-biotin immunohistochemical technique to localize I-1-containing neurons in the hamster suprachiasmatic nucleus and thereby identify potential target neurons for cyclic AMP effects. Numerous densely stained neurons were observed in the hamster SCN. The I-1-immunoreactive cell bodies were intermingled with non-immunoreactive neurons and occupied mostly the ventral half of the nucleus, but cell bodies were found in all compartments of the nucleus. The I-1-immunoreactive neurons located in the ventral SCN sent dendrite-like processes into the underlying optic chiasm, indicating that they are directly innervated from the retina, the intergeniculate leaflet of the thalamus, and/or the dorsal raphe. A few I-1-immunoreactive neurons were observed immediately outside the borders of the SCN, but their pronounced staining intensity and their similar morphology to those found inside the SCN indicate that they belong to the same type of neurons as found in the SCN. Delicate I-1-immunoreactive nerve fibers possessing boutons were found throughout the SCN. Furthermore, axonal fibers were followed dorsally into the subparaventricular area.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of phosphatase inhibitor-1 in rat brain.

The localization of phosphatase inhibitor-1 was investigated in rat brain by use of immunocytochemistry. Studies were performed with an affinity purified IgG raised against purified rabbit skeletal muscle inhibitor-1. In rat brain tissue homogenates, this antibody reacted only with a 29 kDa protein corresponding to inhibitor-1. Immunocytochemical studies with this antibody revealed numerous immunoreactive cell bodies and fibers. The highest concentration of immunoreactive perikarya was observed in the caudate-putamen and nucleus accumbens, and these appeared to be exclusively medium-sized neurons. Other areas containing substantial populations of immunoreactive neurons included the suprachiasmatic nucleus of the hypothalamus, lateral hypothalamus, horizontal limb of the diagonal band of Broca, dentate gyrus of the hippocampal formation, habenula, superior colliculus, claustrum, endopiriform nuclei, and neocortex. The distribution of terminals containing inhibitor-1 coincided with the distribution of terminal fields known to originate from the above regions. Thus, plexuses of immunoreactive axons were seen in the globus pallidus, substantia nigra pars reticulata, paraventricular hypothalamus, dorsal thalamus, CA3 region of the hippocampus, and interpeduncular nucleus. These results demonstrate that phosphatase inhibitor-1, a cyclic AMP-regulated inhibitor of phosphatase-1, is differentially distributed in the rat CNS. Given the widespread role of protein phosphorylation and dephosphorylation in intracellular signal transduction, these results suggest that neurons containing high levels of inhibitor-1 may share common, hitherto unrecognized, properties in terms of neurotransmitter regulation and/or responsiveness.

Noradrenaline and neuropeptide Y innervation of the rat hypothalamus are differentially affected by 6-hydroxydopamine.

The neuropeptide Y (NPY) innervation of the hypothalamus is thought to arise largely from noradrenaline (NA) neurons of the medullary tegmentum in the rat. This view was tested in this study by analyzing the effects of intraventricular injections of 6-hydroxydopamine (6-OHDA) on hypothalamic NA and NPY innervation. 6-OHDA markedly depletes or eliminates the NA innervation of the hypothalamus, as demonstrated by dopamine-beta-hydroxylase immunohistochemistry, but does not affect the NPY innervation of the hypothalamus. These results indicate that the hypothalamic NPY innervation arises in large part from intrinsic NPY-producing neurons rather than from medullary neurons in which NA and NPY coexist.