Rationale: Neuromedin-U (NmU) is an agonist at NMU1R and NMU2R. The brain distribution of NmU and its receptors, in particular NMU2R, suggests widespread central roles for NmU. In agreement, centrally administered NmU affects feeding behaviour, energy expenditure and pituitary output. Further central nervous system (CNS) roles for NmU warrant investigation. Objectives: To investigate the CNS role of NmU by mapping NMU1R and NMU2R mRNA and measuring the behavioural, endocrine, neurochemical and c-fos response to intracerebroventricular (i.c.v.) NmU. Methods: Binding affinity and functional potency of rat NmU was determined at human NMU1R and NMU2R. Expression of NMU1R and NMU2R mRNA in rat and human tissue was determined using semi-quantitative reverse-transcription polymerase chain reaction. In in-vivo studies, NmU was administered i.c.v. to male Sprague-Dawley rats, and changes in grooming, motor activity and pre-pulse inhibition (PPI) were assessed. In further studies, plasma endocrine hormones, v.).Results: NmU bound to NMU1R (K I , 0.11±0.02 nM) and NMU2R (K I , 0.21±0.05 nM) with equal affinity and was equally active at NMU1R (EC 50 , 1.25±0.05 nM) and NMU2R (EC 50 , 1.10±0.20 nM) in a functional assay. NMU2R mRNA expression was found at the highest levels in the CNS regions of both rat and human tissues. NMU1R mRNA expression was restricted to the periphery of both species with the exception of the rat amygdala. NmU caused a marked increase in grooming and motor activity but did not affect PPI. Further, NmU decreased plasma prolactin but did not affect levels of corticosterone, luteinising hormone or thyroid stimulating hormone. NmU elevated levels of 5-HT in the frontal cortex and hypothalamus, with decreased levels of its metabolites in the hippocampus and hypothalamus, but did not affect dopamine function. NmU markedly increased FLI in the nucleus accumbens, frontal cortex and central amygdala.

Endothelins are a family of peptide hormones having profound cardiovascular, mitogenic, and potential neuroregulatory functions. In mammals, the ET 1 peptide family is composed of three members, ET-1, ET-2, and ET-3, that are encoded by three separate genes, which are differentially expressed in the tissues of the periphery and central nervous system (for reviews, see Refs. 1 and 2). Mammalian ETs share high sequence homology and structural similarity with a family of 21 amino acid peptide toxins from the snake Atractaspis engaddensis, the sarafotoxins (3).Two major subtypes of ET receptors (ET A and ET B ) (4) have been identified based on the rank order potency of ET-1, ET-2, and S6c (5,6). ET A receptor is defined by high and equal affinity for ET-1 and ET-2, approximately 70 -100-fold lower affinity for ET-3, and a 1000-fold lower affinity for S6c. In contrast, the ET B receptor subtype displays equal high affinities for all ET-related peptides. Two additional receptors have been cloned and characterized from Xenopus melanophores (ET C ) and heart (ET AX ) (7,8). While ET C receptors display high affinity for ET-3 compared to ET-1, ET AX receptors displayed extremely weak affinity for BQ123 as well as S6c (ET A -and ET B -selective ligands, respectively). Receptors for ET are differentially expressed in a wide variety of tissues and cell types (9, 10). ETs and sarafotoxins bind to a common receptor and initiate a common signal transduction pathway, principally a G-protein-mediated activation of phospholipase C and subsequent inositol triphosphate-mediated increase in Ca 2ϩ levels (1, 11).ET mediates a number of physiological effects including vasoconstriction mitogenesis, and induction of c-fos transcription (12-23). These diverse and complex physiological effects mediated by ET in conjunction with the molecular heterogeneity and differential tissue distribution of the ET-related peptides and their receptors underscores the importance of utilizing molecular biological approaches to dissect the components of ET physiology. Several laboratories have postulated the presence of additional ET receptors to account for the diverse biochemical and physiological activities of various ETs (24). This hypothesis has been supported by binding as well as functional studies. We have previously reported the cloning, functional characterization, and regulation of the human ET A and ET B receptor subtypes (25). In this report, we describe the cloning and functional characterization of a novel ET B receptor splice variant from human placenta. EXPERIMENTAL PROCEDURESConstruction and Screening of the cDNA Libraries-The porcine cerebellum cDNA library (26) in pcDNA vector was screened by hybridization to nitrocellulose replicates using 32 P-labeled porcine ET B -R cDNA coding sequence as a probe in 20% formamide, 5 ϫ SSC (SSC is 150 mM NaCl, 15 mM sodium citrate), 5 ϫ Denhardt's, 0.1% SDS, and 0.2 mg/ml Escherichia coli tRNA at 42°C (27). Filters were washed with 2 ϫ SSC, 0.1% SDS at 42°C. Several positive recombinant clones wer...

Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for 125I-labeled NmU (KD 364 pM and Bmax 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca2+ concentration (EC50 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.

Two endogenous receptors for the potent smooth musclestimulating peptide neuromedin U (NmU) have recently been identified and cloned. Pharmacological, binding, and expression studies were conducted in an attempt to determine the receptor(s) involved in the smooth muscle-stimulating effects of NmU. The NmU peptides caused a concentration-dependent contraction of canine isolated urinary bladder. NmU did not have this same effect in the urinary bladder from rat, guinea pig, rabbit, mouse, or ferret. Although NmU had no effect on canine uterus it did cause contraction of canine stomach, ileum, and colon. As well as causing contraction of canine bladder in vitro, NmU administered systemically resulted in a significant increase in urinary bladder pressure in vivo. High-affinity binding sites for NmU were identified in canine bladder. The four NmU peptides porcine NmU-8, rat NmU-23, human NmU-25, and porcine NmU-25 displaced 125 I-NmU-25 binding with similar K i values (0.08 -0.24 nM). A different binding profile was revealed in human embryonic kidney-293 cells transiently expressed with the canine NmU-2 receptor where porcine NmU-8 (K i ϭ 147.06 nM) was much less potent than the other NmU peptides. Using TaqMan, expression of NmU-1 was detected in human urinary bladder, small intestine, colon, and uterus. Expression of NmU-2 was much lower or absent in these human tissues and undetectable in canine bladder and stomach. The results of this study reveal significant species differences in the activity of NmU. The contractile activity in human and canine smooth muscle seems to be mediated by the recently cloned NmU-1 receptor.

Experiments were designed to compare the distribution and physiological roles of endothelin (ET) receptor subtypes, ETA and ETB, in the kidneys of normotensive Sprague-Dawley (SD) and spontaneously hypertensive (SH) rats. Using [125I] ET-1 and subtype-selective ligands sarafotoxin 6c (S6c, ETB-selective agonist) and BQ123 (ETA-selective antagonist), the distribution of ETA and ETB receptors in SD rat kidney cortex, outer medulla and papilla was calculated to be 50:50, 30:70 and 10:90, respectively. The ET receptor subtypes in outer medulla and papilla of age-matched SH rats were similar to those of SD. However, in the cortex of SH rats, the ratio of ETA to ETB was 25:75 compared to 50:50 in SD rats. In addition, the affinity of the ET receptors was also higher in SH rats (117 pM vs. 235 pM). In the conscious SD rats, bolus i.v. injections of ET-1 and S6c elicited similar dose-dependent decrease in renal blood flow (RBF), which were unaffected by the infusion of the selective ETA receptor antagonist, BQ123. The SH rats were more sensitive to the renal vasoconstrictor effect of S6c and ET-1. Also, the dose-response curve to S6c was shifted to the left when compared to ET-1; however, BQ123 infusion abolished this difference. In renal clearance studies, BQ123 infusions decreased RBF and glomerular filtration rate (GFR) only in SH rats, and the fractional excretion of sodium only in SD rats. The combined data indicate that the distribution and functional roles of ETA and ETB receptor subtypes are altered in the kidneys of SH rats.

The physiological roles of endothelin-B (ETB) receptor subtypes in systemic and renal hemodynamics were assessed in conscious Sprague-Dawley rats. Mean arterial pressure, hindlimb flow, and renal blood flow were measured via an implanted catheter and pulsed Doppler flow probes. Bolus intravenous injections of sarafotoxin 6c (S6c), a selective ETB agonist, elicited transient dose-dependent vasodilation, followed by sustained vasoconstriction in the systemic bed, but only vasoconstriction in the renal bed. RES-701-1, a selective ETB antagonist, blocked the dilator and potentiated the constrictor effect; SB-209670, a mixed ET receptor antagonist, attenuated both responses to S6c. In follow-up studies, the role of endogenous ET was assessed by administration of the antagonists alone: RES-701-1, SB-209670, and the ETA-selective antagonist BQ-123. RES-701-1 unmasked a significant systemic and renal vasoconstriction, which was attenuated by SB-209670 but not by BQ-123. SB-209670 and BQ-123 had no effect on basal hemodynamic parameters. Data from radioligand binding experiments showed that RES-701-1 binds with high affinity to the cloned human ETB receptor but poorly to the ETB receptor predominant in the rat kidney. Collectively, the results indicate that 1) the vascular effects of ET in the rat are mediated by two ETB receptor subtypes: an RES-701-1-sensitive subtype, mediating vasodilation, and an RES-701-1-insensitive subtype, mediating vasoconstriction; 2) the predominant role of endogenous ET is vasodilation; and 3) the ETA receptor plays a negligible role in the control of vascular tone in the rat.

No abstract

Functional studies have demonstrated that adrenoceptor agonist-evoked relaxation is mediated primarily by ␤ 3 -adrenergic receptors (ARs) in human bladder. Thus, the use of selective ␤ 3 -AR agonists in the pharmacological treatment of overactive bladder is being explored. The present studies investigated the effects of a novel selective ␤ 3 -AR agonist, (R)