Renal impairment, hypertension and plasma urotensin II.

BACKGROUND: Human urotensin II (UII) is a potent mammalian vasoconstrictor thought to be produced and cleared by the kidneys. Conflicting data exist regarding the relationship between UII concentrations, kidney function and blood pressure (BP). We measured the associations between kidney function [including end-stage renal disease (ESRD)] and levels of BP with plasma concentrations of UII. METHODS: Ninety-one subjects were enrolled. Thirty-one subjects had ESRD (undergoing haemodialysis), 30 subjects had chronic kidney disease (CKD) and 30 control subjects had no kidney disease. Plasma UII concentrations were measured by radioimmunoassay. RESULTS: Mean plasma UII concentrations were highest in controls, lower in subjects with ESRD and lowest in subjects with non-ESRD CKD (P<0.0001). UII concentrations correlated negatively with serum creatinine (P=0.0012) and CKD stage, and positively with creatinine clearance (P=0.013). In ESRD subjects, plasma UII (P=0.008) increased after dialysis, while SBP (P=0.007), DBP (P=0.009), serum creatinine (P<0.0001) and serum urea nitrogen (P<0.0001) decreased. UII concentrations were lower in patients with a history of hypertension (HTN) (P=0.016). Age, race and gender did not appear to be associated with UII concentration. However, the distribution of African American race and male gender appear to be associated with increasing stages of chronic kidney disease. CONCLUSIONS: These data suggest a potential vasodilatory role of UII in humans with kidney disease or hypertension. The reduction in UII levels in CKD also suggests either reduced production or greater clearance, or both, of UII.

Benzofuran-substituted urea derivatives as novel P2Y(1) receptor antagonists.

Benzofuran-substituted urea analogs have been identified as novel P2Y(1) receptor antagonists. Structure-activity relationship studies around the urea and the benzofuran moieties resulted in compounds having improved potency. Several analogs were shown to inhibit ADP-mediated platelet activation.

Differential effects of p38 mitogen-activated protein kinase and cyclooxygenase 2 inhibitors in a model of cardiovascular disease.

The evidence is compelling for a role of inflammation in cardiovascular diseases; however, the chronic use of anti-inflammatory drugs for these indications has been disappointing. The recent study compares the effects of two anti-inflammatory agents [cyclooxygenase 2 (COX2) and p38 inhibitors] in a model of cardiovascular disease. The vascular, renal, and cardiac effects of 4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib; a COX2 inhibitor) and 6-{5-[(cyclopropylamino)carbonyl]-3-fluoro-2-methylphenyl}-N-(2,2-dimethylpropyl)-3-pyridinecarboxamide [GSK-AHAB, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor], were examined in the spontaneously hypertensive stroke-prone rat (SHR-SP). In SHR-SPs receiving a salt-fat diet (SFD), chronic treatment with GSK-AHAB significantly and dose-dependently improved survival, endothelial-dependent and -independent vascular relaxation, and indices of renal function, and it attenuated dyslipidemia, hypertension, cardiac remodeling, plasma renin activity (PRA), aldosterone, and interleukin-1beta (IL-1beta). In contrast, chronic treatment with a COX2-selective dose of rofecoxib exaggerated the harmful effects of the SFD, i.e., increasing vascular and renal dysfunction, dyslipidemia, hypertension, cardiac hypertrophy, PRA, aldosterone, and IL-1beta. The protective effects of a p38 MAPK inhibitor are clearly distinct from the deleterious effects of a selective COX2 inhibitor in the SHR-SP and suggest that anti-inflammatory agents can have differential effects in cardiovascular disease. The results also suggest a method for evaluating long-term cardiovascular efficacy and safety.

Urotensin-II receptor antagonists: synthesis and SAR of N-cyclic azaalkyl benzamides.

SAR exploration of the central diamine, benzyl, and terminal aminoalkoxy regions of the N-cyclic azaalkyl benzamide series led to the identification of very potent human urotensin-II receptor antagonists such as 1a with a K(i) of 4 nM. The synthesis and structure-activity relationships (SAR) of N-cyclic azaalkyl benzamides are described.

Tetrahydro-4-quinolinamines identified as novel P2Y(1) receptor antagonists.

High-throughput screening of the GSK compound collection against the P2Y(1) receptor identified a novel series of tetrahydro-4-quinolinamine antagonists. Optimal substitution around the piperidine group was pivotal for ensuring activity. An exemplar analog from this series was shown to inhibit platelet aggregation.

Development of potent and selective small-molecule human Urotensin-II antagonists.

This work describes the development of potent and selective human Urotensin-II receptor antagonists starting from lead compound 1, (3,4-dichlorophenyl)methyl{2-oxo-2-[3-phenyl-2-(1-pyrrolidinylmethyl)-1-piperidinyl]ethyl}amine. Several problems relating to oral bioavailability, cytochrome P450 inhibition, and off-target activity at the kappa opioid receptor and cardiac sodium channel were addressed during lead development. hUT binding affinity relative to compound 1 was improved by more than 40-fold in some analogs, and a structural modification was identified which significantly attenuated both off-target activities.

Potent and selective small-molecule human urotensin-II antagonists with improved pharmacokinetic profiles.

Lead compound 1 was successfully redesigned to provide compounds with improved pharmacokinetic profiles for this series of human urotensin-II antagonists. Replacement of the 2-pyrrolidinylmethyl-3-phenyl-piperidine core of 1 with a substituted N-methyl-2-(1-pyrrolidinyl)ethanamine core as in compound 7 resulted in compounds with improved oral bioavailability in rats. The relationship between stereochemistry and selectivity for hUT over the kappa-opioid receptor was also explored.

The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems.

Several peptidic urotensin-II (UT) receptor antagonists exert 'paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4-11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)>>GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human>>rat UT receptor). The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. In contrast to the 'low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5-fold less than seen with urantide). Since GSK248451 (1 mg kg(-1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.

Identification and characterization of binding sites for human urotensin-II in Sprague-Dawley rat renal medulla using quantitative receptor autoradiography.

Urotensin-II (U-II), a ligand for the G-protein-coupled receptor UT, has been characterized as the most potent mammalian vasoconstrictor identified to date. Although circulating levels of U-II are altered in lower species (e.g., fish) upon exposure to hypo-osmotic stress, little is known about the actions of this cyclic undecapeptide within the kidney, an organ that plays a pivotal role in the control of cardiovascular homeostasis, influencing both cardiac preload (plasma volume) and after load (peripheral resistance). The present study reports the identification of specific, high affinity [125I]hU-II binding sites in Sprague-Dawley rat kidney outer medulla by autoradiography and also through membrane radioligand binding (Kd 1.9 +/- 0.9 nM and Bmax 408 +/- 47 amol mm(-2) and Kd 1.4 +/- 0.3 nM and Bmax 51.3 +/- 7.8 fmol mg(-1) protein, respectively). Differences were observed in the binding characteristics within rat strains. Compared to the Sprague-Dawley, Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rat kidney outer medulla displayed low density < 20 fmol mg(-1) protein and low affinity (> 1 microM) [125I]hU-II binding sites. Thus, the relative contribution of specific U-II binding sites to the physiological actions of U-II in the control of cardiorenal homeostasis is worthy of further investigation.

Urotensin-II levels in acute coronary syndromes.

BACKGROUND: Urotensin-II (U-II) is a vasoactive peptide with diffuse expression in human cardiomyocyte and vascular smooth muscle cells. Recent studies have reported increased plasma levels of U-II in patients with congestive heart failure. OBJECTIVE: We sought to determine the plasma levels of U-II in patients with acute coronary syndromes (ACS), stable coronary artery disease (CAD) and healthy controls. METHODS: We prospectively measured plasma U-II levels in 54 patients with ACS, 51 patients with stable coronary disease and 29 healthy volunteers. Monoclonal antibodies against U-II were generated and plasma U-II levels were determined by radioimmunoassay from extracted venous samples. RESULTS: ACS patients had significantly lower levels than patients with stable CAD and healthy controls (2.53+/-1.62 vs. 3.45+/-2.53 vs. 3.3+/-3.9 ng/ml, p=0.008, respectively). In both ACS and stable CAD patients, we found a negative relationship between plasma U-II levels and systemic arterial pressures. The correlation coefficients for systolic and mean arterial pressure were -0.272, p=0.006 and -0.209, p=0.04, respectively. CONCLUSIONS: Plasma U-II levels were significantly decreased in patients with acute coronary syndromes and related negatively to systemic arterial pressures. This finding suggests a down-regulation of U-II expression in patients with acute coronary syndromes. CONDENSED ABSTRACT: Urotensin-II (U-II) is a vasoactive peptide with diffuse staining in human cardiomyocytes and vascular smooth muscle cells. We measured plasma U-II levels in patients with acute coronary syndromes (ACS), stable coronary artery disease (CAD) and healthy controls. We observed lower U-II levels in ACS patients and a negative correlation between U-II levels and systemic arterial pressure. This finding suggests a down-regulation of U-II expression in patients with ACS.

Bucindolol displays intrinsic sympathomimetic activity in human myocardium.

BACKGROUND: Most clinical studies have shown that beta-adrenergic receptor antagonists improve long-term survival in heart failure patients. Bucindolol, a nonselective beta-receptor blocker, however, failed to reduce heart failure mortality in a recent large clinical trial. The reasons for this failure are not known. Bucindolol has partial agonist properties in rat myocardium, but whether it has agonist activity in human heart is controversial. To address this, we measured the ability of bucindolol to increase cAMP accumulation in human myocardium. METHODS AND RESULTS: Myocardial strips ( approximately 1 mm(3)) obtained from rat and nonfailing human hearts were confirmed to be viable for > or = 48 hours in normoxic tissue culture by MTT assay and histology. Freshly isolated strips were exposed to beta-adrenergic antagonists and agonists and assayed for cAMP. In both rat and human strips, the full beta-adrenergic agonist isoproterenol raised cAMP levels by >2.5-fold at 15 minutes. Carvedilol and propranolol had no effect on basal cAMP levels, whereas metoprolol reduced basal cAMP by approximately 25%. In contrast, bucindolol and xamoterol increased cAMP levels in a concentration-dependent manner in both rat and human myocardium (maximum 1.64+/-0.25-fold and 2.00+/-0.27-fold over control, respectively, P<0.01 for human tissue). CONCLUSIONS: Bucindolol exhibits approximately 60% of the beta-adrenergic agonist activity of xamoterol in normal human myocardial tissue.

Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375.

1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and 'native' UT receptors (K(i) 4.7+/-1.5 to 20.7+/-3.6 nM at rodent, feline and primate recombinant UT receptors and K(i) 5.4+/-0.4 nM at the endogenous UT receptor in SJRH30 cells). 2. Prior exposure to SB-706375 (1 microM, 30 min) did not alter [(125)I]hU-II binding affinity or density in recombinant cells (K(D) 3.1+/-0.4 vs 5.8+/-0.9 nM and B(max) 3.1+/-1.0 vs 2.8+/-0.8 pmol mg(-1)) consistent with a reversible mode of action. 3. The novel, nonpeptidic radioligand [(3)H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (K(D) 2.6+/-0.4 nM, B(max) 0.86+/-0.12 pmol mg(-1)) in a manner that was inhibited by both U-II isopeptides and SB-706375 (K(i) 4.6+/-1.4 to 17.6+/-5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. 4. SB-706375 was a potent, competitive hU-II antagonist across species with pK(b) 7.29-8.00 in HEK293-UT receptor cells (inhibition of [Ca(2+)](i)-mobilization) and pK(b) 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (K(app) approximately 20 nM). 5. SB-706375 was a selective U-II antagonist with >/=100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (K(i)/IC(50)>1 microM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 microM). 6. In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals.

Cloning and pharmacological characterization of the cat urotensin-II receptor (UT).

Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds. The purpose of this study was to clone and pharmacologically characterize cat recombinant UT to determine whether this system represents a model for characterizing UT antagonists. Cloned cat UT displayed 74% identity to primate UT, and 77% identity to rodent UT. [(125)I] hU-II bound in a saturable manner to a single site on recombinant cat UT with high affinity (K(D) 288+/-13pM) and high density (B(max) 747+/-66fmol/mg protein). U-II isopeptides displayed equipotent, high affinity binding to cat UT (K(i) 1.8-5.3nM). Cat UT was coupled to intracellular [Ca(2+)] release (EC(50) 0.6+/-0.2nM) and total inositol phosphate (IP) formation (EC(50) 0.4+/-0.1nM). Protein kinase C activation desensitized cat, but not human, UT-mediated IP formation. UT mRNA expression was detected in cat blood vessels, trachea, lung, and kidney, where the medulla (K(D) 815+/-34) and cortex and (K(D) 316+/-39pM) displayed high affinity binding for human U-II (hU-II). The cat urotensin-II receptor represents a suitable in vitro model to examine the role of the U-II/UT system in the etiology of hypertension, assisting in the evaluation of the UT antagonists to help treat cardiovascular disease.

Inhibitory effects of putative peptidic urotensin-II receptor antagonists on urotensin-II-induced contraction of cat isolated respiratory smooth muscle.

Urotensin-II is purported to influence pulmonary function by modulating smooth muscle tone/growth. In the present study, Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated the presence of UT receptor mRNA in cat trachea, bronchi and lung parenchyma. Urotensin-II contracted cat isolated trachea and bronchi with similar potencies (pEC(50)s 8.61+/-0.07-8.81+/-0.10). Contractile efficacies ranged from 19+/-9% to 63+/-11% KCl in the primary and secondary bronchi. The peptidic UT receptor antagonists BIM-23127, SB-710411 and GSK248451 (7.18+/-0.12, 7.52+/-0.08 and 9.05+/-0.16 cat recombinant UT pK(i)s) inhibited urotensin-II-induced contraction of cat isolated trachea with pK(b)s 6.36+/-0.11, 6.74+/-0.07 and 9.27+/-0.12, respectively. As such, feline lung contains significant amounts of UT mRNA and this receptor appears to be functionally coupled to bronchoconstriction (the peptidic tool compound GSK248451 representing a sub-nanomolar inhibitor of such effects). These findings suggest that the cat represents a suitable species for future studies designed to assess the effects of the urotensin-II receptor on pulmonary (patho)physiology.

Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines.

1 In an effort to identify endogenous, native mammalian urotensin-II (U-II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [125I]hU-II binding sites. 2 UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. 3 [(125)I]hU-II bound to a homogenous population of high-affinity, saturable (Kd 67.0+/-11.8 pm, Bmax 9687+/-843 sites cell(-1)) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (< or =15% dissociation 90 min). A lower density of high-affinity U-II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667+/-165 sites cell(-1), Kd 74+/-8 pm). 4 Consistent with the profile recorded in human recombinant UT-HEK293 cells, [125I]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (Kis 0.5+/-0.1-1.2+/-0.3 nm) and related analogues (hU-II[4-11]>[Cys(5,10)]Acm hU-II; Kis 0.4+/-0.1 and 864+/-193 nm, respectively). 5 U-II receptor activation was functionally coupled to phospholipase C-mediated [Ca2+]i mobilization (EC50 6.9+/-2.2 nm) in SJRH30 cells. 6 The present study is the first to identify the presence of 'endogenous' U-II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU-II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function.

Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey.

Urotensin-II (U-II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U-II/UT receptor system. The goal of this study was to clone mouse and primate preproU-II and UT for pharmacological profiling. Monkey and mouse preproU-II genes were identified to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU-II has four exons, while the UT receptor has one exon. Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U-II/UT receptor mRNA in extra-vascular tissue including lung, pancreas, skeletal muscle, kidney and liver. Ligand binding studies showed that [125I]h U-II bound to a single sites to the cloned receptors in a saturable/high affinity manner (Kd 654+/-154 and 214+/-65 pM and Bmax of 1011+/-125 and 497+/-68 fmol mg-1 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high affinity binding of numerous mammalian, amphibian and piscine U-II isopeptides to these receptors (Ki=0.8 - 3 nM). Fluorescein isothiocyanate (FITC) labelled U-II, bound specifically to HEK-293 cells expressing mouse or monkey UT receptor, confirming cell surface expression of recombinant UT receptor. Exposure of these cells to human U-II resulted in an increase in intracellular [Ca2+] concentrations (EC50 3.2+/-0.8 and 1.1+/-0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC50 7.2+/-1.8 and 0.9+/-0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.

Molecular cloning and pharmacological characterization of bovine calcitonin receptor-like receptor from bovine aortic endothelial cells.

A complementary DNA encoding calcitonin receptor-like receptor (CRLR) was isolated from a bovine aortic endothelial cell library. The bovine CRLR has 462 amino acids and 92% homology with the human CRLR. In a reverse transcriptase-polymerase chain reaction assay, bovine CRLR was found to be widely distributed, including in the heart and lungs. Stable transfection of bovine CRLR in human embryonic kidney cells (HEK-293) resulted in specific high-affinity [125I] rat adrenomedulin (rADM)-binding (dissociation constant=145+/-15 pM). ADM-stimulated adenylyl cyclase activity with an EC50 value of 5.0+/-1.2 nM. The human ADM receptor antagonist hADM(22-52) inhibited [125I]rADM-binding and ADM-stimulated adenylyl cyclase activity. Interactions between bovine CRLR and individual receptor activity modifying proteins (RAMPs) were also investigated. Transient co-transfection of bovine CRLR cDNA with human receptor activity modifying protein 1 (hRAMP1) cDNA in HEK-293 cells resulted in the expression of a CRLR that displayed high-affinity binding to calcitonin gene-related peptide. Co-transfection of bovine CRLR with human RAMP2 or RAMP3 cDNAs in HEK-293 cells displayed high-affinity ADM receptors. These observations suggest that in the absence of exogenous RAMPs heterologous expression of bovine CRLR results in an ADM receptor phenotype.

G protein-coupled receptors in drug discovery.

G protein-coupled receptors (GPCRs) represent one of the most important drug discovery targets such that compounds targeted against GPCRs represent the single largest drug class currently on the market. With the revolutionary advances in human genome sciences and the identification of numerous orphan GPCRs, it is even more important to identify ligands for these orphan GPCRs so that their physiological and pathological roles can be delineated. To this end, major pharmaceutical industries are investing enormous amounts of time and money to achieve this object. This review is a bird's eye view on the various aspects of GPCRs in drug discovery.

Localisation of NMU1R and NMU2R in human and rat central nervous system and effects of neuromedin-U following central administration in rats.

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, [DOPAC + HVA]/[dopamine] and [5-HIAA]/[5-HT] ratios and levels of Fos-like immunoreactivity (FLI) were measured 20 min post-NmU (i.c.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. CONCLUSIONS: These data provide further evidence for widespread roles for NmU and its receptors in the brain.

Differential levels of "urotensin-II-like" activity determined by radio-receptor and radioimmuno-assays.

Plasma and urinary levels of "urotensin(U)-II-like" substances determined in healthy human volunteers were 12.4 +/- 0.6 ng/ml and 2.2 +/- 0.3 ng/ml by RIA, an order of magnitude lower than that seen by RRA, 167.5 +/- 9.5 ng/ml and 65.2 +/- 4.3 ng/ml. HPLC demonstrated the existence of at least three prominent activity peaks in plasma and urine, the more hydrophobic of which did not co-elute with U-II, degradation products or URP. RRA and RIA recognized these peaks with contrasting efficacy. As such, published levels of "U-II-like" activity should be interpreted with caution until a better understanding is obtained regarding what species specific RIA and RRA assay reagents interact with.