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.

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.

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.

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.

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.

GW427353 (solabegron), a novel, selective beta3-adrenergic receptor agonist, evokes bladder relaxation and increases micturition reflex threshold in the dog.

Functional studies have demonstrated that adrenoceptor agonist-evoked relaxation is mediated primarily by beta3-adrenergic receptors (ARs) in human bladder. Thus, the use of selective beta3-AR agonists in the pharmacological treatment of overactive bladder is being explored. The present studies investigated the effects of a novel selective beta3-AR agonist, (R)-3'-[[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]ethyl]amino]-[1,1'-biphenyl]-3-carboxylic acid (GW427353; solabegron) on bladder function in the dog using in vitro and in vivo techniques. GW427353 stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human beta3-AR, with an EC50 value of 22 +/- 6 nM and an intrinsic activity 90% of isoproterenol. At concentrations of 10,000 nM, GW427353 produced a minimal response in cells expressing either beta1-ARs or beta2-ARs (maximum response <10% of that to isoproterenol). In dog isolated bladder strips, GW427353 evoked relaxation that was attenuated by the nonselective beta-AR antagonist bupranolol and 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol (SR59230A) (reported to have beta3-AR antagonist activity). The relaxation was unaffected by atenolol, a selective beta1-AR antagonist, or (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118551), a selective beta2-AR antagonist. GW427353 increased the volume required to evoke micturition in the anesthetized dog following acetic acid-evoked bladder irritation, without affecting the ability of the bladder to void. GW427353-evoked effects on bladder parameters in vivo were inhibited by bupranolol. The present study demonstrates that selective activation of beta3-AR with GW427353 evokes bladder relaxation and facilitates bladder storage mechanisms in the dog.

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor.

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.

Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells.

Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A2 and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B4 and 6-keto-prostaglandin F1alpha, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.

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.

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.

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.

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.

Aminoalkoxybenzyl pyrrolidines as novel human urotensin-II receptor antagonists.

High throughput screening of the corporate compound collection led to the discovery of a novel series of substituted aminoalkoxybenzyl pyrrolidines as human urotensin-II receptor antagonists. The synthesis, initial structure-activity relationships, and optimization of the initial hit that led to the identification of a truncated sub-series, represented by SB-436811 (1a), are described.

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.

Novel function for receptor activity-modifying proteins (RAMPs) in post-endocytic receptor trafficking.

RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking.

Neuromedin U elicits cytokine release in murine Th2-type T cell clone D10.G4.1.

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 (125)I-labeled NmU (K(D) 364 pM and B(max) 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 Ca(2+) concentration (EC(50) 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.