Nambi Aiyar

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14

Urotensin-II (U-II) is a vasoactive ‘somatostatin-like’ cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 (refs 4, 5) and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

Differential vasoconstrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey

Urotensin‐II (U‐II) and its G‐protein‐coupled receptor, GPR14, are expressed within mammalian cardiac and peripheral vascular tissue and, as such, may regulate mammalian cardiovascular function. The present study details the vasoconstrictor profile of this cyclic undecapeptide in different vascular tissues isolated from a diverse range of mammalian species (rats, mice, dogs, pigs, marmosets and cynomolgus monkeys). The vasoconstrictor activity of human U‐II was dependent upon the anatomical origin of the vessel studied and the species from which it was isolated. In the rat, constrictor responses were most pronounced in thoracic aortae and carotid arteries: −log[EC50]s 9.09±0.19 and 8.84±0.21, Rmaxs 143±21 and 67±26% 60 mM KCl, respectively (compared, for example, to −log[EC50] 7.90±0.11 and Rmax 142±12% 60 mM KCl for endothelin‐1 [ET‐1] in thoracic aortae). Responses were, however, absent in mice aortae (−log[EC50] <6.50). These findings were further contrasted by the observation that U‐II was a ‘coronary‐selective’ spasmogen in the dog (−log[EC50] 9.46±0.11, Rmax 109±23% 60 mM KCl in LCX coronary artery), yet exhibited a broad spectrum of vasoconstrictor activity in arterial tissue from Old World monkeys (−log[EC50]s range from 8.96±0.15 to 9.92±0.13, Rmaxs from 43±16 to 527±135% 60 mM KCl). Interestingly, significant differences in reproducibility and vasoconstrictor efficacy were seen in tissue from pigs and New World primates (vessels which responded to noradrenaline, phenylephrine, KCl or ET‐1 consistently). Thus, human U‐II is a potent, efficacious vasoconstrictor of a variety of mammalian vascular tissues. Although significant species/anatomical variations exist, the data support the hypothesis that U‐II influences the physiological regulation of mammalian cardiovascular function.

Interleukin-1 Receptor and Receptor Antagonist Gene Expression After Focal Stroke in Rats

BACKGROUND AND PURPOSE The expression of interleukin-1 beta (IL-1 beta) is upregulated after focal brain ischemia, and previous work has demonstrated its involvement in ischemic injury. The IL-1 receptor antagonist (IL-1ra), a natural competitive antagonist of IL-1 receptors (IL-1Rs), has been demonstrated to play a role in attenuating brain ischemic injury. To hypothesize the involvement of the IL-1 system in ischemic injury, we examined other IL-1 components, including IL-1ra, IL-1RI, and IL-1RII for their mRNA expression after focal stroke. METHODS Quantitative reverse transcription and polymerase chain reaction (RT-PCR) technique was used to examine the mRNA expression profile of IL-1ra and two IL-1R isoforms in a temporal fashion (n = 4 for each time point) after permanent occlusion of the middle cerebral artery (MCAO) in spontaneously hypertensive rats. IL-1ra and IL-1R mRNA expression was confirmed by Northern blot analysis using poly(A) RNA isolated after 2 and 12 hours of MCAO. RESULTS Very low levels of IL-1ra mRNA were detected in sham-operated or nonischemic cortex. IL-1ra mRNA in ischemic cortex was greatly increased at 12 hours (16.5-fold increase over sham samples, P < .001) and remained elevated for up to 5 days (17.2-fold increase, P < .01) after MCAO. IL-1RI mRNA was relatively highly expressed in normal cortex and was further elevated late after ischemic injury (3.3-fold increase at day 5, P < .001). In contrast, the low basal expression of IL-1RII mRNA was remarkably elevated at 6 hours (5.3-fold increase, P < .05), reaching peak levels 12 hours (10.3-fold increase, P < .001) after MCAO. CONCLUSIONS Differential expression of IL-1 beta, IL-1ra, IL-1RI, and IL-1RII mRNAs after focal stroke may suggest a distinct role(s) for each component of the IL-1 system in ischemic injury. The data also stress the importance of evaluating all the components of a given cytokine system (eg, agonist, receptors, and natural antagonist) after focal stroke.

CLONING AND CHARACTERIZATION OF A HUMAN ANGIOTENSIN II TYPE 1 RECEPTOR

A human liver cDNA library was screened using a rat type 1 angiotensin II receptor cDNA coding sequence as a probe. cDNA clones were isolated which encoded a protein of 359 amino acids that shared 94.4% and 95.3% identify to rat and bovine type 1 angiotensin II receptors, respectively. Ligand binding studies of the cloned receptor expressed in COS cells suggested that it is pharmacologically a type 1 angiotensin II receptor subtype. Electrophysiological studies of the receptor expressed in Xenopus laevis oocytes revealed that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of calcium. Southern and Northern blot analyses indicated that the cloned receptor is represented as a single copy in the human genome and is expressed in many tissues of different histogenic origin with the exception of brain, where mRNA transcripts were barely detectable.

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.

Monocyte Chemoattractant Protein-1 is Upregulated in Rats With Volume-Overload Congestive Heart Failure

Background —Chemokines are potent proinflammatory and immune modulators. Increased expression of chemokines, eg, monocyte chemoattractant protein-1 (MCP-1), has recently been described in clinical and experimental heart failure. The present report is aimed at exploring the expression, localization, and binding site regulation of MCP-1, a member of the C-C chemokine family, in a rat model of volume-overload congestive heart failure (CHF). Methods and Results —An aortocaval fistula was surgically created between the abdominal aorta and inferior vena cava. Rats with CHF were further subdivided into compensated and decompensated subgroups. Northern blot analysis and real-time quantitative polymerase chain reaction demonstrated upregulation of MCP-1 mRNA expression correlating with the severity of CHF (288±22, 502±62, and 826±138 copies/ng total RNA for sham, compensated, and decompensated animals, respectively; n=5, P <0.05). MCP-1 protein was localized by immunohistochemistry in cardiomyocytes, vascular endothelium and smooth muscle cells, infiltrating leukocytes, and interstitial fibroblasts, and its intensity increased with severity of CHF. In addition, rats with CHF displayed a significant decrease of 125I-labeled MCP-1 binding sites to myocardium-derived membranes (384.3±57.0, 181.3±8.8, and 123.3±14.1 fmol/mg protein for sham, compensated, and decompensated animals, respectively). Conclusions —Volume-overload CHF in rats is associated with alterations in the expression, immunohistochemical localization, and receptor binding of the MCP-1 chemokine in the myocardium. These changes were more pronounced in rats with decompensated CHF. The data suggest that activation of the MCP-1 system may contribute to the progressive cardiac decompensation and development of CHF in rats with aortocaval fistula.

Pharmacological evidence for complex and multiple site interaction of CXCR4 with SDF-1α: implications for development of selective CXCR4 antagonists

The C-X-C chemokine SDF-1 and its receptor CXCR4, mediate a pivotal role in the pathophysiology of HIV-1 infection and vascular inflammatory diseases. In this study, we investigated the pharmacological properties of SDF-1alpha interaction with CXCR4 in human leukemia cell lines. Our data, based on [125I]-SDF-1alpha radioligand binding, SDF-1alpha-induced [35S]-GTPgammaS binding and use of specific CXCR4 antagonist AMD3100 reveals the complex nature of SDF-1alpha-CXCR4 interaction. Firstly, homologous competition with cold SDF-1alpha revealed a bimodal ligand displacement curve and secondly, although AMD3100 inhibited both SDF-1alpha-mediated chemotaxis (IC(50)=4.7 nM) and [35S]-GTPgammaS binding (IC(50)=7.4 nM) with high affinity, it was intriguingly up to 3000-fold less potent (IC(50)=15.2 microM) in the radioligand binding assay. These results provide pharmacological evidence for the recently described two-site model for SDF-1alpha-CXCR4 interaction. Accordingly, inhibition of SDF-1alpha binding to one of the receptor sites is sufficient to antagonize function, without causing its complete displacement from the receptor. Furthermore, these findings have important implications in the development and evaluation of CXCR4-selective small molecule antagonists for therapeutic use.

GW427353 (Solabegron), a Novel, Selective β3-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 β3-adrenergic receptors (ARs) in human bladder. Thus, the use of selective β3-AR agonists in the pharmacological treatment of overactive bladder is being explored. The present studies investigated the effects of a novel selective β3-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 β3-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 β1-ARs or β2-ARs (maximum response <10% of that to isoproterenol). In dog isolated bladder strips, GW427353 evoked relaxation that was attenuated by the nonselective β-AR antagonist bupranolol and 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol (SR59230A) (reported to have β3-AR antagonist activity). The relaxation was unaffected by atenolol, a selective β1-AR antagonist, or (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118551), a selective β2-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 β3-AR with GW427353 evokes bladder relaxation and facilitates bladder storage mechanisms in the dog.

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 F1α, 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.

Bucindolol Displays Intrinsic Sympathomimetic Activity in Human Myocardium

Background—Most clinical studies have shown that &bgr;-adrenergic receptor antagonists improve long-term survival in heart failure patients. Bucindolol, a nonselective &bgr;-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 (≈1 mm3) obtained from rat and nonfailing human hearts were confirmed to be viable for ≥48 hours in normoxic tissue culture by MTT assay and histology. Freshly isolated strips were exposed to &bgr;-adrenergic antagonists and agonists and assayed for cAMP. In both rat and human strips, the full &bgr;-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 ≈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 ≈60% of the &bgr;-adrenergic agonist activity of xamoterol in normal human myocardial tissue.