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 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.

Transcription factors of the nuclear factor–κB/rel (NF-κB) family may be important in cell survival by regulating unidentified, anti-apoptotic genes. One such gene that protects cells from apoptosis induced by Fas or tumor necrosis factor type α (TNF), IEX- 1L, is described here. Its transcription induced by TNF was decreased in cells with defective NF-κB activation, rendering them sensitive to TNF-induced apoptosis, which was abolished by transfection with IEX- 1L. In support, overexpression of antisense IEX- 1L partially blocked TNF-induced expression of IEX -1L and sensitized normal cells to killing. This study demonstrates a key role of IEX- 1L in cellular resistance to TNF-induced apoptosis.

Members of the tumor necrosis factor receptor (TNFR) superfamily are important for cell growth and survival. In addition to providing costimulatory signals for cell proliferation, ligation of both TNFR1 and Fas can result in programmed cell death or apoptosis. The underlying mechanism requires an intact 80-aa stretch present in the cytoplasmic tails of both TNFR1 and Fas, termed the death domain (DD). Here we show that CD27, a member of the TNFR family, expressed on discrete subpopulations of T and B cells and known to provide costimulatory signals for T and B cell proliferation and B cell Ig production, can also induce apoptosis. Co-crosslinking of surface Ig receptors along with ligation of CD27 augments CD27-mediated apoptosis. Unlike TNFR1 and Fas, the cytoplasmic tail of CD27 is relatively short and lacks the DD. Using the yeast two-hybrid system, we have cloned a novel protein (Siva) that binds to the CD27 cytoplasmic tail. It has a DD homology region, a box-B-like ring finger, and a zinc finger-like domain. Overexpression of Siva in various cell lines induces apoptosis, suggesting an important role for Siva in the CD27-transduced apoptotic pathway.

dehydrogenase by phosphorylation involves no long-range conformational change in the free enzyme.

1 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 pro®ling. 2 Monkey and mouse preproU-II genes were identi®ed 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. 3 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. 4 Ligand binding studies showed that [ 125 I]h U-II bound to a single sites to the cloned receptors in a saturable/high a nity manner (K d 654+154 and 214+65 pM and B max of 1011+125 and 497+68 fmol mg 71 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high a nity binding of numerous mammalian, amphibian and piscine U-II isopeptides to these receptors (K i =0.8 ± 3 nM). Fluorescein isothiocyanate (FITC) labelled U-II, bound speci®cally to HEK-293 cells expressing mouse or monkey UT receptor, con®rming cell surface expression of recombinant UT receptor. 5 Exposure of these cells to human U-II resulted in an increase in intracellular [Ca 2+ ] concentrations (EC 50 3.2+0.8 and 1.1+0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC 50 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.

Urotensin-II (U-II), the most potent mammalian vasoconstrictor identified, and its receptor, UT, exhibits increased expression in cardiac tissue and plasma in congestive heart failure (CHF) patients. Cardiomyocyte hypertrophy is primarily responsible for increased myocardial mass associated with cardiac injury. Neurohumoral factors such as angiotensin-II, endothelin-1, catecholamines, and inflammatory cytokines are thought to mediate this response. U-II shares similar biological activities with other hypertrophic G(q)-coupled receptor ligands such as angiotensin-II and endothelin-1, but a role for U-II in cardiomyocyte hypertrophy has not been characterized. The hypothesis of the current study was that U-II, acting through its G(q)-coupled receptor UT plays a hypertrophic role in cardiac hypertrophic remodeling. We report that adenoviral upregulation of the UT receptor "unmasked" U-II-induced hypertrophy in H9c2 cardiomyocytes, with a threshold response of 202+/-8 binding sites/cell. U-II was equally as efficacious as phenylephrine in inducing hypertrophy, measured by a reporter assay (EC(50) 0.7+/-0.2 nM) and [(3)H]-leucine incorporation (EC(50) 150+/-40 nM). A competitive peptidic UT receptor antagonist, BIM-23127, inhibited U-II-induced hypertrophy ( K(B) 34+/-6 nM). U-II did not affect cell proliferation or apoptosis, indicating that U-II is more hypertrophic than apoptotic or hyperplastic in cardiomyocytes. U-II (10 nM) stimulated interleukin-6 release in UT-expressing cardiomyocytes (4.6-fold at 6 h). Finally, in a rat heart failure model, cardiac ventricular mRNA expression of U-II, UT receptor, interleukin-6, and interleukin-1-beta is increased time-dependently following myocardial injury. These results indicate that U-II might play a role in cardiac remodeling associated with CHF by stimulation of cardiomyocyte hypertrophy via UT, and through upregulation of inflammatory cytokines. As such, UT antagonism may represent a novel therapeutic target for the clinical management of heart failure.

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.

Angiotensin II (Ang II) activates p38 mitogen-activated protein kinase (p38 MAPK) and increases reactive oxygen species (ROS), but the nature of the relationship in vivo is not fully understood. We assess the effect of SB239063AN, a highly selective, orally active, p38 MAPK inhibitor, on Ang II-dependent hypertension, target-organ damage and ROS production. Sprague-Dawley rats and MAPKAP kinase-2 knockout mice were infused with Ang II. Ang II infusion increased the levels of phosphorylated p38 MAPK in the heart and aorta. Production of superoxide anion and expression of NAD(P)H oxidase subunit gp91 in the aorta were increased 4- and 5-fold, respectively. In addition, Ang II infusion led to endothelial dysfunction, progressive and sustained hypertension, and cardiac hypertrophy. Treatment with SB239063AN (800 ppm in the diet) significantly attenuated the levels of phosphorylated p38 MAPK in the heart and aorta, reduced superoxide anion generation by 57% (P < 0.01), markedly suppressed gp91 mRNA expression, prevented endothelial dysfunction, and blunted both the hypertension and cardiac hypertrophy. Ang II-dependent hypertension was also significantly attenuated in MAPKAP kinase-2 knockout mice. The results suggest that Ang II induced hypertension, organ damage, and ROS production are possibly mediated by p38 MAPK and inhibition of p38 MAPK may offer a therapeutic approach for cardiovascular disease.