Zhaohui Ao

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.

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.

Effects of p38 MAPK Inhibitor on angiotensin II-dependent hypertension, organ damage, and superoxide anion production.

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 gp91phox 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 gp91phox 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.

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.

Deletion of the UT receptor gene results in the selective loss of urotensin-II contractile activity in aortae isolated from UT receptor knockout mice

Urotensin‐II (U‐II) is among the most potent mammalian vasoconstrictors identified and may play a role in the aetiology of essential hypertension. Currently, only one mouse U‐II receptor (UT) gene has been cloned. It is postulated that this protein is solely responsible for mediating U‐II‐induced vasoconstriction. This hypothesis has been investigated in the present study, which assessed basal haemodynamics and vascular reactivity to hU‐II in wild‐type (UT(+/+)) and UT receptor knockout (UT(−/−)) mice. Basal left ventricular end‐diastolic and end‐systolic volumes/pressures, stroke volumes, mean arterial blood pressures, heart rates, cardiac outputs and ejection fractions in UT(+/+) mice and in UT(−/−) mice were similar. Relative to UT(+/+) mouse isolated thoracic aorta, where hU‐II was a potent spasmogen (pEC50=8.26±0.08) that evoked relatively little vasoconstriction (17±2% 60 mM KCl), vessels isolated from UT(−/−) mice did not respond to hU‐II. However, in contrast, the superior mesenteric artery isolated from both the genotypes did not contract in the presence of hU‐II. Reactivity to unrelated vasoconstrictors (phenylephrine, endothelin‐1, KCl) and endothelium‐dependent/independent vasodilator agents (carbachol, sodium nitroprusside) was similar in the aorta and superior mesenteric arteries isolated from both the genotypes. The present study is the first to directly link hU‐II‐induced vasoconstriction with the UT receptor. Deletion of the UT receptor gene results in loss of hU‐II contractile action with no ‘nonspecific’ alterations in vascular reactivity. However, as might be predicted based on the limited contractile efficacy recorded in vitro, the contribution that hU‐II and its receptor make to basal systemic haemodynamics appears to be negligible in this species.

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

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. UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. [125I]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 (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). 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]>[Cys5,10]Acm hU‐II; Kis 0.4±0.1 and 864±193 nM, respectively). U‐II receptor activation was functionally coupled to phospholipase C‐mediated [Ca2+]i mobilization (EC50 6.9±2.2 nM) in SJRH30 cells. 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.

NADPH oxidase-dependent and -independent mechanisms of reported inhibitors of reactive oxygen generation

NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CβII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.

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.

Targeted disruption of the endothelin-B-receptor gene attenuates inflammatory nociception and cutaneous inflammation in mice.

Endothelin-1 (ET-1) has been suggested to have a potential function as an inflammatory mediator. The study reported here assessed the putative inflammatory/nociceptive actions of the ET isopeptides using endothelin-B (ET(B))-receptor knockout (KO) mice and ET(A)- (SB 234551) and ET(B)- (A192621) selective antagonists. Phenylbenzoquinone (PBQ)-induced algesia was evident in the wild-type (WT) ET(B) (+/+) mice, attenuated by 80% in the heterozygous ET(B) (+/-) mice, and absent in the ET(B) (-/-) homozygotes. This was reproduced pharmacologically in WT ET(B) (+/+) mice where the algesic effect of PBQ was inhibited 74% by A192621, but unaffected by SB 234551 (both at 25 mg/kg p.o.). Similar observations were made in a model of cutaneous inflammation: ET(B) (+/+) mice had a marked inflammatory response to topical arachidonic acid, ET(B) (+/-) and ET(B) (-/-) mice had significantly reduced edema responses (37% and 65% inhibition). Neutrophil infiltration was reduced in the ET(B) (+/-) and ET(B) (-/-) mice (51% and 65% reduction, respectively). Topical administration of A192621 (500 microg/ear) inhibited arachidonic acid-induced swelling (39%) in WT ET(B) (+/+) mice. Collectively, these results support a role for the ET(B)-receptor in the mediation of inflammatory pain and cutaneous inflammatory responses. As such, the development of ET(B)-receptor-selective antagonists may be of therapeutic utility in the treatment of inflammatory disorders.

Differential modulation of endothelin ligand-induced contraction in isolated tracheae from endothelin B (ETB) receptor knockout mice

The role of endothelin B (ETB) receptors in mediating ET ligand‐induced contractions in mouse trachea was examined in ETB receptor knockout animals. Autoradiographic binding studies, using [125I]‐ET‐1, confirmed the presence of ETA receptors in tracheal and bronchial airway smooth muscle from wild‐type (+/+) and homozygous recessive (−/−) ETB receptor knockout mice. In contrast, ETB receptors were not detected in airway tissues from (−/−) mice. In tracheae from (+/+) mice, the rank order of potencies of the ET ligands was sarafotoxin (Stx) S6c>ET‐1>ET‐3; Stx S6c had a lower efficacy than ET‐1 or ET‐3. In tissues from (−/−) mice there was no response to Stx S6c (up to 0.1 μM), whereas the maximum responses and potencies of ET‐1 and ET‐3 were similar to those in (+/+) tracheae. ET‐3 concentration‐response curve was biphasic in (+/+) tissues (via ETA and ETB receptor activation), and monophasic in (−/−) preparations (via stimulation of only ETA receptors). In (+/+) preparations SB 234551 (1 nM), an ETA receptor‐selective antagonist, inhibited the secondary phase, but not the first phase, of the ET‐3 concentration‐response curve, whereas A192621 (100 nM), an ETB receptor‐selective antagonist, had the opposite effect. In (−/−) tissues SB 234551 (1 nM), but not A192621 (100 nM), produced a rightward shift in ET‐3 concentration‐response curves. The results confirm the significant influence of both ETA and ETB receptors in mediating ET‐1‐induced contractions in mouse trachea. Furthermore, the data do not support the hypothesis of atypical ETB receptors. In this preparation ET‐3 is not an ETB receptor‐selective ligand, producing contractions via activation of both ETA and ETB receptors.