RESUMO
The chemokine receptors CXCR1 and CXCR2 are G-protein-coupled receptors (GPCRs) implicated in mediating cellular functions associated with the inflammatory response. Potent CXCR2 receptor antagonists have been discovered, some of which have recently entered clinical development. The aim of this study was to identify key amino acid residue differences between CXCR1 and CXCR2 that influence the relative antagonism by two compounds that have markedly different chemical structures. By investigating the effects of domain switching and point mutations, we found that the second extracellular loop, which contained significant amino acid sequence diversity, was not important for compound antagonism. We were surprised to find that switching the intracellular C-terminal 60 amino acid domains of CXCR1 and CXCR2 caused an apparent reversal of antagonism at these two receptors. Further investigation showed that a single amino acid residue, lysine 320 in CXCR2 and asparagine 311 in CXCR1, plays a predominant role in describing the relative antagonism of the two compounds. Homology modeling studies based on the structure of bovine rhodopsin indicated a potential intracellular antagonist binding pocket involving lysine 320. We conclude that residue 320 in CXCR2 forms part of a potential allosteric binding pocket on the intracellular side of the receptor, a site that is distal to the orthosteric site commonly assumed to be the location of antagonist binding to GPCRs. The existence of a common intracellular allosteric binding site at GPCRs related to CXCR2 may be of value in the design of novel antagonists for therapeutic intervention.
Assuntos
Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Linhagem Celular , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ensaio Radioligante , Receptores de Interleucina-8A/química , Receptores de Interleucina-8A/efeitos dos fármacos , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/efeitos dos fármacos , Receptores de Interleucina-8B/genética , Homologia de Sequência de AminoácidosRESUMO
We have discovered that endothelin-1 (ET-1) vasoconstriction is significantly enhanced in aortas of young (8-16-week-old) apolipoprotein E-deficient (ApoE-/-) mice devoid of atherosclerotic lesions (maximum response expressed as a percentage of the mean response to 100 mM KCl (E(MAX)) = 55.7% +/- 19.5% KCl, n = 5) compared to age-matched C57BL/6/J control animals (E(MAX) = 12.6% +/- 2.5% KCl, n = 8), indicating that alterations in the endothelin system may contribute to disease progression, at least in this animal model. There was no difference in the potency of ET-1 to contract aorta from the two groups (C57BL/6/J pD2 = 8.74 +/- 0.30; ApoE-/- pD2 = 8.50 +/- 0.15, P > 0.05). This increased response was specific to ET-1, as it was not observed with phenylephrine or U46619, nor was it due to a non-receptor mediated increase in contractile sensitivity, as there was no change in response to KCl between the two groups. [125I]ET-1 bound with subnanomolar affinity (K(D)) to aorta (K(D) = 0.018 +/- 0.002 nM, n = 4) and, with an order of magnitude lower affinity, to heart (K(D) = 0.47 +/- 0.05, n = 5) of C57BL/6/J mice with binding densities (B(MAX)) of 9.3 +/- 2.4 fmol mg(-1)protein and 100 +/- 14 fmol mg(-1) protein, respectively. Alterations in vascular reactivity to ET-1 could not be explained by increased endothelin receptor density or affinity, as these were not altered in aorta (K(D) = 0.011 +/- 0.003 nM; B(MAX) = 10.1 +/- 3.9 fmol mg(-1), n = 4) and heart (K(D) = 0.43 +/- 0.04 nM; B(MAX) = 115 +/- 26 fmol mg(-1), n == 6) of ApoE-/- animals. The ratio of ET(A) to ET(B) receptors in heart of control and ApoE-/- mice was similar, comprising 89% and 85% ET(A) receptors, respectively. In isolated aorta from ApoE-/- mice on the Western diet, which more closely resembled more advanced stages of the disease in man, the augmented ET-1 vasoconstrictor response was maintained (E(MAX) = 25.2% +/- 6.8% KCl, n = 9); however, it was completely prevented in animals that had received 10 weeks of oral atorvastatin (30 mg kg(-1) day(-1)) (E(MAX) = 4.0% +/- 1.5% KCl, n = 5), a concentration that was chosen because it did not affect plasma cholesterol and triglyceride levels. Therefore, this protective prevention of enhanced ET-1 vasoconstriction in ApoE-/- mice by atorvastatin was independent of its lipid-lowering properties.
Assuntos
Apolipoproteínas E/genética , Arteriosclerose/metabolismo , Endotelinas/farmacologia , Ácidos Heptanoicos/farmacologia , Pirróis/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Arteriosclerose/genética , Arteriosclerose/fisiopatologia , Atorvastatina , Feminino , Homozigoto , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de TempoRESUMO
1. Systemic infusions of urocortin 1 produce a decrease in mean arterial pressure. This effect may be mediated by a direct action on novel corticotropin-releasing factor type 2 (CRF(2)) receptors predicted to be expressed in blood vessels and the heart. Our objectives were to determine the presence of CRF(2) receptors in the human cardiovascular system using the selective radioligand [(125)I]antisauvagine 30. We also investigated the potential functional roles of novel CRF(2) ligands in the regulation of vascular tone in human arteries in vitro. 2. Radioligand binding techniques were used to characterise the CRF(2) receptor. [(125)I]antisauvagine 30 bound specifically, saturably, reversibly and with high affinity to CRF(2) receptors in human left ventricle (K(D) 0.21+/-0.03 nm, B(MAX) 0.80+/-0.18 fmol mg(-1) protein), and no change in receptor density or affinity was observed in the dilated cardiomyopathy group. 3. Autoradiographical studies revealed highly localised binding of [(125)I]antisauvagine 30 to intramyocardial blood vessels. Binding sites were also detected in the myocardium and in the medial layer of internal mammary arteries. 4. In endothelium-denuded human internal mammary artery in vitro, all peptides tested produced a potent and sustained vasodilator response reversing endothelin-1-induced constrictions (10 nm) (urocortin 1: pD(2) 8.39+/-0.32, E(MAX) 46+/-7.7%; urocortin 2: pD(2) 8.27+/-0.17, E(MAX) 60+/-8.5%; urocortin 3: pD(2) 8.61+/-0.25, E(MAX) 61+/-7.2%; CRF: pD(2) 8.28+/-0.27, E(MAX): 40+/-10%). 5. We have demonstrated the presence of CRF(2) receptors in the human cardiovascular system and a direct, endothelium-independent vasodilator action of urocortins 2 and 3, which may counter-balance the centrally mediated pressor effects of CRF and urocortin 1.
Assuntos
Sistema Cardiovascular/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Vasodilatadores/farmacologia , Adolescente , Adulto , Idoso , Sistema Cardiovascular/efeitos dos fármacos , Hormônio Liberador da Corticotropina/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Ligantes , Masculino , Artéria Torácica Interna/efeitos dos fármacos , Artéria Torácica Interna/metabolismo , Pessoa de Meia-Idade , Miocárdio/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Hormônio Liberador da Corticotropina/agonistas , Urocortinas , Vasodilatadores/metabolismoRESUMO
1 The potential vasodilator function of the peptide ghrelin, recently identified as the endogenous ligand of the growth hormone secretagogue orphan receptor (GHS-R), was investigated in human endothelium-denuded internal mammary artery. The peptide endothelin-1 (ET-1) is a potent and long-lasting vasoconstrictor. Comparisons were made with established and putative endogenous vasodilators to determine if any could reverse ET-1-induced vasoconstriction in this vessel. 2 Ghrelin (0.1-300 nM) potently dilated 10 nM ET-1-induced constrictions (pD(2) 8.39+/-0.29; E(MAX) 63+/-5.6%; n=9/14, responders/total). 3 ANP (pD(2) 7.75+/-0.14; E(MAX) 106+/-2.0; n=5/5) and CGRP (pD(2) 8.08+/-0.17; E(MAX) 76+/-15% n=5/6) both produced complete reversal of the constrictor response to ET-1 (E(MAX) not significantly different from 100%, P>0.05 one-sample t-test). 4 The following caused partial reversal of the ET-1 response: Adrenomedullin (n=9/9) and two peptides derived from proadrenomedullin, PAMP-12 (n=6/7) and PAMP-20 (n=9/9) (pD(2) values 7.63+/-0.28, 7.97+/-0.23 and 8.51+/-0.29; E(MAX) 58+/-7.3, 54+/-10 and 51+/-7.8% respectively). Unexpectedly, amylin was only 2 fold less potent than CGRP, although there was less than 50% reversal of the ET-1 constriction (pD(2) 7.86+/-0.30; E(MAX) 41+/-5.4%; n=7/9). CNP (n=6/6) also partially reversed constrictions to ET-1 (E(MAX) 53+/-6.3; pD(2) 8.07+/-0.38). 5 BNP (n=4/5) and PGI(2) (n=6/8) were weak vasodilators, since concentration-response curves failed to reach a maximum within the range tested. PGE(2) caused a small dilatation in some vessels (E(MAX) 17+/-2.1%; pD(2) 8.63+/-0.36; n=4/8). 6 We have demonstrated ghrelin to be an effective, endothelium-independent vasodilator of the long-lasting constrictor ET-1 in human arteries producing responses similar to those of adrenomedullin (P>0.05, ANOVA). British Journal of Pharmacology (2002) 136, 1146-1152
Assuntos
Endotelina-1/antagonistas & inibidores , Artéria Torácica Interna/efeitos dos fármacos , Hormônios Peptídicos/fisiologia , Vasodilatação/fisiologia , Adulto , Idoso , Feminino , Grelina , Humanos , Técnicas In Vitro , Masculino , Artéria Torácica Interna/fisiologia , Pessoa de Meia-Idade , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Hormônios Peptídicos/farmacologia , Vasodilatadores/farmacologiaRESUMO
We detected urotensin-II-like immunoreactivity in the endothelium of normal human blood vessels from heart, kidney, placenta, adrenal, thyroid and umbilical cord. Immunoreactivity was also detected in endocardial endothelial and kidney epithelial cells. In atherosclerotic coronary artery, immunoreactivity localized to regions of macrophage infiltration. Urotensin-II constricted human atherosclerotic epicardial coronary arteries with pD2=10.58 +/- 0.46 (mean +/- S.E.M.) and Emax=11.4 +/- 4.2% KCl and small coronary arteries with pD2=9.25 +/- 0.38 and Emax=77 +/- 16% KCl. Small coronary arteries clearly exhibited a greater maximum response to urotensin-II than epicardial vessels. This enhanced responsiveness may be of importance in heart failure, where circulating concentrations of U-II are increased, or in atherosclerosis where focally up-regulated urotensin-II production may act down stream to produce significant vasospasm, compromising blood flow to the myocardium. We conclude that urotensin-II is a locally released vasoactive mediator that may be an important regulator of blood flow particularly to the myocardium and may have a specific role in human atherosclerosis.
Assuntos
Vasos Coronários/fisiologia , Urotensinas/fisiologia , Vasoconstritores , Arteriosclerose/fisiopatologia , Doença das Coronárias/fisiopatologia , Vasos Coronários/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Urotensinas/genética , Urotensinas/farmacologiaRESUMO
The endothelin system was characterized in the C57BL/6J mouse, a strain commonly used in genetically manipulated models of cardiovascular disease. Functional responses to endothelin-1 (ET-1) were measured in segments of aorta mounted in wire myographs. Endothelin-1 produced a potent vasoconstriction [EC50: 0.49 nM (0.18-1.3 nM), n = 5], and the maximum response was 11 +/- 2.9% KCl response. Competition binding assays (0.1 nM [I]-ET-1 vs 2 pM to 100 microM PD156707) were conducted on cryostat sections of mouse heart and brain. The endothelin-A (ETA) selective antagonist bound to mouse heart with two affinities, yielding a KDETA of 1.1 +/- 0.2 nM (BMAX 130 +/- 16 fmol/mg protein) and a KDETB (endothelin-B) of 0.51 +/- 0.09 microM (BMAX 14.0 +/- 1.1 fmol/mg protein) (n = 5). ETA receptors were predominant in the heart, with competition assays yielding receptor ratios of 90:10 ETA:ETB. Contrastingly, mouse brain was ETB-rich, although PD156707 also competed with two affinities (ETA: KD 0.97 +/- 0.87 nM, BMAX 61.7 +/- 10 fmol/mg protein; ETB: KD 0.87 +/- 0.08 microM, BMAX 132 +/- 6.7 fmol/mg protein). In conclusion, we have shown that endothelin-1 is a potent constrictor of mouse aorta and endothelin receptors are highly expressed in mouse heart and brain.
Assuntos
Dioxóis/metabolismo , Endotelina-1/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Animais , Aorta/metabolismo , Ligação Competitiva , Encéfalo/metabolismo , Dioxóis/farmacologia , Antagonistas do Receptor de Endotelina A , Antagonistas do Receptor de Endotelina B , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Ratos , Especificidade da Espécie , VasoconstriçãoRESUMO
In conscious male Sprague-Dawley rats, we compared regional hemodynamic actions of the selective corticotropin-releasing factor type 2 (CRF(2)) receptor ligands human and mouse urocortin 2 (hUCN2 and mUCN2, respectively) with those of CRF. Bolus i.v. doses of 3 and 30 pmol kg(-1) hUCN2, mUCN2, or CRF had no significant hemodynamic actions, but at doses of 300 and 3000 pmol kg(-1), all three peptides caused dose-dependent tachycardia and hypotension, with rapid-onset, short-duration, mesenteric vasodilatation and slower-onset, more prolonged hindquarters vasodilatation but little or no change in renal vascular conductance. Pretreatment with the nonselective CRF receptor antagonist astressin or the selective CRF(2) receptor antagonist antisauvagine 30 abolished all the cardiovascular actions of all three peptides. Indomethacin had no effect on responses to hUCN2, and there was no evidence for any involvement of nitric oxide (NO) in the vasodilator actions of hUCN2. There was no evidence that recruitment of angiotensin- and endothelin-mediated vasoconstrictor mechanisms counteracted the vascular actions of hUCN2. The results indicate that the hemodynamic effects of i.v. hUCN2, mUCN2, and CRF depend on activation of CRF(2) receptors and do not involve NO or prostanoids.
Assuntos
Hormônio Liberador da Corticotropina/farmacologia , Hemodinâmica/efeitos dos fármacos , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Animais , Interações Medicamentosas , Indanos/farmacologia , Indometacina/farmacologia , Losartan/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Hormônio Liberador da Corticotropina/metabolismo , UrocortinasRESUMO
The effect of previous nitrate therapy on vascular responses to endothelin-1 (ET-1) and NO was investigated in human internal mammary artery (IMA) in vitro. Cumulative concentration-response curves to ET-1 were constructed in rings of IMA and the data grouped into IMA from patients given nitrates prior to the bypass graft operation (nitrate group) and IMA from patients who were not prescribed nitrates (control group). No significant differences were observed between the two groups, either in EC(50) value [P>0.05; 3.5 nM (2.4-5.3 nM; 95% confidence interval) and 4.8 (2.2-10 nM), nitrate and control groups respectively] or E(max) (P>0.05; 78+/-7.5% and 85+/-9.5%, nitrate and control group respectively). No significant differences in concentration-response curves to the NO-donor diethylamine NONOate (DEA/NO) in rings of IMA pre-constricted with 10 nM ET-1 were observed between control and nitrate groups [P>0.05; EC(50) values 0.59 (0.21-1.7) microM and 0.17 (0.03-0.87) microM; E(max) 110+/-5.7% and 112+/-4.5%, nitrate and control groups respectively]. Concentration-response curves to DEA/NO constructed in normal coronary artery were not significantly different from those in coronary artery obtained from patients with ischaemic heart disease (IHD) [P>0.05; E(max) 124+/-11% and 138+/-20%; EC(50) 0.08 (0.02-0.30) microM and 0.23 (0.02-24) microM, normal and IHD respectively]. These data indicate that nitrate therapy does not induce long-term changes in the ET signalling pathway. Furthermore, the tolerance to nitrate therapy is likely to be because of impaired bio-transformation of the drug rather than reduced sensitivity of the media to NO. The similar responses to DEA/NO in normal and atherosclerotic coronary artery suggests that the reduced vasodilator responses in IHD is because of a dysfunctional endothelium and is not mediated by changes in the NO signalling pathway of the smooth muscle.
Assuntos
Arteriosclerose/tratamento farmacológico , Hidrazinas/farmacologia , Músculo Liso Vascular/fisiopatologia , Nitratos/uso terapêutico , Doadores de Óxido Nítrico/farmacologia , Arteriosclerose/metabolismo , Vasos Coronários , Relação Dose-Resposta a Droga , Endotelina-1/farmacologia , Humanos , Técnicas In Vitro , Artéria Torácica Interna , Músculo Liso Vascular/efeitos dos fármacos , Óxidos de Nitrogênio , Estatísticas não Paramétricas , Vasoconstritores/farmacologiaRESUMO
Results from over a dozen prostate cancer susceptibility genome-wide scans, encompassing some 1,500 hereditary prostate cancer families, indicate that prostate cancer is an extremely heterogeneous disease with multiple loci contributing to overall susceptibility. In an attempt to reduce locus heterogeneity, we performed a genomewide linkage scan for prostate cancer susceptibility genes with 36 Jewish families, which represent a stratification of hereditary prostate cancer families with potentially increased locus homogeneity. The 36 Jewish families represent a combined dataset of 17 Jewish families from the Fred Hutchinson Cancer Research Center-based Prostate Cancer Genetic Research Study dataset and 19 Ashkenazi Jewish families collected at Johns Hopkins University. All available family members, including 94 affected men, were genotyped at markers distributed across the genome with an average interval of <10 centimorgans. Nonparametric multipoint linkage analyses were the primary approach, although parametric analyses were performed as well. Our strongest signal was a significant linkage peak at 7q11-21, with a nonparametric linkage (NPL) score of 3.01 (P = 0.0013). Simulations indicated that this corresponds to a genomewide empirical P = 0.006. All other regions had NPL P values >/=0.02. After genotyping additional markers within the 7q11-21 peak, the NPL score increased to 3.35 (P = 0.0004) at D7S634 with an allele-sharing logarithm of odds of 3.12 (P = 0.00007). These studies highlight the utility of analyzing defined sets of families with a common origin for reducing locus heterogeneity problems associated with studying complex traits.