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1.
J Renin Angiotensin Aldosterone Syst ; 16(1): 79-91, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23559668

RESUMO

HYPOTHESIS: Cardiac hypertrophy in myocytes is in part regulated by changes in expression of a novel Ang II type 2 receptor (AT2-receptor) interacting protein identified as ATIP. INTRODUCTION: The role of the AT2-receptor in cardiac hypertrophy is controversial, with some reports indicating that AT2-receptor activation has detrimental effects on disease progression, whereas others indicate that it has a beneficial role. MATERIALS AND METHODS: In an effort to unravel this paradox, we examined the expression and function of ATIP in cell-based models of cardiac hypertrophy using QPCR, immunohistochemistry, cell proliferation, morphological and transfection techniques in H9c2 cardio-myoblast and myotubules. RESULTS: These studies indicate that in cultured cardio-myoblast and myotubules, Ang II mediates cellular hypertrophy and proliferation solely via the AT1-receptor, the ATIP variants are abundantly expressed and that ATIP3 may play an anti-proliferative/hypertrophic role in these cells in the absence of AT2-receptor expression or activation. CONCLUSIONS: Previously ATIP has been shown to inhibit growth factor signalling in cancerous cells via an interaction with the AT2-receptor. This is the first report to identify that ATIP may have a similar role in other disease states characterised by excessive growth and indicates that for ATIP3, at least, an interaction with the AT2-receptor may not be necessary.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mioblastos Cardíacos/metabolismo , Animais , Cardiomegalia/metabolismo , Linhagem Celular , Proliferação de Células , Tamanho Celular , Vetores Genéticos , Fibras Musculares Esqueléticas/metabolismo , Fosforilação , Ratos , Receptor Tipo 2 de Angiotensina/metabolismo , Timidina/metabolismo
2.
Cancers (Basel) ; 3(4): 3824-37, 2011 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-24213113

RESUMO

Angiotensin II (Ang II), the main effector of the renin angiotensin system, acts upon two distinct transmembrane receptors, the Ang II type 1 and the type 2 (AT2-) receptor, to induce promotion and inhibition of ERK2 phosphorylation. The AT2-receptor, through an interaction with its putative signaling partner MTUS1/ATIP (AT2-receptor interacting protein), inhibits the mitogenic effects of EGF in prostate cancer cell lines representing both early and late stage disease. This is the first report on the expression of ATIP in normal and malignant human prostatic biopsies. The expression of ATIP and its major isoforms, ATIP1 and ATIP3, in normal prostatic cells and three prostate cancer cell lines was examined using QPCR and immunohistochemistry. Human biopsies containing benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and well, moderately and poorly differentiated prostate cancer were also examined. Overall, ATIP1 and ATIP3 mRNA expression was increased in malignant compared to normal tissues and cell lines. ATIP immunostaining was low or absent in both the basal and columnar epithelial cell layers surrounding BPH acini; however, it was observed in high concentration in neoplastic epithelial cells of HGPIN and was clearly evident in cytoplasms of malignant cells in all prostate cancer grades. ATIP immunostaining was also identified in the cytoplasms of LNCaP and PC3 prostate cancer cells. As the AT2-receptor/ATIP inhibitory signaling pathway exists in malignant cells in all grades of prostate cancer, enhancement of this pathway may be a therapeutic target even after the development of androgen-independence.

3.
Prostate ; 70(14): 1563-74, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20687230

RESUMO

BACKGROUND: We have previously demonstrated Ang II type 2 (AT(2)-) receptor-mediated inhibition of EGF-induced prostate cancer cell growth in androgen-dependent (LNCaP) and independent (PC3) prostate cancer cell lines. METHODS: To explore the signaling pathways involved in this inhibitory effect, we examined the interaction of the AT(2)-receptor with its novel regulatory partner ATIP using real time PCR, over-expression, siRNA and [(3)H]thymidine incorporation assays. RESULTS: The results in human prostate cancer cell lines demonstrate the presence of ATIP in both cell lines examined, and suggest that (i) the AT(2)-receptor through an interaction with ATIP mediates an anti-growth factor effect in both androgen-dependent and androgen-independent cell lines; (ii) ATIP expression decreases as the rate of cell growth and androgen-independence increase; and (iii) EGF may act on cell growth in part by reducing the content of ATIP present in the cells. CONCLUSIONS: The results support our earlier proposal in normal cell lines that ATIP is an important component of the cellular response to AT(2)-receptor activation. The results further suggest that a critical level of ATIP is required to mediate the effect of AT(2)-receptor activation to inhibit EGF mediated increases in cell growth. They also suggest that EGF may in part induce cell growth by suppressing the level of ATIP expression.


Assuntos
Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Primers do DNA , Fator de Crescimento Epidérmico/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Reação em Cadeia da Polimerase , Neoplasias da Próstata/induzido quimicamente , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Timidina/metabolismo , Proteínas Supressoras de Tumor/metabolismo
4.
Cancer Detect Prev ; 31(5): 391-5, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18031950

RESUMO

BACKGROUND: Despite increasing interest in the renin-angiotensin system in cancer, little is known about angiotensin II (Ang II) expression in human prostate tumors. METHODS: Using immunohistochemistry, we examined Ang II expression in prostate cancer (Gleason grades 2-5), benign prostatic hyperplasia (BPH), and high-grade prostatic intraepithelial neoplasia (HGPIN). RESULTS: Ang II was present in proliferating neoplastic cells in HGPIN, in malignant cells in all grades of prostate cancer examined, in basal but not luminal epithelial cells in BPH, and in the cytoplasm of LNCaP, DU145, and PC3 prostate cancer cells. CONCLUSIONS: The data establishes the presence of Ang II in pre-malignant and malignant prostate cells, suggests Ang II staining in non-basal epithelial cells is an early sign of malignant change, and supports suggestions that HGPIN and malignant prostate cells both arise from transformed basal cells. Using immunohistochemistry we examined Ang II expression in proliferative disorders of the prostate and concluded that Ang II staining in non-basal epithelial cells is evidence of early malignant change.


Assuntos
Angiotensina II/biossíntese , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/metabolismo , Idoso , Biomarcadores Tumorais/análise , Western Blotting , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica , Masculino , Hiperplasia Prostática/patologia , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/patologia
5.
J Med Chem ; 49(12): 3467-77, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16759089

RESUMO

Site-directed mutagenesis and photoaffinity labeling experiments suggest the existence of at least two distinct binding orientations for aryloxypropanolamine competitive antagonists in the beta-adrenergic receptor (beta-AR), one where the aryloxy moiety is located near transmembrane alpha-helix 7 (tm 7) and another where it is near tm 5. To explore a hydrophobic pocket involving tms 1, 2, 3, and 7 for potential aryloxy interaction sites, we selected Tyr(356(7.43)) and Trp(134(3.28)) in the rat beta(1)-AR for site-directed mutagenesis studies. Ser(190(4.57)) was also investigated, as the equivalent residues are known antagonist interaction sites in the muscarinic M(1) and the dopamine D(2) receptors. Binding affinities (pK(i)) of a series of structurally diverse aryloxypropanolamine competitive antagonists were determined for wild type and Y356A, Y356F, W134A, and S190A mutant rat beta(1)-ARs stably expressed in Chinese hamster ovary cells. To visualize possible antagonist/receptor interactions, the compounds were docked into a three-dimensional model of the wild-type rat beta(1)-AR. The results indicate that Tyr(356(7.43)) is an important aromatic interaction site for five of the eight competitive antagonists studied, whereas none of the compounds appeared to interact directly with Trp(134(3.28)). Only two of the competitive antagonists interacted with Ser(190(4.57)) on tm 4. Overall, the results extend our understanding of how beta(1)-AR competitive antagonists bind to the hydrophobic pocket involving tms 1, 2, 3, and 7; highlight the importance of Tyr(356(7.43)) in this binding pocket; and demonstrate the involvement of tm 4 in competitive antagonist binding.


Assuntos
Antagonistas de Receptores Adrenérgicos beta 1 , Antagonistas Adrenérgicos beta/farmacologia , Serina/genética , Tirosina/genética , Animais , Sítios de Ligação , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , Modelos Moleculares , Mutagênese Sítio-Dirigida , Propanolaminas/química , Propanolaminas/farmacologia , Ratos , Receptores Adrenérgicos beta 1/genética , Estereoisomerismo , Transfecção
6.
Biochem Pharmacol ; 68(4): 675-88, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15276075

RESUMO

We investigated the role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43)) in agonist binding to, and activation of, the rat beta(1)-adrenergic receptor by comparing pK(i)s and functional responses of W134A, S190A and Y356F mutant receptors to wild type, all stably expressed in CHO cells. All three mutations significantly (P < 0.05) reduced adenylyl cyclase intrinsic activity (IA) compared to wild type in response to stimulation with both (-)-isoprenaline (53-88%) and (-)-RO363 (46-61%), and there was no significant correlation either between IA or pD(2) and pK(i) (P > 0.4), suggesting that changes in pK(i) were not sufficient to explain the fall in adenylyl cyclase activity. The most pronounced reduction in affinity (126-fold, P < 0.01) was displayed by xamoterol for the Y356F mutation, suggesting that xamoterol is able to directly interact with Tyr(356(7.43)). For the other agonists, the change in pK(i) values for the mutant receptors ranged from a 20-fold decrease to a 2-fold increase compared to the wild type. In a three-dimensional model of the rat beta(1)-adrenergic receptor, Trp(134(3.28)) and Tyr(356(7.43)) form part of a hydrophobic binding pocket involving residues in transmembrane helices 1, 2, 3 and 7. Our results suggest that Trp(134(3.28)) and Tyr(356(7.43)), together with Trp(353(7.40)), are able to interact via pi-pi interactions to stabilize the extracellular ends of transmembrane helices 3 and 7. Ser(190(4.57)) appears to be involved in a hydrogen bonding network, which maintains the spatial relationship between transmembrane helices 3 and 4. These interhelical interactions suggest that the three mutated residues stabilize the active receptor state by maintaining the proper packing of their respective transmembrane helix within the helix bundle, facilitating the appropriate movement and rotation of the transmembrane regions during the activation process.


Assuntos
Agonistas Adrenérgicos/farmacologia , Receptores Adrenérgicos beta 1/metabolismo , Serina/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Ensaio Radioligante , Ratos , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 1/genética , Homologia de Sequência de Aminoácidos
7.
Eur J Med Chem ; 39(7): 625-31, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15236843

RESUMO

To determine the role played by Tyr(356 (7.43)) in the rat beta(1)-adrenoceptor in binding the antagonists (+/-)cyanopindolol (4-[3-(t-butylamino]-3-(2'-cyano-indoloxy)-2-propanolol) and its iodinated analogue (+/-)[(125)Iodo]cyanopindolol (1-(t-butylamino]-3-(2'-cyano-3'-iodo-indoloxy)-2-propanolol), Tyr(356 (7.43)) was mutated to either Phe or Ala and binding affinities determined for wild type and mutant rat beta(1)-adrenoceptors. Our results indicate that Tyr(356 (7.43)) is important for (+/-)cyanopindolol, but not (+/-)[(125)Iodo]cyanopindolol, binding and that (+/-)cyanopindolol adopts a "reverse" binding orientation whereas (+/-)[(125)Iodo]cyanopindolol cannot be accommodated in this binding mode. We define a "reverse" antagonist binding mode as one where the aryloxy moiety interacts with residues on transmembrane helices 1, 2, 3 and 7. The beta(1)-adrenoceptor site-directed mutagenesis results are the first to support a "reverse" antagonist binding orientation and the involvement of Tyr(356 (7.43)) in this binding mode.


Assuntos
Antagonistas Adrenérgicos beta/metabolismo , Pindolol/análogos & derivados , Pindolol/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Tirosina/metabolismo , Antagonistas Adrenérgicos beta/química , Antagonistas Adrenérgicos beta/farmacologia , Animais , Radioisótopos do Iodo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pindolol/química , Pindolol/farmacologia , Ligação Proteica , Conformação Proteica , Ensaio Radioligante , Ratos , Receptores Adrenérgicos beta 1/genética , Tirosina/química , Tirosina/genética
8.
Eur J Med Chem ; 37(9): 731-41, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12350290

RESUMO

The synthesis of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride (D140S.HCl 6), a novel short acting beta(1)-specific adrenoceptor antagonist, has been described. The antagonist potency for D140S.HCl 6 has been compared with esmolol, another short acting agent, and other well known beta-adrenoceptor antagonists in isolated rat tissue preparations. The pharmacokinetics of D140S.HCl 6 in 7 day continuous intravenous infusions and 4 weeks intravenous bolus injection studies in conscious rats and dogs have been examined in toxicology studies. The effect on the isoprenaline-induced heart rate increase and the pharmacodynamic half-life of D140S.HCl 6 has been compared with esmolol in a conscious rat model. In addition, the results of a range of toxicological studies are presented. The results indicate that D140S.HCl 6 is a highly specific beta(1)-adrenoceptor antagonist (pA(2) = 8.15+/-0.22, beta(1)/beta(2) selectivity > 4400). The in vitro studies suggest D140S.HCl is ca. ten times more potent and 60 times more beta(1)-specific than racemic esmolol. Pharmacokinetic non-linearity was seen when given as a 7 day intravenous infusion at toxicological doses above 10 mg kg(-1) h(-1) in the rat and 2.5 mg kg(-1) h(-1) in the dog. Both D140S.HCl 6 and esmolol have very short durations of action after intravenous infusion in the rat (pharmacodynamic half-life is < 15 min for D140S.HCl and 10 min for esmolol). The toxicological tests indicate that D140S.HCl 6 shows no unexpected toxicity and none of the tissue irritancy problems reported for esmolol formulations.


Assuntos
Agonistas de Receptores Adrenérgicos beta 1 , Agonistas Adrenérgicos beta/síntese química , Agonistas Adrenérgicos beta/farmacologia , Propano/síntese química , Propano/farmacologia , Agonistas Adrenérgicos beta/toxicidade , Antagonistas Adrenérgicos beta/farmacologia , Animais , Atenolol/farmacologia , Cromatografia Líquida de Alta Pressão , Cães , Feminino , Meia-Vida , Átrios do Coração/efeitos dos fármacos , Técnicas In Vitro , Infusões Intravenosas , Espectroscopia de Ressonância Magnética , Contração Miocárdica/efeitos dos fármacos , Propano/análogos & derivados , Propanolaminas/farmacologia , Ratos , Ratos Sprague-Dawley , Traqueia/efeitos dos fármacos
9.
Eur J Med Chem ; 37(2): 111-25, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11858844

RESUMO

A series of 36 phenoxypropanolamines was examined to determine the structure--activity relationships of beta-adrenoceptor (beta-AR) antagonists for the human beta(1)-AR. The binding affinities of all the compounds were determined for human beta(1)-ARs expressed in Chinese hamster ovary cells and the antagonist potency for rat atrial beta(1)-ARs was determined for 32 of these compounds for comparative purposes. The compounds, based upon a phenoxypropanolamine core structure with various meta-, ortho-, para- and amine-substituents, displayed binding affinities (pK(i)) for the human beta(1)-AR ranging from 5.49 to 9.35. Antagonist potencies (pA(2)) in the rat ranged from 5.52 to 9.56 and correlated with the human binding affinities (r(2)=0.86). Twenty-six compounds were used as the training set for comparative molecular field analysis (CoMFA) of antagonist binding affinity at the human beta(1)-AR and also of antagonist potency for rat atrial beta(1)-ARs. The CoMFA models were derived using both the CoMFA electrostatic and steric field parameters. The initial human beta(1)-AR model (n=26, q(2)=0.59, ONC=6, SE(CV)=0.70, r(2)=0.98, SE(non-CV)=0.16, F(6,19)=148) predicted the binding affinities of seven out of ten test compounds, not included in the training set, with residual pK(i) values less-than-or-equal0.50. The final human beta(1)-AR model (n=36, q(2)=0.66, ONC=5, SE(CV)=0.61, r(2)=0.95, SE(non-CV)=0.24, F(5,30)=107), consisting of the training set plus the test set of compounds, may prove useful in the design of new phenoxypropanolamine type beta(1)-AR antagonists. The initial rat beta(1)-AR model (n=26, q(2)=0.42, ONC=6, SE(CV)=0.76, r(2)=0.94, SE(non-CV)=0.25, F(6,19)=47) predicted the affinities of five out of six test compounds with residual pA(2) values less-than-or-equal0.50. The final rat beta(1)-AR model (i.e. training set plus test set of compounds) (n=32, q(2)=0.38, ONC=5, SE(CV)=0.69, r(2)=0.93, SE(non-CV)=0.24, F(5,26)=67) in particular has a low q(2) value, indicating that, at least for the rat, the biologically active phenoxypropanolamine conformation may be quite different to the low energy extended conformation chosen for this CoMFA study.


Assuntos
Propanolaminas/síntese química , Propanolaminas/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Antagonistas de Receptores Adrenérgicos beta 1 , Animais , Células CHO , Cricetinae , Átrios do Coração , Humanos , Modelos Moleculares , Estrutura Molecular , Propanolaminas/química , Propanolaminas/farmacologia , Ratos , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Relação Estrutura-Atividade
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