RESUMO
Background: Radiotherapy is an effective treatment of intermediate/high-risk locally advanced prostate cancer, however, >30% of patients relapse within 5 years. Clinicopathological parameters currently fail to identify patients prone to systemic relapse and those whom treatment intensification may be beneficial. The purpose of this study was to independently validate the performance of a 70-gene Metastatic Assay in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Patients and methods: A bridging cohort of prostate cancer diagnostic biopsy specimens was profiled to enable optimization of the Metastatic Assay threshold before further independent clinical validation in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Multivariable Cox proportional hazard regression analysis was used to assess assay performance in predicting biochemical failure-free survival (BFFS) and metastasis-free survival (MFS). Results: Gene expression analysis was carried out in 248 patients from the independent validation cohort and the Metastatic Assay applied. Ten-year MFS was 72% for Metastatic Assay positive patients and 94% for Metastatic Assay negative patients [HR = 3.21 (1.35-7.67); P = 0.003]. On multivariable analysis the Metastatic Assay remained predictive for development of distant metastases [HR = 2.71 (1.11-6.63); P = 0.030]. The assay retained independent prognostic performance for MFS when assessed with the Cancer of the Prostate Assessment Score (CAPRA) [HR = 3.23 (1.22-8.59); P = 0.019] whilst CAPRA itself was not significant [HR = 1.88, (0.52-6.77); P = 0.332]. A high concordance [100% (61.5-100)] for the assay result was noted between two separate foci taken from 11 tumours, whilst Gleason score had low concordance. Conclusions: The Metastatic Assay demonstrated significant prognostic performance in patients treated with radical radiotherapy both alone and independent of standard clinical and pathological variables. The Metastatic Assay could have clinical utility when deciding upon treatment intensification in high-risk patients. Genomic and clinical data are available as a public resource.
Assuntos
Biópsia/métodos , Neoplasias da Próstata/patologia , Neoplasias da Próstata/radioterapia , Idoso , Estudos de Coortes , Intervalo Livre de Doença , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Neoplasias da Próstata/genética , Reprodutibilidade dos Testes , Estudos Retrospectivos , Medição de Risco/métodos , Fatores de RiscoRESUMO
Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.
Assuntos
Antineoplásicos/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Mesotelioma , Mimetismo Molecular , Neoplasias Pleurais , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , VorinostatRESUMO
BACKGROUND: The CXC-chemokine expression is linked with colorectal cancer (CRC) progression but their significance in resected CRC is unclear. We explored the prognostic impact of such expression in stage II and III CRC. METHODS: Tissue microarrays were constructed from stage II and III CRC biopsies (n=254), and the expression of CXCL1 and CXCL8, and their receptors CXCR1 and CXCR2, in malignant and adjacent normal tissue was graded by immunohistochemistry and was correlated with prognostic factors. RESULTS: Expression of CXCL1, CXCR1 and CXCR2 was elevated in tumour epithelium relative to normal adjacent tissue (P<0.001). CXCL8 expression was detectable in the peritumoural inflammatory infiltrate. There was no overall association between CXCL1, CXCR1 or CXCR2 expression and prognostic endpoints; however, univariate subgroup survival analysis demonstrated an inverse association between CXCL1 and recurrence-free survival (RFS) in stage III patients (P=0.041). The CXCL8 positivity in the tumour infiltrate, however, correlated with earlier disease stage (P<0.001) and improved relapse-free survival across the cohort (P<0.001). Disease stage (P<0.001) and tumour infiltrate CXCL8 positivity (P=0.007) were associated with enhanced RFS in multivariate Cox regression analysis. CONCLUSION: Autocrine CXC-chemokine signalling may have adverse prognostic effects in early CRC. Conversely, CXCL8 positivity within the immune infiltrate may have good prognostic significance.
Assuntos
Quimiocinas CXC/biossíntese , Neoplasias Colorretais/metabolismo , Mucosa Intestinal/metabolismo , Células Estromais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Interleucina-8/biossíntese , Estadiamento de Neoplasias , PrognósticoRESUMO
BACKGROUND: We determined how CXC-chemokine signalling and necrosis factor-kappaB (NF-kappaB) activity affected heat-shock protein 90 (Hsp90) inhibitor (geldanamycin (GA) and 17-allylamino-demethoxygeldanamycin (17-AAG)) cytotoxicity in castrate-resistant prostate cancer (CRPC). METHODS: Geldanamycin and 17-AAG toxicity, together with the CXCR2 antagonist AZ10397767 or NF-kappaB inhibitor BAY11-7082, was assessed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay in two CRPC lines, DU145 and PC3. Flow cytometry quantified apoptotic or necrosis profiles. Necrosis factor-kappaB activity was determined by luciferase readouts or indirectly by quantitative PCR and ELISA-based determination of CXCL8 expression. RESULTS: Geldanamycin and 17-AAG reduced PC3 and DU145 cell viability, although PC3 cells were less sensitive. Addition of AZ10397767 increased GA (e.g., PC3 IC(20): from 1.67+/-0.4 to 0.18+/-0.2 nM) and 17-AAG (PC3 IC(20): 43.7+/-7.8 to 0.64+/-1.8 nM) potency in PC3 but not DU145 cells. Similarly, BAY11-7082 increased the potency of 17-AAG in PC3 but not in DU145 cells, correlating with the elevated constitutive NF-kappaB activity in PC3 cells. AZ10397767 increased 17-AAG-induced apoptosis and necrosis and decreased NF-kappaB activity/CXCL8 expression in 17-AAG-treated PC3 cells. CONCLUSION: Ansamycin cytotoxicity is enhanced by inhibiting NF-kappaB activity and/or CXC-chemokine signalling in CRPC cells. Detecting and/or inhibiting NF-kappaB activity may aid the selection and treatment response of CRPC patients to Hsp90 inhibitors.
Assuntos
Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , NF-kappa B/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Receptores de Interleucina-8B/antagonistas & inibidores , Rifabutina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Humanos , Interleucina-8/genética , Masculino , NF-kappa B/fisiologia , Necrose , Nitrilas/farmacologia , Orquiectomia , Neoplasias da Próstata/patologia , Receptores de Interleucina-8B/fisiologia , Transdução de Sinais , Sulfonas/farmacologiaRESUMO
We sought to characterise whether dexamethasone (DEX) may enhance tumour response to docetaxel in in vitro and in vivo models of metastatic prostate cancer (CaP). In vitro experiments conducted on PC3 and human bone marrow endothelial cells (hBMECs) determined that administration of DEX (10 nM) reduced constitutive nuclear factor-kappaB (NF-kappaB) activity, decreasing interleukin (IL)-8, CXCL1 and VEGF gene expression in PC3 cells. Dexamethasone also attenuated docetaxel-induced NF-kappaB and activator protein-1 transcription and reduced docetaxel-promoted expression/secretion of IL-8 and CXCL1 in PC3 and hBMECs. Although DEX failed to enhance docetaxel cytotoxicity on PC3 cells, DEX potentiated the antiangiogenic activity of docetaxel in vitro, further reducing vessel area and vessel length in developing endothelial tubes (P<0.05). Docetaxel had a potent antiangiogenic activity in the dorsal skin flap-implanted PC3 tumours in vivo. Small blood vessel formation was further suppressed in tumours co-treated with docetaxel and DEX, substantiated by an increased average vessel diameter and segment length and a decreased number of branch points in the residual tumour vasculature (P<0.001). Our data show that DEX potentiates the antiangiogenic activity of docetaxel, suggesting a putative mechanism for the palliative and survival benefits of these agents in metastatic CaP.
Assuntos
Inibidores da Angiogênese/farmacologia , Dexametasona/farmacologia , Orquiectomia , Neoplasias da Próstata/irrigação sanguínea , Taxoides/farmacologia , Animais , Linhagem Celular Tumoral , Docetaxel , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Interleucina-8/biossíntese , Interleucina-8/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Taxoides/uso terapêutico , Fator de Transcrição AP-1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hypoxic cancer cells are resistant to treatment, leading to the selection of cells with a more malignant phenotype. The expression of interleukin-8 (IL-8) plays an important role in the tumorigenesis and metastasis of solid tumors including prostate cancer. Recently, we detected elevated expression of IL-8 and IL-8 receptors in human prostate cancer tissue. The objective of the current study was to determine whether hypoxia increases IL-8 and IL-8 receptor expression in prostate cancer cells and whether this contributes to a survival advantage in hypoxic cells. IL-8, CXCR1 and CXCR2 messenger RNA (mRNA) expression in PC3 cells was upregulated in response to hypoxia in a time-dependent manner. Elevated IL-8 secretion following hypoxia was detected by enzyme-linked immunosorbent assay, while immunoblotting confirmed elevated receptor expression. Attenuation of hypoxia-inducible factor (HIF-1) and nuclear factor-kappaB (NF-kappaB) transcriptional activity using small interfering RNA (siRNA), a HIF-1 dominant-negative and pharmacological inhibitors, abrogated hypoxia-induced transcription of CXCR1 and CXCR2 in PC3 cells. Furthermore, chromatin-IP analysis demonstrated binding of HIF-1 and NF-kappaB to CXCR1. Finally, inhibition of IL-8 signaling potentiated etoposide-induced cell death in hypoxic PC3 cells. These results suggest that IL-8 signaling confers a survival advantage to hypoxic prostate cancer cells, and therefore, strategies to inhibit IL-8 signaling may sensitize hypoxic tumor cells to conventional treatments.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , NF-kappa B/metabolismo , Neoplasias da Próstata/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8B/genética , Sobrevivência Celular , Imunoprecipitação da Cromatina , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Immunoblotting , Imunoprecipitação , Interleucina-8/metabolismo , Masculino , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Neoplasias da Próstata/patologia , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição Gênica , Regulação para CimaRESUMO
Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFkappaB mechanism, since pharmacological inhibition of IkappaKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).
Assuntos
Medula Óssea/patologia , Neoplasias da Mama/patologia , Adesão Celular/fisiologia , Cortactina/fisiologia , Endotélio/patologia , Receptores de Hialuronatos/fisiologia , Invasividade Neoplásica , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Primers do DNA , Humanos , Imuno-Histoquímica , Transdução de Sinais , Transcrição GênicaRESUMO
Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the alpha(1a)-adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4-1200-fold) for the antagonists prazosin, WB4101, BMY7378, (+) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/metabolismo , Membrana Celular/metabolismo , Imidazóis/metabolismo , Fenilalanina/metabolismo , Sequência de Aminoácidos , Animais , Clonidina/metabolismo , Sequência Conservada , Cricetinae , Di-Hidropiridinas/metabolismo , Dioxanos/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oximetazolina/metabolismo , Piperazinas/metabolismo , Prazosina/metabolismo , Estrutura Secundária de Proteína , Ratos , Receptores Adrenérgicos alfa 1/metabolismo , Relação Estrutura-AtividadeRESUMO
alpha(1)-Adrenergic receptors (alpha(1A), alpha(1B), and alpha(1D)) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the alpha(1A) and alpha(1D) subtypes is thought to be mainly responsible for this activity, the role of the alpha(1B)-adrenergic receptor (alpha(1B)AR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active alpha(1B)ARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by alpha(1B)AR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of alpha(1)ARs that is important in systemic regulation of blood pressure.
Assuntos
Cardiomegalia/genética , Hipotensão/genética , Receptores Adrenérgicos alfa 1/biossíntese , Receptores Adrenérgicos alfa 1/genética , Animais , Pressão Sanguínea/genética , Peso Corporal , Bradicardia/genética , Relação Dose-Resposta a Droga , Ecocardiografia , Epinefrina/sangue , Artéria Femoral/patologia , Frequência Cardíaca , Septos Cardíacos/patologia , Humanos , Hidrocortisona/sangue , Inositol 1,4,5-Trifosfato/biossíntese , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Norepinefrina/sangue , Técnicas de Cultura de Órgãos , Tamanho do Órgão , Fenótipo , Fenilefrina/sangue , Regiões Promotoras Genéticas , Fatores de TempoRESUMO
Progress toward elucidating the function of alpha1B-adrenergic receptors (alpha1BARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate alpha1B-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active alpha1BARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in alpha1B-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the alpha1AR antagonist terazosin, indicating that alpha1AR signaling participated directly in the pathology. Our results indicate that overstimulation of alpha1BAR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.
Assuntos
Apoptose , Atrofia/etiologia , Doenças Neurodegenerativas/etiologia , Receptores Adrenérgicos alfa 1/biossíntese , Fatores Etários , Animais , Córtex Cerebral/patologia , Membro Posterior/patologia , Camundongos , Camundongos Transgênicos , Doença de Parkinson/etiologia , Fenótipo , Receptores Adrenérgicos alfa 1/genética , Convulsões/etiologia , Substância Negra/patologiaRESUMO
We examined the role that aromatic residues located in the transmembrane helices of the alpha(1a)-adrenergic receptor play in promoting antagonist binding. Since alpha(1)-antagonists display low affinity binding at beta(2)-adrenergic receptors, two phenylalanine residues, Phe-163 and Phe-187, of the alpha(1a)-AR were mutated to the corresponding beta(2)-residue. Neither F163Q nor F187A mutations of the alpha(1a) had any effect on the affinity of the alpha(1)-antagonists. However, the affinity of the endogenous agonist epinephrine was reduced 12.5- and 8-fold by the F163Q and F187A mutations, respectively. An additive loss in affinity (150-fold) for epinephrine was observed at an alpha(1a) containing both mutations. The loss of agonist affinity scenario could be reversed by a gain of affinity with mutation of the corresponding residues in the beta(2) to the phenylalanine residues in the alpha(1a). We propose that both Phe-163 and Phe-187 are involved in independent aromatic interactions with the catechol ring of agonists. The potency but not the efficacy of epinephrine in stimulating phosphatidylinositol hydrolysis was reduced 35-fold at the F163Q/F187A alpha(1a) relative to the wild type receptor. Therefore, Phe-163 and Phe-187 represent novel binding contacts in the agonist binding pocket of the alpha(1a)-AR, but are not involved directly in receptor activation.
Assuntos
Agonistas alfa-Adrenérgicos/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Cricetinae , Efedrina/metabolismo , Epinefrina/metabolismo , Fosfatos de Inositol/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/genética , Fenilalanina/metabolismo , Conformação Proteica , Ratos , Receptores Adrenérgicos alfa 1/genética , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D)-AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR-expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose-dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling.
Assuntos
Agonistas Adrenérgicos/farmacologia , Agonistas de Receptores Adrenérgicos alfa 1 , Transdução de Sinais , Antagonistas Adrenérgicos/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 1 , Animais , Células Cultivadas , Humanos , Imuno-Histoquímica , Fosfatos de Inositol , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Receptores Adrenérgicos alfa 1/metabolismo , Receptores de Superfície Celular/metabolismo , TransfecçãoRESUMO
Benzodiazepines, a class of drugs commonly used to induce anesthesia and sedation, can attenuate intracellular calcium oscillations evoked by alpha(1)-adrenergic receptor (alpha(1)-AR) stimulation in pulmonary artery smooth muscle cells. We postulated a direct action of benzodiazepines in modulating alpha(1)-AR function at the receptor level. Benzodiazepines bound to each of the cloned alpha(1)-AR subtypes (alpha(1a)-, alpha(1b)-, or alpha(1d)-AR) on COS-1 cell membranes transiently transfected to express a single population of alpha(1)-AR subtype. The ability of benzodiazepines to alter alpha(1)-AR signal transduction was investigated by measuring total inositol phosphate generation in rat-1 fibroblast cells, stably transfected to express a single alpha(1)-AR subtype. By themselves, benzodiazepines displayed partial agonism. At alpha(1b)-ARs and alpha(1d)-ARs, the maximal inositol phosphate response to phenylephrine was potentiated almost 2-fold by either midazolam or lorazepam (100 microM). At alpha(1a)-ARs, diazepam, lorazepam, and midazolam all increased the maximal response of the partial agonist clonidine at these receptors, whereas the response to the full agonist phenylephrine was unaltered or inhibited. The potentiating actions of midazolam and its partial agonism at alpha(1)-ARs was blocked by the addition of 1 microM prazosin, an alpha(1)-AR antagonist, and not by a gamma-aminobutyric acid(A)-receptor antagonist. These studies show that benzodiazepines modulate the function of alpha(1)-ARs in vitro, and this is the first report of a potential allosteric site on alpha(1)-ARs that may be therapeutically useful for drug design.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Benzodiazepinas/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Benzodiazepinas/metabolismo , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Epinefrina/farmacologia , Fibroblastos , Fosfatos de Inositol/metabolismo , Ligantes , Conformação Molecular , Fenilefrina/farmacologia , Ensaio Radioligante , Ratos , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Calcitonin gene-related peptide (CGRP) is an endogenous vasodilator peptide that produces its effects by activation of CGRP1 and CGRP2 receptor subtypes. These receptor subtypes are characterized in functional studies using the agonist Cys(Acm)2, 7-human-alpha-calcitonin gene-related peptide (Cys(ACM)2, 7-h-alpha-CGRP), which activates CGRP2 receptors, and the antagonist h-alphaCGRP(8-37) which has a high affinity for CGRP1 receptors and a low affinity for CGRP2 receptors. Our aim was to identify factors that may limit the use of these drugs to characterize CGRP receptor subtypes. We studied CGRP receptors using isolated ring segments of pig coronary and basilar arteries studied in vitro. The affinity of the antagonist h-alphaCGRP(8-37) for inhibiting h-alphaCGRP-induced relaxation of coronary arteries (log10 of the antagonist equilibrium dissociation constant = -5.33) was determined from Schild plots that had steep slopes. Therefore, we used capsaicin to investigate the role of endogenous CGRP in confounding affinity measurements for h-alphaCGRP(8-37). After capsaicin treatment, the slopes of the Schild plots were not different from one, and a higher affinity of h-CGRP(8-37) in blocking relaxation was obtained (log10 of the antagonist equilibrium dissociation constant = -6.01). We also investigated the agonist activity of the putative CGRP2 receptor selective agonist Cys(Acm)2,7-h-alphaCGRP. We found that maximal relaxation of coronary arteries caused by Cys(Acm)2,7-h-alphaCGRP was dependent upon the level of contractile tone induced by KCl. We also determined the KA for Cys(Acm)2,7-h-alphaCGRP and found that the KA (817 nM) was not significantly different from the EC50 (503 nM) for this drug in causing relaxation, indicating that Cys(Acm)2, 7-h-alphaCGRP is a partial agonist. Because experimental conditions affect the actions of h-CGRP(8-37) and Cys(Acm)2,7-h-alphaCGRP, the conditions must be carefully controlled to reliably identify CGRP receptor subtypes.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/análogos & derivados , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Músculo Liso Vascular/fisiologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologia , Animais , Artéria Basilar/fisiologia , Capsaicina/farmacologia , Vasos Coronários/fisiologia , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Cloreto de Potássio/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/efeitos dos fármacos , Reprodutibilidade dos Testes , Substância P/farmacologia , Suínos , Vasodilatação/efeitos dos fármacosRESUMO
Previous structure-activity studies have shown that the disulphide bridge of calcitonin gene-related peptide (CGRP) is important for the highly potent, CGRP receptor-mediated effects of this peptide. In this study penicillamine (Pen) was substituted for one or both of the cysteinyl residues to determine conformational and topographical properties of the disulphide bridge favourable for binding to CGRP receptors and/or receptor activation. Pen constrains the conformational flexibility of disulphide bridges in other peptides. Binding affinities were measured using a radioligand binding assay with membranes prepared from pig coronary arteries and 125I-h-alpha-CGRP. Functional effects were characterized using a previously reported pig coronary artery relaxation bioassay. The binding affinity of [Pen2]h-alpha-CGRP was not significantly different from that of h-alpha-CGRP. All other analogues showed reduced affinity for CGRP receptors. [Pen2]h-alpha-CGRP also caused relaxation of coronary arteries. The remaining analogues either caused relaxation with significantly reduced potency or failed to relax the arteries at concentrations up to 1 x 10(-5)M. All analogues that did not relax coronary arteries contained a D-Pen in position 7 and inhibited CGRP-induced relaxation. [D-Pen2,7]h-alpha-CGRP was the most potent antagonist with a K8 value of 630 nM. This affinity is similar to that of the classical CGRP receptor antagonist, h-alpha-CGRP(8-37), on these arteries (KBs 212 nM). These studies show that modifying the topography of the disulphide bridge can cause large and variable effects on ligand binding and activation of CGRP receptors. The contribution of position 7 to the conformation and topography of the disulphide bridge of h-alpha-CGRP is crucial to the future design of agonists of CGRP receptors. Furthermore, position 7 is important for the development of new CGRP receptor antagonists with structures based on the whole sequence of h-alpha-CGRP.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Peptídeos/síntese química , Vasodilatação/efeitos dos fármacos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Vasos Coronários/efeitos dos fármacos , Dissulfetos , Peptídeos/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Relação Estrutura-Atividade , Suínos , Vasodilatadores/farmacologiaRESUMO
Alpha 1-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this alpha 1-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster alpha 1b-AR and in Rat-1 fibroblasts stably transfected with the human alpha 1a-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial alpha 1-AR agonists and this effect was fully inhibited by the alpha 1-AR antagonist prazosin. However, this synergistic potentiation was not observed for full alpha 1-AR agonists. Instead, TEA caused a parallel rightward shift of the dose-response curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of alpha 1-ARs with a Ki of 28.7 +/- 4.7 mM. In addition, the site of binding by TEA to the alpha 1-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating alpha 1-AR activation.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1 , Etilaminas/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Cloreto de Sódio/química , Animais , Sítios de Ligação/genética , Células COS , Linhagem Celular , Cricetinae , Sinergismo Farmacológico , Etilaminas/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Ratos , Receptores Adrenérgicos alfa 1/química , Receptores Adrenérgicos alfa 1/genética , Cloreto de Sódio/farmacologia , Solubilidade , Relação Estrutura-AtividadeRESUMO
A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala14]-h-alpha-CGRP, [Aib14]-h-alpha-CGRP, [Asp14]-h-alpha-CGRP, [Asn14]-h-alpha-CGRP, and [Pro14]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala14]-h-alpha-CGRP, [Aib14]-h-alpha-CGRP, [Asp14]-h-alpha-CGRP, and [Asn14]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly14 of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro14]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro14]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.