RESUMO
Intermittent hypoxia (IH) is a feature of obstructive sleep apnea (OSA), a condition highly associated with hypertension-related cardiovascular diseases. Repeated episodes of IH contribute to imbalance of angiogenic growth factors in the hypertrophic heart, which is key in the progression of cardiovascular complications. In particular, the interaction between vascular endothelial growth factor (VEGF) and the kallikrein-kinin system (KKS) is essential for promoting angiogenesis. However, researchers have yet to investigate experimental models of IH that reproduce OSA, myocardial angiogenesis, and expression of KKS components. We examined temporal changes in cardiac angiogenesis in a mouse IH model. Adult male C57BI/6 J mice were implanted with Matrigel plugs and subjected to IH for 1-5 weeks with subsequent weekly histological evaluation of vascularization. Expression of VEGF and KKS components was also evaluated. After 3 weeks, in vivo myocardial angiogenesis and capillary density were decreased, accompanied by a late increase of VEGF and its type 2 receptor. Furthermore, IH increased left ventricular myocardium expression of the B2 bradykinin receptor, while reducing mRNA levels of B1 receptor. These results suggest that in IH, an unexpected response of the VEGF and KKS systems could explain the reduced capillary density and impaired angiogenesis in the hypoxic heart, with potential implications in hypertrophic heart malfunction.
Assuntos
Cardiomegalia/metabolismo , Hipóxia/metabolismo , Cininas/metabolismo , Miocárdio/metabolismo , Neovascularização Fisiológica , Apneia Obstrutiva do Sono/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Capilares/metabolismo , Capilares/fisiologia , Cardiomegalia/complicações , Vasos Coronários/metabolismo , Vasos Coronários/fisiologia , Hipóxia/complicações , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Apneia Obstrutiva do Sono/complicações , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The regulation of the kallikrein-kinin system is an important mechanism controlling vasodilation and promoting inflammation. We aimed to investigate the role of Toll-like receptor 2 (TLR2) in regulating kinin B1 and B2 receptor expression in human gingival fibroblasts and in mouse gingiva. Both P. gingivalis LPS and the synthetic TLR2 agonist Pam2CSK4 increased kinin receptor transcripts. Silencing of TLR2, but not of TLR4, inhibited the induction of kinin receptor transcripts by both P. gingivalis LPS and Pam2CSK4. Human gingival fibroblasts (HGF) exposed to Pam2CSK4 increased binding sites for bradykinin (BK, B2 receptor agonist) and des-Arg10-Lys-bradykinin (DALBK, B1 receptor agonist). Pre-treatment of HGF for 24 h with Pam2CSK4 resulted in increased PGE2 release in response to BK and DALBK. The increase of B1 and B2 receptor transcripts by P. gingivalis LPS was not blocked by IL-1ß neutralizing antibody; TNF-α blocking antibody did not affect B1 receptor up-regulation, but partially blocked increase of B2 receptor mRNA. Injection of P. gingivalis LPS in mouse gingiva induced an increase of B1 and B2 receptor mRNA. These data show that activation of TLR2 in human gingival fibroblasts as well as in mouse gingival tissue leads to increase of B1 and B2 receptor mRNA and protein.
Assuntos
Receptores da Bradicinina/genética , Receptor 2 Toll-Like/metabolismo , Adulto , Animais , Bradicinina/metabolismo , Feminino , Fibroblastos/metabolismo , Gengiva/metabolismo , Humanos , Inflamação/metabolismo , Cininas/metabolismo , Lipopeptídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/genética , Receptores da Bradicinina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Endometriosis is a gynaecological disease exhibiting severe pelvic pain, but the mechanism of pain production remains unknown. Bradykinin (BK) is known as an inflammatory mediator, and shows elevated levels in inflammatory diseases such as rheumatoid arthritis. In the present study, we evaluated whether BK is involved in endometriosis-related pain. METHODS: Endometriotic lesions were used for immunohistochemistry. Primary cultures of endometriotic stromal cells (ESC) were stimulated with IL-1ß and/or BK. Quantitative RT-PCR was used to evaluate the mRNA expressions of BK receptors (BKR) and endothelin-1 in ESC. The concentration of endothelin-1 in cystic fluid of endometrioma or non-endometrioma was measured with ELISA. The conditioned medium of ESC stimulated with IL-1ß and/or BK was injected intraplantarly in mice, and evaluated whether pain-related licking behaviour was elicited. RESULTS: The expressions of BK and BKR in endometriotic lesions were observed by immunohistochemistry. In vitro experiments showed that IL-1ß induced BKR-B1 and B2 on ESC. Activation of these receptors by BK significantly induced endothelin-1 expression in ESC, which was negated completely by HOE-140, a BKR-B2 antagonist. The cystic fluid of endometrioma contained higher amount of endothelin-1 compared to non-endometrioma. Intraplantar injection of the conditioned medium of ESC treated with IL-1ß and BK significantly induced licking behaviour, which was suppressed with BQ-123, an endothelin type-A receptor antagonist. CONCLUSIONS: The present study demonstrated the presence and the function of the BK axis in endometriosis, and established a potential new therapy target for endometriosis-related pain. SIGNIFICANCE: The present study demonstrated (1) the presence and the function of the BK system in endometriosis, (2) activation of BKR induced endothelin-1 in endometriotic lesion and (3) blocking endothelin-1 was effective to decrease pain.
Assuntos
Bradicinina/metabolismo , Endometriose/metabolismo , Endotelina-1/metabolismo , Dor/metabolismo , Receptores da Bradicinina/metabolismo , Células Estromais/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Líquido Cístico/metabolismo , Endotelina-1/efeitos dos fármacos , Endotelina-1/genética , Endotelina-1/farmacologia , Feminino , Humanos , Interleucina-1beta/farmacologia , Camundongos , Doenças Ovarianas/metabolismo , Doenças Peritoneais/metabolismo , RNA Mensageiro/metabolismo , Receptores da Bradicinina/efeitos dos fármacos , Receptores da Bradicinina/genética , Células Estromais/efeitos dos fármacosRESUMO
Chronic vulvar pain is alarmingly common in women of reproductive age and is often accompanied by psychological distress, sexual dysfunction, and a significant reduction in quality of life. Localized provoked vulvodynia (LPV) is associated with intense vulvar pain concentrated in the vulvar vestibule (area surrounding vaginal opening). To date, the origins of vulvodynia are poorly understood, and treatment for LPV manages pain symptoms, but does not resolve the root causes of disease. Until recently, no definitive disease mechanisms had been identified; our work indicates LPV has inflammatory origins, although additional studies are needed to understand LPV pain. Bradykinin signaling is one of the most potent inducers of inflammatory pain and is a candidate contributor to LPV. We report that bradykinin receptors are expressed at elevated levels in LPV patient versus healthy control vestibular fibroblasts, and patient vestibular fibroblasts produce elevated levels of proinflammatory mediators with bradykinin stimulation. Inhibiting expression of one or both bradykinin receptors significantly reduces proinflammatory mediator production. Finally, we determined that bradykinin activates nuclear factor (NF)κB signaling (a major inflammatory pathway), whereas inhibition of NFκB successfully ablates this response. These data suggest that therapeutic agents targeting bradykinin sensing and/or NFκB may represent new, more specific options for LPV therapy. PERSPECTIVE: There is an unmet need for the development of more effective vulvodynia therapies. As we explore the mechanisms by which human vulvar fibroblasts respond to proinflammatory/propain stimuli, we move closer to understanding the origins of chronic vulvar pain and identifying new therapeutic targets, knowledge that could significantly improve patient care.
Assuntos
Bradicinina/metabolismo , Dor Pélvica/metabolismo , Transdução de Sinais/fisiologia , Adulto , Bradicinina/análogos & derivados , Bradicinina/genética , Bradicinina/farmacologia , Antagonistas dos Receptores da Bradicinina/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Dor Crônica , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Dor Pélvica/tratamento farmacológico , Dor Pélvica/patologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismoRESUMO
Infraorbital nerve constriction (CION) causes hypersensitivity to facial mechanical, heat and cold stimulation in rats and mice and is a reliable model to study trigeminal neuropathic pain. In this model there is evidence that mechanisms operated by kinin B1 and B2 receptors contribute to heat hyperalgesia in both rats and mice. Herein we further explored this issue and assessed the role of kinin receptors in mechanical hyperalgesia after CION. Swiss and C57Bl/6 mice that underwent CION or sham surgery or dynorphin A (1-17) administration were repeatedly submitted to application of either heat stimuli to the snout or mechanical stimuli to the forehead. Treatment of the animals on the fifth day after CION surgery with DALBK (B1 receptor antagonist) or HOE-140 (B2 receptor antagonist), both at 0.01-1µmol/kg (i.p.), effectively reduced CION-induced mechanical hyperalgesia. Knockout mice for kinin B1, B2 or B1/B2 receptors did not develop heat or mechanical hyperalgesia in response to CION. Subarachnoid dynorphin A (1-17) delivery (15nmol/5µL) also resulted in orofacial heat hyperalgesia, which was attenuated by post-treatment with DALBK (1 and 3µmol/kg, i.p.), but was not affected by HOE-140. Additionally, treatment with an anti-dynorphin A antiserum (200µg/5µL, s.a.) reduced CION-induced heat hyperalgesia for up to 2h. These results suggest that both kinin B1 and B2 receptors are relevant in orofacial sensory nociceptive changes induced by CION. Furthermore, they also indicate that dynorphin A could stimulate kinin receptors and this effect seems to contribute to the maintenance of trigeminal neuropathic pain.
Assuntos
Bradicinina/metabolismo , Dinorfinas/metabolismo , Dor Facial/metabolismo , Neuralgia/metabolismo , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Modelos Animais de Doenças , Dinorfinas/farmacologia , Temperatura Alta , Hiperalgesia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurotransmissores/farmacologia , Medição da Dor , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , TatoRESUMO
OBJECTIVES: Clinical and experimental observations have suggested that bradykinin, a major activation product of the plasma kallikrein-kinin system, is involved in the pathogenesis of arthritis, but the pathogenic role of bradykinin receptors remains inconclusive. In this study we examined whether bradykinin receptors are important in the pathogenesis of anti-collagen antibody-induced arthritis (CAIA) using double receptor-deficient (B1RB2R(-/-)) mice. METHODS: CAIA was induced in B1RB2R(+/+) and B1RB2R(-/-) mice by injection of an anti-collagen antibody cocktail on day 0 and lipopolysaccharide on day 3. Severity of disease was evaluated by measurement of joint diameter and histological analysis. The expression of proinflammatory cytokines in joint tissue and peripheral mononuclear cells was determined by ELISA and real-time RT-PCR. RESULTS: The absent expression of B1R and B2R mRNA in B1RB2R(-/-) mice was confirmed by RT-PCR. Although B1RB2R(+/+) mice developed severe CAIA, the severity of the disease was significantly attenuated in B1RB2R(-/-) mice. In B1RB2R(+/+) mice bearing CAIA, both B1R and B2R mRNA levels were increased in joint tissue and peripheral mononuclear cells. Compared with B1RB2R(+/+) mice, the production of IL-1ß and IL-6 in joint tissue and their mRNA expression in peripheral mononuclear cells were remarkably reduced in B1RB2R(-/-) mice. CONCLUSION: These observations provide genetic evidence that bradykinin plays an important role in the pathogenesis of CAIA. B1R, whose expression is induced in inflamed joint tissue and peripheral inflammatory cells, is important in the development of CAIA.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/fisiopatologia , Bradicinina/fisiologia , Receptores da Bradicinina/fisiologia , Animais , Anticorpos Anti-Idiotípicos/efeitos adversos , Artrite Experimental/metabolismo , Colágeno/efeitos adversos , Colágeno/imunologia , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Articulações/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores da Bradicinina/deficiência , Receptores da Bradicinina/genéticaRESUMO
Kinins, a group of important pro-inflammatory peptides, are abundantly found in tissues and biological fluids of cancer patients. Bradykinin, the major representative of kinins, induces vascular permeability and, in consequence, promotes tumor expansion. Additionally, the kinin-induced inflammatory responses, especially those mediated by kinin metabolites without the C-terminal arginine residue, lead to enhanced tumor growth. The present study aimed at analyzing the ability of the human glioblastoma cell line U-373, derived from a malignant tumor, to produce kinin peptides. The proteins involved in kinin generation, i.e., the kininogens and the kallikreins, were shown to be expressed in these cells. Moreover, tumor necrosis factor α, a proinflammatory cytokine that mediates tumorigenesis, was found to enhance the expression of enzymes associated with kinin production. The strong binding of kininogen to the cell surface and the enzymatic degradation of this protein by cells suggest the activation of kinin-generating systems. Indeed, glioblastoma cells, pre-treated with tumor necrosis factor α, released kinin peptides from exogenous kininogen. The expression of kinin receptors in these cells was also shown to increase under the influence of this cytokine. Our results suggest that the human glioblastoma cell line U-373 constitutes a good cellular model that can be helpful in cancer research focused on kinin-induced inflammation. Furthermore, our findings can contribute to new approaches in cancer treatment with the use of kinin receptor antagonists and inhibitors of kinin production.
Assuntos
Bradicinina/genética , Neoplasias Encefálicas/genética , Citocinas/genética , Glioblastoma/genética , Calicreínas/genética , Cininogênios/genética , Receptores da Bradicinina/genética , Bradicinina/imunologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Citocinas/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glioblastoma/imunologia , Glioblastoma/patologia , Humanos , Inflamação , Calicreínas/imunologia , Cininogênios/imunologia , Modelos Biológicos , Receptores da Bradicinina/imunologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.
Assuntos
Angiotensina II/agonistas , Bradicinina/agonistas , Peptídeos/química , Receptor Tipo 1 de Angiotensina/química , Receptores da Bradicinina/química , Sequência de Aminoácidos , Angiotensina II/genética , Angiotensina II/metabolismo , Sítios de Ligação , Bradicinina/genética , Bradicinina/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismoRESUMO
The kallikrein-kinin system (KKS) has been previously linked to glucose homeostasis. In isolated muscle or fat cells, acute bradykinin (BK) stimulation was shown to improve insulin action and increase glucose uptake by promoting glucose transporter 4 translocation to plasma membrane. However, the role for BK in the pathophysiology of obesity and type 2 diabetes remains largely unknown. To address this, we generated genetically obese mice (ob/ob) lacking the BK B2 receptor (obB2KO). Despite similar body weight or fat accumulation, obB2KO mice showed increased fasting glycemia (162.3 ± 28.2 mg/dl vs 85.3 ± 13.3 mg/dl), hyperinsulinemia (7.71 ± 1.75 ng/ml vs 4.09 ± 0.51 ng/ml) and impaired glucose tolerance when compared with ob/ob control mice (obWT), indicating insulin resistance and impaired glucose homeostasis. This was corroborated by increased glucose production in response to a pyruvate challenge. Increased gluconeogenesis was accompanied by increased hepatic mRNA expression of forkhead box protein O1 (FoxO1, four-fold), peroxisome proliferator-activated receptor gamma co-activator 1-alpha (seven-fold), phosphoenolpyruvate carboxykinase (PEPCK, three-fold) and glucose-6-phosphatase (eight-fold). FoxO1 nuclear exclusion was also impaired, as the obB2KO mice showed increased levels of this transcription factor in the nucleus fraction of liver homogenates during random feeding. Intraportal injection of BK in lean mice was able to decrease the hepatic mRNA expression of FoxO1 and PEPCK. In conclusion, BK modulates glucose homeostasis by affecting hepatic glucose production in obWT. These results point to a protective role of the KKS in the pathophysiology of type 2 diabetes mellitus.
Assuntos
Bradicinina/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Gluconeogênese/fisiologia , Glucose/metabolismo , Fígado/metabolismo , Receptores da Bradicinina/metabolismo , Análise de Variância , Animais , Bradicinina/administração & dosagem , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Gluconeogênese/efeitos dos fármacos , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina/genética , Sistema Calicreína-Cinina/fisiologia , Leptina/metabolismo , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/genética , Obesidade/metabolismo , Obesidade/fisiopatologia , PPAR gama/genética , PPAR gama/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Ratos , Receptores da Bradicinina/genéticaRESUMO
The standard chemotherapy for epithelial ovarian cancer (EOC) patients is currently a combination of taxane and platinum. However, most EOC patients still suffer relapses, and there is an immediate need for the development of novel and more effective therapeutic modalities against this deadly disease. Recently, the nonpeptide bradykinin (BK) antagonist 2,3,4,5,6-pentafluorocinnamoyl-(o-2,6-dichlorobenzyl)-l-tyrosine-N-(4-amino-2,2,6,6-tetramethyl-piperidyl) amide (BKM-570) was shown to cause impressive growth inhibition of lung and prostate tumors, displaying superior in vivo inhibitory effects than convential chemotherapeutic drugs. Here, we investigated BKM-570 cytotoxic effects in two EOC cell lines, derived from different EOC histopathologies: a clear cell carcinoma (TOV-21), and an endometrioid carcinoma (TOV-112). We showed that BKM-570 effectively inhibited the growth of ovarian cancer cells, as its cytotoxic effects were comparable to those of cisplatin, and were independent of the functional status of BK receptors. Moreover, BKM-570 synergized with cisplatin in inhibiting EOC cell growth. To better understand the molecular mechanisms of the antiproliferative action of this BK antagonist in EOC cells, we performed gene expression profiling in TOV-21 and TOV-112 cells following treatment with 10 µM BKM-570 for 24 h. BKM-570 displayed similar cytotoxic effects in the two cell lines analyzed, as genes with previously shown involvement in apoptosis/antiapoptosis and cell adhesion were proportionally upregulated and downregulated in both cell lines, whereas genes involved in basic cellular mechanisms, including cell growth and maintenance, metabolism, cell cycle control, inflammatory and immune response, signal transduction, protein biosynthesis, transcription regulation, and transport, were predominantly downregulated upon treatment. Our data are indicative of the therapeutic potential of BKM-570 and related compounds in EOC management.
Assuntos
Antineoplásicos/farmacologia , Bradicinina/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Tirosina/análogos & derivados , Linhagem Celular Tumoral , Cisplatino/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/metabolismo , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/farmacologiaRESUMO
UNLABELLED: An upregulation of the endogenous opioid, dynorphin A, in the spinal cord is seen in multiple experimental models of chronic pain. Recent findings implicate a direct excitatory action of dynorphin A at bradykinin receptors to promote hyperalgesia in nerve injured rats, and its upregulation may promote, rather than counteract, enhanced nociceptive input due to injury. Here we examined a model of inflammatory pain by unilateral injection of complete Freund's adjuvant (CFA) into the rat hind paw. Rats exhibited tactile hypersensitivity and thermal hyperalgesia in the inflamed paw by 6 hours after CFA injection, whereas a significant elevation of prodynorphin transcripts in the lumbar spinal cord was seen at day 3 but not at 6 hours. Thermal hyperalgesia at day 3, but not at 6 hours, after CFA injection was blocked by intrathecal administration of anti-dynorphin antiserum or by bradykinin receptor antagonists. The antihyperalgesic effect of the latter was not due to de novo production of bradykinin or upregulation of spinal bradykinin receptors. These data suggest that elevated spinal dynorphin on peripheral inflammation mediates chronic inflammatory hyperalgesia. The antihyperalgesic effect of bradykinin receptor antagonists requires the presence of upregulated spinal dynorphin but not of de novo production of bradykinin, supporting our hypothesis that pathological levels of dynorphin may activate spinal bradykinin receptors to mediate inflammatory hyperalgesia. PERSPECTIVE: This study shows that chronic peripheral inflammation induces a significant upregulation of the endogenous opioid peptide dynorphin. Elevated levels of spinal dynorphin and activation of spinal bradykinin receptors are essential to maintain inflammatory hyperalgesia. The results suggest that blockade of spinal bradykinin receptors may have therapeutic potential in chronic inflammatory pain.
Assuntos
Dinorfinas/genética , Inflamação/genética , Dor/genética , Receptores da Bradicinina/genética , Medula Espinal/metabolismo , Adjuvantes Imunológicos , Análise de Variância , Animais , Bradicinina/administração & dosagem , Bradicinina/metabolismo , Bradicinina/farmacologia , Dinorfinas/metabolismo , Adjuvante de Freund , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Injeções Espinhais , Calidina/metabolismo , Cininogênios/genética , Masculino , Dor/induzido quimicamente , Dor/fisiopatologia , Medição da Dor/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores da Bradicinina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/efeitos dos fármacos , Trítio , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Kinins increase vascular permeability as well as mitogenesis and proliferation, hence they have a potential to promote neoplasmatic transformation. In the present study we investigated the expression profile and localization of kinin B1 and B2 receptors in colorectal polyps. The biopsy samples from various polyps were obtained during endoscopy in tubular (n=18), villous (n=15) and hyperplasic polyps (n=15). The expression of genes encoding B1 and B2 was estimated by QRT-PCR TaqMan analysis. In second series B1 and B2 receptors were visualized by immunohistochemical staining in tissue specimens from colonic polyps and adjacent normal tissue. We found the highest expression of gene encoding B1 in tubular adenomas (1891 number of copies mRNA/microg total RNA+/-312 SE) which is significantly higher as compared with controls (683+/-197 SE, p<0.013). In contrast, the expression of gene for B2 was significantly increased in hyperplastic polyps (3852+/-936 SE) as compared with controls (843+/-263 SE, p<0.0016). In normal colon a well as in hyperplasic polyps B1 and B2 receptors were immunohistochemically localized in enterocytes, however in hyperplastic polyps the intensity of staining was more prominent for B2 comparing to the control group. In contrast, in tubular adenomas staining reaction for B1 was more intense than in control samples. Increased level of B1 in adenoma suggests that kinins may play a role in abnormal cellular transformation; whereas higher B2 level in hyperplasic polyp suggests its protective role. Our data may indicate that the overall effect of kinins on cellular proliferation depends on the relative level of B1 and B2 receptor expression.
Assuntos
Pólipos do Colo/patologia , Expressão Gênica/genética , Receptores da Bradicinina/genética , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Adenoma Viloso/genética , Adenoma Viloso/metabolismo , Adenoma Viloso/patologia , Pólipos do Colo/genética , Pólipos do Colo/metabolismo , Enterócitos/química , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Humanos , Hiperplasia , Masculino , Pessoa de Meia-Idade , Receptor B1 da Bradicinina/análise , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/análise , Receptor B2 da Bradicinina/genética , Receptores da Bradicinina/análiseRESUMO
Kinins, the vasoactive peptides proteolytically liberated from kininogens, were recently recognized as signals alerting the innate immune system. Here we demonstrate that Leishmania donovani and Leishmania chagasi, two etiological agents of visceral leishmaniasis (VL), activate the kinin system. Intravital microscopy in the hamster cheek pouch showed that topically applied promastigotes induced macromolecular leakage (FITC-dextran) through postcapillary venules. Peaking at 15 min, the parasite-induced leakage was drastically enhanced by captopril (Cap), an inhibitor of angiotensin-converting enzyme (ACE), a kinin-degrading metallopeptidase. The enhanced microvascular responses were cancelled by HOE-140, an antagonist of the B2 bradykinin receptor (B2R), or by pre-treatment of promastigotes with the irreversible cysteine proteinase inhibitor N-methylpiperazine-urea-Phe-homoPhe-vinylsulfone-benzene (N-Pip-hF-VSPh). In agreement with the above-mentioned data, the promastigotes vigorously induced edema in the paw of Cap-treated J129 mice, but not Cap-B2R-/- mice. Analysis of parasite-induced breakdown of high molecular weight kininogens (HK), combined with active site-affinity-labeling with biotin-N-Pip-hF-VSPh, identified 35-40 kDa proteins as kinin-releasing cysteine peptidases. We then checked if macrophage infectivity was influenced by interplay between these kinin-releasing parasite proteases, kininogens, and kinin-degrading peptidases (i.e. ACE). Our studies revealed that full-fledged B2R engagement resulted in vigorous increase of L. chagasi uptake by resident macrophages. Evidence that inflammatory macrophages treated with HOE-140 became highly susceptible to amastigote outgrowth, assessed 72 h after initial macrophage interaction, further suggests that the kinin/B2R activation pathway may critically modulate inflammation and innate immunity in visceral leishmaniasis.
Assuntos
Permeabilidade Capilar/fisiologia , Cisteína Endopeptidases/metabolismo , Cininas/metabolismo , Leishmania donovani/enzimologia , Leishmania infantum/enzimologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Animais , Cricetinae , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptidil Dipeptidase A/metabolismo , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , Fatores de TempoRESUMO
The angiotensin-converting enzyme (ACE) is a membrane-bound peptidyl dipeptidase known to act on a variety of peptide substrates in the extracellular space. Its most notable functions are the formation of angiotensin II and the degradation of bradykinin. In the current experiments, we found that exogenous ACE added to vascular smooth muscle cell culture strongly induces and upregulates the genes of bradykinin receptors B1 and B2. This transcriptional regulatory property of ACE was shown to be unrelated to its known enzymatic properties. Indeed, ACE at 3.75 microg/ml added in the culture medium of vascular smooth muscle cells was found to cause marked upregulation of the mRNA expression of the genes for the B1 and B2 receptors of bradykinin by 22- and 11-fold, respectively. This phenomenon was not altered by the addition of specific angiotensin II antagonists for the AT1 or AT2 receptors. Moreover, the ACE inhibitor captopril, which inhibited ACE enzymatic activity, did not block its effect at the bradykinin receptor gene transcription level. Expression of both receptor genes was completely abolished by actinomycin D. Furthermore, transcriptional upregulation was inhibited by curcumin, suggesting involvement of different transcriptional factors in this phenomenon. Electrophoretic mobility shift assay revealed increase in NF-kappaB and activator protein-1 protein binding for consensus sequences, between ACE-treated cells versus untreated cells. The data indicate a novel biological function of the ACE unrelated to its well-known enzymatic function as a peptidyl dipeptidase.
Assuntos
Regulação da Expressão Gênica/fisiologia , Peptidil Dipeptidase A/fisiologia , Receptores da Bradicinina/biossíntese , Receptores da Bradicinina/genética , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Western Blotting , Núcleo Celular/química , Células Cultivadas , AMP Cíclico/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , NF-kappa B/metabolismo , RNA/biossíntese , RNA/isolamento & purificação , Ratos , Ratos Wistar , Receptor B1 da Bradicinina/biossíntese , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/biossíntese , Receptor B2 da Bradicinina/genética , Fator de Transcrição AP-1/metabolismo , Regulação para CimaRESUMO
Kinins are proinflammatory peptides that mediate numerous vascular and pain responses to tissue injury. Two pharmacologically distinct kinin receptor subtypes have been identified and characterized for these peptides, which are named B1 and B2 and belong to the rhodopsin family of G protein-coupled receptors. The B2 receptor mediates the action of bradykinin (BK) and lysyl-bradykinin (Lys-BK), the first set of bioactive kinins formed in response to injury from kininogen precursors through the actions of plasma and tissue kallikreins, whereas the B(1) receptor mediates the action of des-Arg9-BK and Lys-des-Arg9-BK, the second set of bioactive kinins formed through the actions of carboxypeptidases on BK and Lys-BK, respectively. The B2 receptor is ubiquitous and constitutively expressed, whereas the B1 receptor is expressed at a very low level in healthy tissues but induced following injury by various proinflammatory cytokines such as interleukin-1beta. Both receptors act through G alpha(q) to stimulate phospholipase C beta followed by phosphoinositide hydrolysis and intracellular free Ca2+ mobilization and through G alpha(i) to inhibit adenylate cyclase and stimulate the mitogen-activated protein kinase pathways. The use of mice lacking each receptor gene and various specific peptidic and nonpeptidic antagonists have implicated both B1 and B2 receptors as potential therapeutic targets in several pathophysiological events related to inflammation such as pain, sepsis, allergic asthma, rhinitis, and edema, as well as diabetes and cancer. This review is a comprehensive presentation of our current understanding of these receptors in terms of molecular and cell biology, physiology, pharmacology, and involvement in human disease and drug development.
Assuntos
Receptor B1 da Bradicinina , Receptor B2 da Bradicinina , Receptores da Bradicinina , Animais , Antagonistas de Receptor B1 da Bradicinina , Antagonistas de Receptor B2 da Bradicinina , Humanos , Biologia Molecular , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/metabolismo , Receptores da Bradicinina/classificação , Receptores da Bradicinina/genética , Receptores da Bradicinina/fisiologia , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Kinins, which are produced by the action of kallikrein enzymes, are blood-derived local-acting peptides that have broad effects mediated by two related G-protein-coupled receptors termed the bradykinin receptors. The endogenous kallikrein-kinin system controls blood circulation and kidney function, and promotes inflammation and pain in pathological conditions, which has led to interest in developing modulators of bradykinin receptors as potential therapeutics. This review discusses recent progress in our understanding of the genetics, molecular biology and pathophysiology of kinins and their receptors, as well as developments in medicinal chemistry, which have brought us closer to therapeutic applications of kinin receptor ligands in various indications. The potential of kinin receptor antagonists as novel analgesic agents that do not result in tolerance or have a liability for abuse has attracted particular interest.
Assuntos
Mediadores da Inflamação/fisiologia , Cininas/fisiologia , Manejo da Dor , Dor/metabolismo , Receptores da Bradicinina/fisiologia , Animais , Humanos , Cininas/química , Cininas/genética , Cininas/metabolismo , Ligantes , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismoRESUMO
G-protein-coupled receptor 100 (GPR100) was discovered by searching the human genome database for novel G-protein-coupled peptide receptors. Full-length GPR100 was amplified from a cDNA library of the neuroendocrine cell line BON, which is derived from a human pancreas carcinoid. The open-reading frame, present on a single exon, coded for a protein of 374 amino acids with highest sequence identity (43%) to the human orphan somatostatin- and angiotensin-like peptide receptor. The analysis of chromosomal localisation mapped the GPR100 gene to chromosome 1q21.2-q21.3. The stable expression of GPR100 in Chinese hamster ovary cells together with aequorin as calcium sensor and the promiscuous G-protein subunit alpha16 as signal transducer revealed bradykinin and kallidin as effectors to elicit a calcium response. Dose-response curves yielded EC50 values for both ligands in the low nanomolar range, while the respective analogues without arginine at the carboxy-terminus were inactive. Calcium mobilisation was inhibited by the phospholipase C blocker U73122, but not by pertussis toxin, suggesting the involvement of the G-protein subunit alphaq and not alphai or alphao in signal transduction. In line with the main function of kinins as peripheral hormones, we found that GPR100 was expressed predominantly in tissues like pancreas, heart, skeletal muscle, salivary gland, bladder, kidney, liver, placenta, stomach, jejunum, thyroid gland, ovary, and bone marrow, but smaller amounts were also detected in the brain and in cell lines derived from tumours of various origins.
Assuntos
Receptores da Bradicinina/química , Receptores Acoplados a Proteínas G/química , Sequência de Aminoácidos/genética , Animais , Bradicinina/farmacologia , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Dados de Sequência Molecular , Receptores da Bradicinina/agonistas , Receptores da Bradicinina/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genéticaRESUMO
Roles for the kallikrein-kinin system in inflammation have been investigated extensively, and many reviews on this topic have been published during the 50 years since the discovery of bradykinin in 1949. Recent progress in the field has been remarkable with the help of experiments using gene-targetted transgenic or knockout mice, which have added further valuable information in addition to previous results obtained from pharmacological and biochemical studies using purified and isolated components of the system. Furthermore, much knowledge has been accumulated as a result of the development of various bradykinin agonists and antagonists. In this review, we focused on the data obtained from the kininogen-deficient rat, which is a natural mutant, and discuss the results in comparison with those from bradykinin receptor knockout mice. These data have clarified that endogenous bradykinin exerts a most important role in inflammatory exudation along with prostanoids, preferentially to histamine, serotonin, or neuropeptides. In inflammatory pain perception also, bradykinin produced in the local perivascular spaces stimulates polymodal pain receptors in conjunction with co-helpers such as prostanoids, vanilloids, and neuropeptides. These important roles are concluded based on consistent results obtained from experiments using several antagonists of bradykinin, kininogen-deficient rats, and bradykinin receptor knockout mice.
Assuntos
Inflamação/metabolismo , Sistema Calicreína-Cinina/fisiologia , Cininogênios/deficiência , Cininogênios/genética , Dor/metabolismo , Ratos Endogâmicos BN/genética , Animais , Humanos , Inflamação/enzimologia , Inflamação/genética , Sistema Calicreína-Cinina/genética , Dor/enzimologia , Dor/genética , Ratos , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismoRESUMO
BACKGROUND & AIMS: The components of the kinin system, including kinongens, kininogenases, and B(2) and B(1) receptors, are expressed and activated during inflammation. Here, we investigated the expression of the kinin B(2) receptor messenger RNA, kininogen and kallikrein immunoreactivity, and the ability of kinins to contract control and inflamed gallbladders in vitro. METHODS: Human gallbladders, obtained from patients undergoing cholecystectomy either for acute cholecystitis secondary to gallstone disease or during elective gastro-entero-pancreatic surgery (controls), were processed for reverse-transcription polymerase chain reaction analysis, kallikrein and kininogen immunohistochemistry, binding studies, and in vitro contractility studies. RESULTS: Tissue expression of B(2) receptor messenger RNA and specific binding of [(3)H]-bradykinin increased significantly in acute cholecystitis compared to controls. Kallikrein immunoreactivity was detected in the epithelium and infiltrating leukocytes, whereas kininogen immunoreactivity in the lumen of blood vessels and interstitial space. Bradykinin contracted isolated strips of control and acute cholecystitis gallbladders. In acute cholecystitis tissue, efficacy of bradykinin was higher than that of control gallbladders and similar to that of cholecystokinin. The contraction induced by bradykinin was significantly attenuated by B(2) receptor antagonism but not by cyclooxygenase inhibition and B(1), muscarinic, or tachykinin receptor antagonism. CONCLUSIONS: All the components of the kinin system are expressed in the human gallbladder. Bradykinin is a powerful spasmogen via B(2) receptor activation in the normal and, especially, in the inflamed human gallbladder.
Assuntos
Bradicinina/análogos & derivados , Bradicinina/metabolismo , Esvaziamento da Vesícula Biliar/fisiologia , Vesícula Biliar/fisiologia , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Adulto , Idoso , Antipsicóticos/farmacologia , Atropina/farmacologia , Benzamidas/farmacologia , Bradicinina/farmacologia , Antagonistas dos Receptores da Bradicinina , Inibidores de Ciclo-Oxigenase/farmacologia , Feminino , Esvaziamento da Vesícula Biliar/efeitos dos fármacos , Expressão Gênica , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Indometacina/farmacologia , Masculino , Pessoa de Meia-Idade , Antagonistas Muscarínicos/farmacologia , Piperidinas/farmacologia , Quinuclidinas/farmacologia , Receptor B1 da Bradicinina , Receptor B2 da Bradicinina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TrítioRESUMO
Although p53 is known to have dual functions as a transcriptional activator and repressor, there has not been an example where both p53-activating and -repressing elements reside within one target promoter. Previous work from this laboratory defined two different p53 response elements, termed P1 and P2, located at nucleotide positions -70 and -707, respectively, in the rat bradykinin B2 receptor promoter. In this study, through manipulation of the DNA sequence and context, we demonstrate opposing roles for P1 and P2 as transcriptional activator and repressor, respectively. Deletion of P1 abrogates p53-mediated activation. P1 maintains its role as an activator upon relocation to the P2 site and activates transcription from a heterologous promoter construct. Thus, P1 is a bona fide positive p53-response element. In contrast, deletion of P2 enhances P1-induced activation. P2 represses transcription when substituted for P1 or when relocated midway between P1 and P2. P2-mediated repression is sequence-dependent, because it is reversed to activation when P2 is substituted by the P1 or p53 consensus sequences. Moreover, site-directed mutagenesis that converts P2 to a higher affinity p53-binding site results in transcriptional activation rather than repression. Surprisingly, P2 strongly activates a heterologous promoter. Thus, P2-mediated transcriptional repression is both sequence- and context-dependent. Investigations into the mechanisms of P2-mediated repression indicate that it is trichostatin-insensitive and unaffected by CBP or mutation of the minimal repression C-terminal domain of p53. However, gel shift assays suggest that p53 competes with other transcriptional activators for binding to overlapping binding sequences within the P2 element. In conclusion, this study provides a rare example of a transcription factor having two divergent functional effects that are sequence- and context-dependent. The interplay of P1 and P2 may be important in the regulation of bradykinin B2 receptor gene expression in response to inflammatory stress and during development.