RESUMEN
Cumulative evidence has established that macrophages orchestrate inflammatory responses that crucially contribute to the pathogenesis of insulin-resistant obesity and type 2 diabetes. In the present study, we examined the impact of hyperglycemia on macrophage pro-inflammatory responses under an inflammatory stimulus. To conduct this study, RAW264.7 macrophages were cultured under normal- (5.5 mM) or high-glucose (22 or 40 mM) conditions for 7 days and stimulated with lipopolysaccharide (LPS). Long-term exposure to high glucose significantly enhanced the increase in the production of pro-inflammatory cytokines, including tumor necrosis-α, interleukin (IL)-1ß, and IL-6, when macrophages were stimulated with LPS. The LPS-induced increases in inducible nitric oxide (NO) synthase (iNOS) expression and NO production were also significantly enhanced by long-term exposure of macrophages to high glucose. Treatment with N-acetyl-L-cysteine, a widely used thiol-containing antioxidant, blunted the enhancement of the LPS-induced upregulation of pro-inflammatory cytokine production, iNOS expression, and NO production in macrophages. When intracellular reactive oxygen species (ROS) were visualized using the fluorescence dye 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein diacetate, acetyl ester, a significant increase in ROS generation was found after stimulation of macrophages with LPS, and this increased ROS generation was exacerbated under long-term high-glucose conditions. LPS-induced translocation of phosphorylated nuclear factor-κB (NF-κB), a transcription factor regulating many pro-inflammatory genes, into the nucleus was promoted under long-term high-glucose conditions. Altogether, the present results indicate that a long-term high-glucose environment can enhance activation of NF-κB in LPS-stimulated macrophages possibly due to excessive ROS production, thereby leading to increased macrophage pro-inflammatory responses.
Asunto(s)
Glucosa/farmacología , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Citocinas/metabolismo , Hemo-Oxigenasa 1/metabolismo , Hiperglucemia/inducido químicamente , Hiperglucemia/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Células RAW 264.7RESUMEN
Vascular endothelial growth factor (VEGF) is a prime regulator of vascular permeability. Acute lung injury (ALI) is characterized by high-permeability pulmonary edema in addition to refractory hypoxemia and diffuse pulmonary infiltrates. In this study, we examined whether VEGF can be implicated as a pulmonary vascular permeability factor in sepsis-associated ALI. We found that a great increase in lung vascular leak occurred in mice instilled intranasally with lipopolysaccharide (LPS), as assessed by IgM levels in bronchoalveolar lavage fluid. Treatment with the VEGF-neutralizing monoclonal antibody bevacizumab significantly reduced this hyperpermeability response, suggesting active participation of VEGF in non-cardiogenic lung edema associated with LPS-induced ALI. However, this was not solely attributable to excessive levels of intrapulmonary VEGF. Expression levels of VEGF were significantly reduced in lung tissues from mice with both intranasal LPS administration and cecal ligation and puncture (CLP)-induced sepsis, which may stem from decreases in non-endothelial cells-dependent VEGF production in the lungs. In support of this assumption, stimulation with LPS and interferon-γ (IFN-γ) significantly increased VEGF in human pulmonary microvascular endothelial cells (HPMECs) at mRNA and protein levels. Furthermore, a significant rise in plasma VEGF levels was observed in CLP-induced septic mice. The increase in VEGF released from HPMECs after LPS/IFN-γ challenge was completely blocked by either specific inhibitor of mitogen-activated protein kinase (MAPK) subgroups. Taken together, our results indicate that VEGF can contribute to the development of non-cardiogenic lung edema in sepsis-associated ALI due to increased VEGF secretion from pulmonary vascular endothelial cells through multiple MAPK-dependent pathways.
Asunto(s)
Lesión Pulmonar Aguda/sangre , Permeabilidad Capilar/fisiología , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Sepsis/sangre , Factor A de Crecimiento Endotelial Vascular/sangre , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/patología , Animales , Línea Celular Transformada , Humanos , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Sepsis/complicaciones , Sepsis/patologíaRESUMEN
Pituitary tumors (PTs) can cause significant mortality and morbidity due to limited therapeutic options. L-type amino acid transporters (LATs), in particular, the LAT1 isoform, is expressed in a variety of tumor cells. Pharmacological inhibition or genetic ablation of LAT1 can suppress leucine transport into cancer cells, resulting in suppression of cancer cell growth. However, roles of LAT1 in PTs have not been elucidated. Therefore, we assessed LAT1 expression in PTs and evaluated a LAT1-specific inhibitor, JPH203, on rat somatomammotroph tumor cells, GH4 cells. GH4 cells dominantly express LAT1 mRNA rather than other LAT isoforms, whereas LAT2 transcripts were most abundant in normal rat pituitary tissues. JPH203 inhibited leucine uptake and cell growth in GH4 cells in a concentration-dependent manner, and appeared to be independent of the mechanistic target, the rapamycin pathway. Although JPH203 did not induce apoptosis, it suppressed growth hormone production in GH4 cells. Also, genetic downregulation of LAT1 showed similar effects on cell growth and hormone production. These results indicated that restriction of LAT1 substrates by JPH203 modulated both cell growth and hormone production. In conclusion, LAT1 may be a new therapeutic target for PTs because its inhibition leads to suppression of cell growth as well as hormone production. JPH203 may represent a promising drug for clinical use in patients with PTs, with the potential of hormonal control and tumor suppression.
Asunto(s)
Hormona del Crecimiento/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Neoplasias Hipofisarias/metabolismo , Animales , Apoptosis/fisiología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/fisiología , Regulación hacia Abajo/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Células HEK293 , Humanos , Hipófisis/metabolismo , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , RatasRESUMEN
Pulmonary endothelial injury is central in the pathogenesis of acute lung injury (ALI). The MAPK signaling cascades are generally thought to be involved in the molecular mechanism underlying the ALI development, but their roles in pulmonary endothelial injury is poorly understood. We thus examined the involvement of the MAPK family member in inflammatory responses of human pulmonary microvascular endothelial cells (HPMVECs) stimulated with LPS and IFN-γ. HPMVECs were found to exhibit the upregulation of expression of Toll-like receptor 4 by IFN-γ, resulting in potentiation of inflammatory cytokine release by LPS stimulation. All MAPKs, ERK1/2, JNK, and p38, were activated by simultaneous stimulation with LPS/IFN-γ. JNK activation in cells stimulated with LPS/IFN-γ was significantly potentiated by the two different p38 inhibitors, SB203580 and RWJ67657, suggesting the negative regulation of JNK activation by p38 in HPMVECs. The mRNA and protein expression levels of ICAM-1 were eliminated by the JNK inhibitor, suggesting that ICAM-1 expression is positively regulated by JNK. The p38 inhibitor significantly enhanced ICAM-1 expression. ERK1/2 activation was not responsible for the LPS/IFN-γ-induced ICAM-1 upregulation in HPMVECs. THP-1 monocyte adhesion to HPMVECs under LPS/IFN-γ stimulation was inhibited by the JNK inhibitor and enhanced by the p38 inhibitor. We conclude that, in HPMVECs stimulated with LPS/IFN-γ, JNK mediates ICAM-1 expression that can facilitate leukocyte adherence and transmigration, while p38 MAPK negatively regulates the upregulation of ICAM-1 through inhibition of JNK activation.
Asunto(s)
Células Endoteliales/metabolismo , Interferón gamma/metabolismo , Lipopolisacáridos/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Biomarcadores , Adhesión Celular/inmunología , Línea Celular , Citocinas/inmunología , Citocinas/metabolismo , Células Endoteliales/efectos de los fármacos , Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Neumonía/etiología , Neumonía/metabolismo , Neumonía/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Levosimendan and milrinone may be used in place of dobutamine to increase cardiac output in septic patients with a low cardiac output due to impaired cardiac function. The effects of the two inotropic agents on cardiac inflammation and left ventricular (LV) performance were examined in mice with cecal ligation and puncture (CLP)-induced sepsis. CLP mice displayed significant cardiac inflammation, as indicated by highly increased pro-inflammatory cytokines and neutrophil infiltration in myocardial tissues. When continuously given, levosimendan prevented but milrinone exaggerated cardiac inflammation, but they significantly reduced the elevations in plasma cardiac troponin-I and heart-type fatty acid-binding protein, clinical markers of cardiac injury. Echocardiographic assessment of cardiac function showed that the effect of levosimendan, given by an intravenous bolus injection, on LV performance was impaired in CLP mice, whereas milrinone produced inotropic responses equally in sham-operated and CLP mice. A lesser effect of levosimendan on LV performance after CLP was also found in spontaneously beating Langendorff-perfused hearts. In ventricular myocytes isolated from control and CLP mice, levosimendan, but not milrinone, caused a large increase in the L-type calcium current. This study represents that levosimendan and milrinone have cardioprotective properties but provide different advantages and drawbacks to cardiac inflammation/dysfunction in sepsis.
Asunto(s)
Cardiotónicos/uso terapéutico , Milrinona/uso terapéutico , Sepsis/tratamiento farmacológico , Simendán/uso terapéutico , Animales , Calcio/fisiología , Ciego/cirugía , Citocinas/genética , Ligadura , Masculino , Ratones Endogámicos BALB C , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Sepsis/etiología , Sepsis/genética , Sepsis/fisiopatología , Función Ventricular Izquierda/efectos de los fármacos , Heridas Penetrantes/complicacionesRESUMEN
Despite advances in overall medical care, sepsis and its sequelae continue to be an embarrassing clinical entity with an unacceptably high mortality rate. The central reason for high morbidity and high mortality of sepsis and its sequelae is the lack of an effective treatment. Previous clinical trials have largely failed to identify an effective therapeutic target to improve clinical outcomes in sepsis. Thus, the key goal favoring the outcome of septic patients is to devise innovative and evolutionary therapeutic strategies. Gene therapy can be considered as one of the most promising novel therapeutic approaches for nasty disorders. Since a number of transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), play a pivotal role in the pathophysiology of sepsis that can be characterized by the induction of multiple genes and their products, sepsis may be regarded as a gene-related disorder and gene therapy may be considered a promising novel therapeutic approach for treatment of sepsis. In this review article, we provide an up-to-date summary of the gene-targeting approaches, which have been developed in animal models of sepsis. Our review sheds light on the molecular basis of sepsis pathology for the development of novel gene therapy approaches and leads to the conclusion that future research efforts may fully take into account gene therapy for the treatment of sepsis.
Asunto(s)
Terapia Genética/métodos , MicroARNs/genética , FN-kappa B/genética , Sepsis/terapia , Factor de Transcripción AP-1/genética , Animales , Apoptosis/genética , Regulación de la Expresión Génica/genética , Humanos , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/uso terapéutico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Sepsis/epidemiología , Sepsis/genética , Resultado del TratamientoRESUMEN
Sepsis is the leading cause of death in critically ill patients, and its incidence continues to rise. Sepsis was defined as a systemic inflammatory response syndrome with an identifiable focus of infection, but therapeutic strategies aimed at eliminating the inflammatory response have only modest clinical benefit. The development of a failure of one or more organs poses a major threat to the survival of patients with sepsis, and mortality in sepsis is most often attributed to multiple organ dysfunction. Accordingly, sepsis has been recently redefined as life-threatening organ dysfunction due to a dysregulated host response to infection. Cardiac dysfunction is a well-recognized important component of septic multiple organ failure and can compromise the balance between oxygen supply and demand, ultimately leading to the development of multiple organ failure. The existence of cardiac dysfunction in sepsis is associated with much higher mortality when compared with septic patients without heart problems. Dobutamine, a ß1-selective adrenoceptor agonist, has been used in septic shock for many years as an only inotrope, but limited clinical outcome measures have been provided as to advisability of the usefulness of dobutamine in septic shock management. Here we provide an overview on the possible mechanisms underlying intrinsic myocardial depression during sepsis and discuss the perspective of several inotropes for sepsis-associated cardiac dysfunction.
Asunto(s)
Cardiomiopatías/dietoterapia , Cardiomiopatías/metabolismo , Choque Séptico/metabolismo , Animales , Cardiomiopatías/etiología , Cardiomiopatías/fisiopatología , Humanos , Terapia Molecular Dirigida , Choque Séptico/etiologíaRESUMEN
Dobutamine has been used in septic shock for many years as an only inotrope, but its benefit has been questioned. We weighed the effects of dobutamine and milrinone as inotropes in mice with cecal ligation and puncture (CLP)-induced polymicrobial sepsis. CLP-induced septic mice exhibited significant cardiac inflammation, as indicated by greatly increased mRNAs of proinflammatory cytokines and robust infiltration of inflammatory cells in the ventricular myocardium. Elevations of plasma cardiac troponin-I showed cardiac injury in CLP mice. Noninvasive echocardiographic assessment of cardiac function revealed that despite preserved left ventricular function in the presence of fluid replacement, the dobutamine inotropic response was significantly impaired in CLP mice compared with sham-operated controls. By contrast, milrinone exerted inotropic effects in sham-operated and CLP mice in an equally effective manner. Surface expression levels of ß1-adrenoceptors and α-subunits of three main G protein families in the myocardium were unaffected by CLP-induced sepsis. Plasma cAMP levels were significantly elevated in both sham-operated and CLP mice in response to milrinone but only in sham-operated controls in response to dobutamine. Of phosphodiesterase (PDE) isoforms, PDE4D, but not PDE3A, both of which are responsible for cardiac cAMP hydrolysis, was significantly upregulated in CLP mouse myocardium. We define a novel mechanism for the impaired responsiveness to dobutamine as an inotrope in sepsis, and understanding the role of PDE4D in modulating cardiac functional responsiveness in sepsis may open the potential of a PDE4D-targeted therapeutic option in septic patients with low cardiac output who have a need for inotropic support.NEW & NOTEWORTHY Advisability of the usefulness of dobutamine in septic shock management is limited. Here, we reveal that the effect of dobutamine as a positive inotrope is impaired in mice with cecal ligation and puncture-induced sepsis without changes in cardiac ß1-adrenoceptor signaling as a result of cAMP breakdown achieved by upregulated phosphodiesterase 4D.
Asunto(s)
Cardiotónicos/farmacología , Ciego/cirugía , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Dobutamina/farmacología , Milrinona/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocardio/enzimología , Inhibidores de Fosfodiesterasa 3/farmacología , Sepsis/tratamiento farmacológico , Sepsis/enzimología , Adenilil Ciclasas/metabolismo , Animales , Ciego/microbiología , AMP Cíclico/sangre , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Hidrólisis , Mediadores de Inflamación/metabolismo , Ligadura , Masculino , Ratones Endogámicos BALB C , Punciones , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Sepsis/microbiología , Sepsis/fisiopatología , Transducción de Señal , Regulación hacia ArribaRESUMEN
The effect of resveratrol on various human cancer cells was investigated with special focus on apoptotic cell death, in an attempt to further characterize its mechanism of action. There were great differences in the anti-viability effect of resveratrol between different types of human cancer cells. While the inhibition of cell viability by resveratrol was marked in U937 and MOLT-4 leukemia cells, resveratrol moderately inhibited cell viability in MCF-7 breast, HepG2 liver, and A549 lung cancer cells, and the effect was slight on cell viability in Caco-2, HCT116, and SW480 colon cancer cells. Following resveratrol treatment, U937 and MOLT-4 markedly increased the population of late apoptotic cells but MCF-7 and HepG2 underwent apoptosis with an increased population of early apoptosis, and resveratrol-induced DNA fragmentation was observed only in leukemic cells. Activation of sirtuin 1 and adenosine-monophosphate-activated protein kinase was not responsible for resveratrol-induced cancer cell death. Instead, resveratrol significantly reduced Akt activation with the downregulation of H-Ras, resulting in facilitation of Bax translocation to mitochondria in leukemic cells. This study suggests that resveratrol can induce apoptotic cell death in human leukemic cells to a greater extent than in human solid tumor cells via reducing Akt activation due to Ras downregulation.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Estilbenos/farmacología , Células A549 , Células CACO-2 , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células HCT116 , Células Hep G2 , Humanos , Células MCF-7 , Mitocondrias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Resveratrol , Sirtuina 1/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Sepsis is one of the most common reasons for critically ill patients to be admitted to an intensive care unit and, despite advances in overall medical care, it represents a major clinical problem and remains the leading cause of death in the critically ill patient population. Although sepsis has been defined as a systemic inflammatory syndrome, in which there is an identifiable focus of infection, clinical trials aimed at anti-inflammatory therapeutic approaches have largely failed to identify an effective therapeutic target to improve clinical outcomes in sepsis. Very recently, the third international consensus definitions have been advocated for sepsis and septic shock. Thus, sepsis is now defined as life-threatening organ dysfunction due to a dysregulated host response to infection. A better understanding of the molecular mechanisms involved in the pathogenesis of sepsis and its resultant organ failure has been sought, and the development of therapies targeted at preventing or limiting molecular events associated with the progress of fatal organ failure, hence leading to improvement of outcomes, is urgently needed. This review article provides an overview of possible pathogenic mechanisms underlying the development of multiple organ dysfunction in sepsis and discusses pharmacological agents regarded as promising in treatment of this disorder.
Asunto(s)
Insuficiencia Multiorgánica , Sepsis , Animales , Apoptosis , Citocinas/inmunología , Células Endoteliales/metabolismo , Humanos , Insuficiencia Multiorgánica/tratamiento farmacológico , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/inmunología , Insuficiencia Multiorgánica/metabolismo , FN-kappa B/metabolismo , Sepsis/complicaciones , Sepsis/tratamiento farmacológico , Sepsis/inmunología , Sepsis/metabolismoRESUMEN
Glycemic fluctuations may play a critical role in the pathogenesis of diabetic complications, such as cardiovascular disease. We investigated whether the oxycarotenoid astaxanthin can reduce the detrimental effects of fluctuating glucose on vascular endothelial cells. Human umbilical venous endothelial cells were incubated for 3 days in media containing 5.5mM glucose, 22 mM glucose, or 5.5mM glucose alternating with 22 mM glucose in the absence or presence of astaxanthin or N-acetyl-L-cysteine (NAC). Constant high glucose increased reactive oxygen species (ROS) generation, but such an effect was more pronounced in fluctuating glucose. This was associated with up-regulated p22(phox) expression and down-regulated peroxisome proliferator activated receptor-γ coactivator (PGC-1α) expression. Astaxanthin inhibited ROS generation, p22(phox) up-regulation, and PGC-1α down-regulation by the stimuli of glucose fluctuation. Fluctuating glucose, but not constant high glucose, significantly decreased the endothelial nitric oxide synthase (eNOS) phosphorylation level at Ser-1177 without affecting total eNOS expression, which was prevented by astaxanthin as well as by the anti-oxidant NAC. Transferase-mediated dUTP nick end labeling (TUNEL) showed increased cell apoptosis in fluctuating glucose. Glucose fluctuation also resulted in up-regulating gene expression of pro-inflammatory mediators, interleukin-6 and intercellular adhesion molecule-1. These adverse changes were subdued by astaxanthin. The phosphorylation levels of c-Jun N-terminal kinase (JNK) and p38 were significantly increased by glucose fluctuations, and astaxanthin significantly inhibited the increase in JNK and p38 phosphorylation. Taken together, our results suggest that astaxanthin can protect vascular endothelial cells against glucose fluctuation by reducing ROS generation.
Asunto(s)
Antioxidantes/farmacología , Glucosa/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Estrés Oxidativo/efectos de los fármacos , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Glucosa/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Inflamación/genética , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/genética , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/genética , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Xantófilas/farmacologíaRESUMEN
G protein-coupled receptor kinase 2(GRK2) is a ubiquitous member of the family of GRKs that are serine/threonine kinases originally discovered for their role in the process of desensitization of agonist-activated G protein-coupled receptors (GPCRs). However, emerging evidence suggests that GRK2 can phosphorylate a large number of non-GPCR substrates and interact with a plethora of proteins involved in signaling and trafficking, suggesting that GRK2 would participate in the regulation of diverse cellular responses in a phosphorylation-dependent and -independent manner. Alternations in GRK2 levels and/or activity are demonstrated in an array of relevant cardiovascular, metabolic, inflammatory, or cancer pathologies. These changes are assumed to contribute to the onset and/or development of such pathologies. Thus, GRK2 may serve as a potentially interesting therapeutic target and those drugs targeted for GRK2 may constitute a novel therapeutic strategy for several intractable diseases, including sepsis.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , HumanosRESUMEN
Adenosine and its precursors, ATP and ADP, exert various physiological effects via binding to purinergic receptors. We previously used co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and immunoelectron microscopy to demonstrate the hetero-oligomerization of purinergic receptor subtypes. Furthermore, pharmacological studies found significant changes in receptor-mediated signaling in human embryonic kidney (HEK) 293T cells co-transfected with these receptors. These findings suggest that heterodimers of purinergic receptors may have distinct pharmacological profiles, possibly due to dimerization-induced conformational changes, further suggesting that hetero-dimerization may be employed to "fine-tune" purinergic receptor signaling. Adenosine A(2A) receptor (A(2A)R), P2Y1 receptor (P2Y1R) and P2Y12 receptor (P2Y12R) are predominantly expressed on human platelets. ADP activates human platelets by stimulating both P2Y1R and P2Y12R, which act sequentially and in concert to achieve complete platelet aggregation. In contrast, adenosine stimulates Gs-coupled A(2A)R, followed by activativation of adenylate cyclase, leading to an increase in intracellular cAMP levels, which potently inhibits platelet activation. We examined the hetero-oligomerization and functional interactions of A(2A)R, P2Y1R, and P2Y12R. In HEK293T cells triply expressing all three receptors, hetero-oligomerization was observed among the three receptors. Additionally, P2Y1R agonist-evoked Ca(2+) signaling was significantly inhibited by co-treatment with an A(2A)R antagonist in HEK293T cells. In human platelets, we identified endogenous A(2A)R/P2Y1R and A(2A)R/P2Y12R heterodimers. We also observed functional Ca(2+)-signaling-related cross-talk similar to those found in HEK293T cells, and found that they appeared to affect platelet shape. These results collectively suggest that intermolecular signal transduction and specific conformational changes occur among components of the hetero-oligomers formed by these three receptors.
Asunto(s)
Plaquetas/química , Plaquetas/citología , Receptores Purinérgicos/fisiología , Adenosina Difosfato/fisiología , Dimerización , Humanos , Agregación Plaquetaria/fisiología , Receptor Cross-Talk/fisiología , Receptor de Adenosina A2A/fisiología , Receptores Purinérgicos/análisis , Receptores Purinérgicos P2Y1/fisiología , Receptores Purinérgicos P2Y12/fisiologíaRESUMEN
G protein-coupled receptors (GPCRs) are classified into a family of seven transmembrane receptors. Receptor oligomerization may be the key to the expression and function of these receptors, for example, ligand binding, desensitization, membrane trafficking, and signaling. The accumulation of evidence that GPCRs form an oligomerization with a functional alternation may change the strategy for the discovery of novel drugs targeting GPCRs. Identification of the oligomer is essential to elucidate GPCR oligomerization. GPCR oligomerizations have been demonstrated using various biochemical approaches, which include the coimmunoprecipitation method, fluorescence resonance energy transfer assay, and bioluminescence RET assay. Thus, various assays are useful for the study of GPCR oligomerization, and we should choose the best method to match the purpose. We previously targeted adenosine A1 and thromboxane A2 (TP) receptors to form a functionally novel hetero-oligomer, since both receptors function in the same cells. This chapter describes the methods used to detect GPCR oligomerization and alterations in the signaling pathways, principally according to our findings on oligomerization between adenosine A1 and TPα receptors.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Proteínas Fluorescentes Verdes/metabolismo , Luciferasas de Renilla/metabolismo , Receptor de Adenosina A1/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Unión Competitiva , AMP Cíclico/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Inmunoprecipitación , Cinética , Luciferasas de Renilla/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Plásmidos , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , Receptor de Adenosina A1/química , Receptor de Adenosina A1/genética , Receptores de Tromboxano A2 y Prostaglandina H2/química , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Transducción de Señal , TransfecciónRESUMEN
The formation of homo- and hetero-oligomers between various G protein-coupled receptors (GPCRs) has been demonstrated over the past decade. In most cases, GPCR heterodimerization increases the diversity of intracellular signaling. GPCR-type purinergic receptors (adenosine and P2Y receptors) are actively reported to form hetero-oligomers with each other, with GPCRs belonging to the same group (type 1, rhodopsin-like), and even with GPCRs from another group. This chapter describes common strategies to identify dimerization of purinergic receptors (coimmunoprecipitation, bioluminescence resonance energy transfer (BRET), and immunoelectron microscopy) and to assess the alteration of their pharmacology (ligand binding, intracellular cAMP, and intracellular Ca(2+) assays). We have reported dimerization of purinergic receptors using these strategies in transfected human embryonic kidney 293T cells and native brain tissue. Our data suggest that homo- and hetero-oligomerization between purinergic receptors exert unique pharmacology in this receptor group. According to these discoveries, heterodimerization is likely to be employed for the "fine-tuning" of purinergic receptor signaling.
Asunto(s)
Receptores Purinérgicos/análisis , Receptores Purinérgicos/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación/métodos , Mediciones Luminiscentes/métodos , Microscopía Electrónica/métodos , Multimerización de Proteína , Transducción de SeñalRESUMEN
Growing evidence indicates that G protein-coupled receptors can form homo- and hetero-oligomers to diversify signal transduction. However, the molecular mechanisms and physiological significance of G protein-coupled receptor-oligomers are not fully understood. Both ADOR1 (adenosine A(1) receptor) and TBXA2R (thromboxane A(2) receptor α; TPα receptor), members of the G protein-coupled receptor family, act on astrocytes and renal mesangial cells, suggesting certain functional correlations. In this study, we explored the possibility that adenosine A(1) and TPα receptors form hetero-oligomers with novel pharmacological profiles. We showed that these receptors hetero-oligomerize by conducting coimmunoprecipitation and bioluminescence resonance energy transfer (BRET(2)) assays in adenosine A(1) receptor and TPα receptor-cotransfected HEK293T cells. Furthermore, coexpression of the receptors affected signal transduction including the accumulation of cyclic AMP and phosphorylation of extracellular signal-regulated kinase-1 and -2 was significantly increased by high and low concentrations of adenosine A(1) receptor agonist and TPα agonists, respectively. Our study provides evidence of hetero-oligomerization between adenosine A(1) and TPα receptors for the first time, and suggests that this oligomerization affects signal transduction responding to different concentrations of receptor agonists.
Asunto(s)
Agonistas del Receptor de Adenosina A1/farmacología , Multimerización de Proteína/efectos de los fármacos , Receptor de Adenosina A1/química , Receptor de Adenosina A1/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/agonistas , Receptores de Tromboxano A2 y Prostaglandina H2/química , Transducción de Señal/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Estructura Cuaternaria de Proteína , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismoRESUMEN
A(2A) adenosine receptor (A(2A)R), P2Y(1) receptor (P2Y(1)R) and P2Y(12) receptor (P2Y(12)R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca(2+) signaling evoked by the P2Y(1)R agonist, 2-methylthioladenosine 5' diphosphate (2MeSADP) was significantly inhibited by the A(2A)R antagonist (ZM241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-α][1,3,5]triazin-5-yl amino]ethyl) phenol) and SCH442416) and the P2Y(12)R antagonist (ARC69931MX) (N6-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-ß,γ-dichloromethylene-ATP)) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y(1)R signaling by A(2A)R and P2Y(12)R antagonists was indeed mediated through A(2A)R and P2Y(12)R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.
Asunto(s)
Receptor de Adenosina A2A/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Antagonistas del Receptor de Adenosina A2/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Ligandos , Unión Proteica , Agonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Triazinas/farmacología , Triazoles/farmacologíaRESUMEN
BACKGROUND: Purines such as adenosine and ATP are now generally recognized as the regulators of many physiological functions, such as neurotransmission, pain, cardiac function, and immune responses. Purines exert their functions via purinergic receptors, which are divided into adenosine and P2 receptors. Recently, we demonstrated that the Gi/o-coupled adenosine A1 receptor (A1R) and Gq/11-coupled P2Y2 receptor (P2Y2R) form a heteromeric complex with unique pharmacology in co-transfected human embryonic kidney cells (HEK293T). However, the heteromeric interaction of A1R and P2Y2R in situ in brain is still largely unknown. FINDINGS: In the present study, we visualized the surface expression and co-localization of A1R and P2Y2R in both transfected HEK293T cells and in rat brain by confocal microscopy and more precisely by immunogold electron microscopy. Immunogold electron microscopy showed the evidence for the existence of homo- and hetero-dimers among A1R and P2Y2R at the neurons in cortex, cerebellum, and particularly cerebellar Purkinje cells, also supported by co-immunoprecipitation study. CONCLUSION: The results suggest that evidence for the existence of homo- and hetero-dimers of A1R and P2Y2R, not only in co-transfected cultured cells, but also in situ on the surface of neurons in various brain regions. While the homo-dimerization ratios displayed similar patterns in all three regions, the rates of hetero-dimerization were prominent in hippocampal pyramidal cells among the three regions.
RESUMEN
It is well accepted that G protein-coupled receptors (GPCRs) arrange into dimers or higher-order oligomers that may modify various functions of GPCRs. GPCR-type purinergic receptors (i.e. adenosine and P2Y receptors) tend to form heterodimers with GPCRs not only of the different families but also of the same purinergic receptor families, leading to alterations in functional properties. In the present review, we focus on current knowledge of the formation of heterodimers between metabotropic purinergic receptors that activate novel functions in response to extracellular nucleosides/nucleotides, revealing that the dimerization seems to be employed for 'fine-tuning' of purinergic signaling. Thus, the relationship between adenosine and adenosine triphosphate is likely to be more and more intimate than simply being a metabolite of the other.