RESUMEN
Brain capillary pericytes have been suggested to play a role in the regulation of cerebral blood flow under physiological and pathophysiological conditions. ATP has been shown to cause constriction of capillaries under ischemic conditions and suggested to be involved in the "no-reflow" phenomenon. To investigate the effects of extracellular ATP on pericyte cell contraction, we studied purinergic receptor activation of cultured bovine brain capillary pericytes. We measured intracellular Ca2+ concentration ([Ca2+]i) responses to purinergic agonists with the fluorescent indicators fura-2 and Cal-520 and estimated contraction of pericytes as relative change in cell area, using real-time confocal imaging. Addition of ATP caused an increase in cytosolic calcium and contraction of the brain capillary pericytes, both reversible and inhibited by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Furthermore, we demonstrated that ATP-induced contraction could be eliminated by intracellular calcium chelation with BAPTA, indicating that the contraction was mediated via purinergic P2-type receptor-mediated [Ca2+]i signaling. ATP stimulation induced inositol triphosphate signaling, consistent with the notion of P2Y receptor activation. Receptor profiling studies demonstrated the presence of P2Y1 and P2Y2 receptors, using ATP, UTP, ADP, and the subtype specific agonists MRS2365 (P2Y1) and 2-thio-UTP (P2Y2). Addition of specific P2X agonists only caused an [Ca2+]i increase at high concentrations, attributed to activation of inositol triphosphate signaling. Our results suggest that contraction of brain capillary pericytes in vitro by activation of P2Y-type purinergic receptors is caused by intracellular calcium release. This adds more mechanistic understanding of the role of pericytes in vessel constriction and points toward P2Y receptors as potential therapeutic targets.NEW & NOTEWORTHY The study concerns brain capillary pericytes, which have been suggested to play a role in the regulation of cerebral blood flow. We show that extracellular ATP causes contraction of primary brain pericytes by stimulation of purinergic receptors and subsequent release of intracellular Ca2+ concentration ([Ca2+]i). The contraction is mainly mediated through activation of P2Y-receptor subtypes, including P2Y1 and P2Y2. These findings add more mechanistic understanding of the role of pericytes in regulation of capillary blood flow. ATP was earlier suggested to be involved in capillary constriction in brain pathologies, and our study gives a detailed account of a part of this important mechanism.
Asunto(s)
Adenosina Trifosfato/farmacología , Encéfalo/irrigación sanguínea , Señalización del Calcio/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Pericitos/efectos de los fármacos , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y/efectos de los fármacos , Animales , Capilares/citología , Bovinos , Células Cultivadas , Inositol 1,4,5-Trifosfato/metabolismo , Pericitos/metabolismo , Fenotipo , Receptores Purinérgicos P2Y/metabolismo , Receptores Purinérgicos P2Y1/efectos de los fármacos , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y2/efectos de los fármacos , Receptores Purinérgicos P2Y2/metabolismoRESUMEN
Esophageal cancer is a prominent worldwide illness that is divided into two main subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Mortality rates are alarming, and the understanding of the mechanisms involved in esophageal cancer development, becomes essential. Purinergic signaling is related to many diseases and among these various types of tumors. Here we studied the effects of the P2Y2 receptor activation in different types of esophageal cancer. Esophageal tissue samples of healthy controls were used for P2Y2R expression quantification. Two human esophageal cancer cell lines Kyse-450 (squamous cell carcinoma) and OE-33 (adenocarcinoma) were used to perform in vitro analysis of cell proliferation, migration, adhesion, and the signaling pathways involved in P2Y2R activation. Data showed that P2Y2R was expressed in biopsies of patients with ESCC and adenocarcinoma, as well as in the two human esophageal cancer cell lines studied. The RT-qPCR analysis demonstrated that OE-33 cells have higher P2RY2 expression than Kyse-450 squamous cell line. Results showed that P2Y2R activation, induced by ATP or UTP, promoted esophageal cancer cells proliferation and colony formation. P2Y2R blockage with the selective antagonist, AR-C 118925XX, led to decreased proliferation, colony formation and adhesion. Treatments with ATP or UTP activated ERK 1/2 pathway in ESCC and ECA cells. The P2Y2R antagonism did not alter the migration of esophageal cancer cells. Interestingly, the esophageal cancer cell lines presented a distinct profile of nucleotide hydrolysis activity. The modulation of P2Y2 receptors may be a promising target for esophageal cancer treatment.
Asunto(s)
Adenocarcinoma/enzimología , Carcinoma de Células Escamosas/enzimología , Proliferación Celular/efectos de los fármacos , Neoplasias Esofágicas/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adenosina Trifosfato/farmacología , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosforilación , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal , Uridina Trifosfato/farmacologíaRESUMEN
Extracellular nucleotides mediate multiple physiological effects such as proliferation, differentiation, or induction of apoptosis through G protein-coupled P2Y receptors or P2X ion channels. Evaluation of the complete physiological role of nucleotides has long been hampered by a lack of potent and selective ligands for all P2 subtypes. Meanwhile, for most of the P2 receptors, selective ligands are available, but only a few potent and selective P2Y2 receptor antagonists are described. This limits the understanding of the role of P2Y2 receptors. The purpose of this study was to search for P2Y2 receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y2 receptors recombinantly expressed in human 1321N1 astrocytoma cells identified NF272 [8-(4-methyl-3-(3-phenoxycarbonylimino-benzamido)benzamido)-naphthalene-1,3,5-trisulfonic acid trisodium salt] as a competitive P2Y2 receptor antagonist with a Ki of 19 µM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA2 value of 5.03 ± 0.22 (mean ± SEM) with a slope not significantly different from unity. Among uracil-nucleotide-preferring P2Y receptors, NF272 shows a moderate selectivity over P2Y4 (3.6-fold) and P2Y6 (5.7-fold). However, NF272 is equipotent at P2Y1, and even more potent at P2Y11 and P2Y12 receptors. Up to 250 µM, NF272 showed no cytotoxicity in MTT cell viability assays in 1321N1, HEK293, and OVCAR-3 cells. Further, NF272 was able to inhibit the ATP-induced calcium signal in OVCAR-3 cells demonstrated to express P2Y2 receptors. In conclusion, NF272 is a competitive but non-selective P2Y2 receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y2 receptor antagonists.
Asunto(s)
Descubrimiento de Drogas , Naftalenos/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Animales , Humanos , Naftalenos/química , Antagonistas del Receptor Purinérgico P2Y/química , Suramina/análogos & derivadosRESUMEN
Stimulation of metabotropic Gq-coupled purinergic P2Y2 receptors decreases activity of the epithelial Na+ channel (ENaC) in renal principal cells of the distal nephron. The physiological consequences of P2Y2 receptor signaling disruption in the P2Y2 receptor knockout mouse are decreased Na+ excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and distal nephron compared with that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure is obscure. Moreover, it is uncertain whether stimulation of P2Y2 receptor inhibition of ENaC is sufficient to drive renal (urinary) Na+ excretion (UNaV). Here, using a pharmacogenetic approach and selective agonism of the P2Y2 receptor, we test the sufficiency of targeted stimulation of Gq signaling in principal cells of the distal nephron and P2Y2 receptors to increase UNaV. Selective stimulation of the P2Y2 receptor with the ligand MRS2768 decreased ENaC activity in freshly isolated tubules (as assessed by patch-clamp electrophysiology) and increased UNaV (as assessed in metabolic cages). Similarly, selective agonism of hM3Dq-designer receptors exclusively activated by designer drugs (DREADD) restrictively expressed in principal cells of the distal nephron with clozapine- N-oxide decreased ENaC activity and, consequently, increased UNaV. Clozapine- N-oxide, when applied to control littermates, failed to affect ENaC and UNaV. This study represents the first use of pharmacogenetic (DREADD) technology in the renal tubule and demonstrated that selective activation of the P2Y2 receptor and Gq signaling in principal cells is sufficient to promote renal salt excretion.
Asunto(s)
Riñón/metabolismo , Farmacogenética , Receptores Purinérgicos P2Y2/efectos de los fármacos , Receptores Purinérgicos P2Y2/genética , Sodio/orina , Animales , Canales Epiteliales de Sodio/efectos de los fármacos , Canales Epiteliales de Sodio/genética , Femenino , Túbulos Renales/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Masculino , Ratones , Ratones Noqueados , Nefronas/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Transducción de Señal/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Cloruro de Sodio/metabolismoRESUMEN
The human P2Y2 receptor ( hP2Y2R) is a G-protein-coupled receptor that shows promise as a therapeutic target for many important conditions, including for antimetastatic cancer and more recently for idiopathic pulmonary fibrosis. As such, there is a need for new hP2Y2R antagonists and molecular probes to study this receptor. Herein, we report the development of a new series of non-nucleotide hP2Y2R antagonists, based on the known, non-nucleotide hP2Y2R antagonist AR-C118925 (1), leading to the discovery of a series of fluorescent ligands containing different linkers and fluorophores. One of these conjugates, 98, displayed micromolar affinity for hP2Y2R (p Kd = 6.32 ± 0.10, n = 17) in a bioluminescence-energy-transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane labeling of astrocytoma cells expressing untagged hP2Y2R. These properties make 98 one of the first tools for studying hP2Y2R distribution and organization.
Asunto(s)
Dibenzocicloheptenos/farmacología , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Antagonistas del Receptor Purinérgico P2/síntesis química , Antagonistas del Receptor Purinérgico P2/farmacología , Pirimidinonas/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Astrocitoma/metabolismo , Línea Celular , Dibenzocicloheptenos/química , Humanos , Ligandos , Microscopía Confocal , Sondas Moleculares , Unión Proteica , Pirimidinonas/química , Proteínas Recombinantes/química , Relación Estructura-ActividadRESUMEN
A homology model of the nucleotide-activated P2Y2R was created based on the X-ray structures of the P2Y1 receptor. Docking studies were performed, and receptor mutants were created to probe the identified binding interactions. Mutation of residues predicted to interact with the ribose (Arg110) and the phosphates of the nucleotide agonists (Arg265, Arg292) or that contribute indirectly to binding (Tyr288) abolished activity. The Y114F, R194A, and F261A mutations led to inactivity of diadenosine tetraphosphate and to a reduced response of UTP. Significant reduction in agonist potency was observed for all other receptor mutants (Phe111, His184, Ser193, Phe261, Tyr268, Tyr269) predicted to be involved in agonist recognition. An ionic lock between Asp185 and Arg292 that is probably involved in receptor activation interacts with the phosphate groups. The antagonist AR-C118925 and anthraquinones likely bind to the orthosteric site. The updated homology models will be useful for virtual screening and drug design.
Asunto(s)
Agonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Receptores Purinérgicos P2Y2/química , Receptores Purinérgicos P2Y2/metabolismo , Homología de Secuencia de Aminoácido , Análisis Espectral/métodosRESUMEN
RATIONALE: Autologous stem cell therapy using human c-Kit+ cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged patients with HF with genetic predispositions and comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impair overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with proregenerative molecules ex vivo is crucial for improving the therapeutic outcome in patients with HF. OBJECTIVE: To improve hCPC proliferation and migration responses that are critical for regeneration by targeting proregenerative P2Y2 nucleotide receptor (P2Y2R) activated by extracellular ATP and UTP molecules released following injury/stress. METHODS AND RESULTS: c-Kit+ hCPCs were isolated from cardiac tissue of patients with HF undergoing left ventricular assist device implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y2R, in slow-growing hCPCs compared with fast growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y2R. Mechanistically, P2Y2R-induced proliferation and migration were dependent on activation of YAP (yes-associated protein)-the downstream effector of Hippo signaling pathway. CONCLUSIONS: Proliferation and migration of functionally impaired hCPCs are enhanced by P2Y2R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling-a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y2R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in patients with HF.
Asunto(s)
Células Madre Adultas/metabolismo , Proliferación Celular , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfato/farmacología , Células Madre Adultas/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Vía de Señalización Hippo , Humanos , Cinética , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/efectos de los fármacos , Fenotipo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Receptores Purinérgicos P2Y2/genética , Regeneración , Transducción de Señal/efectos de los fármacos , Factores de Transcripción , Transfección , Uridina Trifosfato/farmacología , Proteínas Señalizadoras YAPRESUMEN
We recently reported that natriuresis produced by renal medullary salt loading is dependent on endothelin (ET)-1 and purinergic (P2) receptors in male rats. Because sex differences in ET-1 and P2 signaling have been reported, we decided to test whether ovarian sex hormones regulate renal medullary ET-1 and P2-dependent natriuresis. The effect of medullary NaCl loading on Na+ excretion was determined in intact and ovariectomized (OVX) female Sprague-Dawley rats with and without ET-1 or P2 receptor antagonism. Isosmotic saline (284 mosmol/kgH2O) was infused in the renal medullary interstitium of anesthetized rats during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) infusion. Medullary NaCl loading significantly enhanced Na+ excretion in intact and OVX female rats. ETA+B or P2 receptor blockade did not attenuate the natriuretic effect of medullary NaCl loading in intact females, whereas ETA+B or P2 receptor blockade attenuated the natriuretic response to NaCl loading in OVX rats. Activation of medullary P2Y2 and P2Y4 receptors by UTP infusion had no significant effect in intact females but enhanced Na+ excretion in OVX rats. Combined ETA+B receptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data demonstrate that medullary NaCl loading induces ET-1 and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis, suggesting that ovarian hormones may regulate the interplay between the renal ET-1 and P2 signaling systems to facilitate Na+ excretion.
Asunto(s)
Endotelina-1/metabolismo , Médula Renal/metabolismo , Natriuresis , Ovariectomía , Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2/metabolismo , Eliminación Renal , Sodio/orina , Animales , Antagonistas de los Receptores de Endotelina/farmacología , Endotelina-1/genética , Femenino , Médula Renal/efectos de los fármacos , Natriuresis/efectos de los fármacos , Agonistas del Receptor Purinérgico P2/farmacología , Antagonistas del Receptor Purinérgico P2/farmacología , Ratas Sprague-Dawley , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y2/efectos de los fármacos , Eliminación Renal/efectos de los fármacos , Transducción de Señal , Cloruro de Sodio/administración & dosificación , Cloruro de Sodio/metabolismo , Factores de TiempoRESUMEN
OBJECTIVES: Extracellular nucleotide release at the site of arterial injury mediates the proliferation and migration of vascular smooth muscle cells. Our aim was to investigate the role of the P2Y2 nucleotide receptor (P2Y2R) in neointimal hyperplasia. Approach and Results: Vascular injury was induced by the implantation of a polyethylene cuff around the femoral artery in wild-type and P2Y2R-deficient mice (P2Y2R-/-). Electron microscopy was used to analyze monocyte and lymphocyte influx to the intima 36 h after injury. Compared to wild-type littermates, P2Y2R-/- mice exhibited a 3-fold decreased number of mononuclear leukocytes invading the intima (p < 0.05). Concomitantly, the migration of smooth muscle cells was decreased by more than 60% (p < 0.05), resulting in a sharp inhibition of intimal thickening formation in P2Y2R-/- mice (n = 15) 14 days after cuff placement. In vitro, loss of P2Y2R significantly impaired monocyte migration in response to nucleotide agonists. Furthermore, transgenic rats overexpressing the P2Y2R developed accelerated intimal lesions resulting in more than 95% luminal stenosis (p < 0.05, n = 10). CONCLUSIONS: Loss- and gain-of-function approaches established direct evidence for P2Y2R involvement in neointimal hyperplasia. Specific anti-P2Y2R therapies may be used against restenosis and bypass graft failure.
Asunto(s)
Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Receptores Purinérgicos P2Y2/metabolismo , Lesiones del Sistema Vascular/metabolismo , Animales , Células Cultivadas , Quimiotaxis de Leucocito , Constricción Patológica , Modelos Animales de Enfermedad , Arteria Femoral/lesiones , Arteria Femoral/metabolismo , Arteria Femoral/ultraestructura , Predisposición Genética a la Enfermedad , Hiperplasia , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/ultraestructura , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/ultraestructura , Fenotipo , Agonistas del Receptor Purinérgico P2Y/farmacología , Ratas Sprague-Dawley , Ratas Transgénicas , Receptores Purinérgicos P2Y2/deficiencia , Receptores Purinérgicos P2Y2/efectos de los fármacos , Receptores Purinérgicos P2Y2/genética , Factores de Tiempo , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patología , Lesiones del Sistema Vascular/prevención & controlRESUMEN
OBJECTIVE: A solid body of evidence supports a role of extracellular ATP and its P2 receptors in innate and adaptive immunity. It promotes inflammation as a danger signal in various chronic inflammatory diseases. Thus, we hypothesize contribution of extracellular ATP and its receptor P2Y2 in vascular inflammation and atherosclerosis. APPROACH AND RESULTS: Extracellular ATP induced leukocyte rolling, adhesion, and migration in vivo as assessed by intravital microscopy and in sterile peritonitis. To test the role of extracellular ATP in atherosclerosis, ATP or saline as control was injected intraperitoneally 3× a week in low-density lipoprotein receptor(-/-) mice consuming high cholesterol diet. Atherosclerosis significantly increased after 16 weeks in ATP-treated mice (n=13; control group, 0.26 mm2; ATP group, 0.33 mm2; P=0.01). To gain into the role of ATP-receptor P2Y2 in ATP-induced leukocyte recruitment, ATP was administered systemically in P2Y2-deficient or P2Y2-competent mice. In P2Y2-deficient mice, the ATP-induced leukocyte adhesion was significantly reduced as assessed by intravital microscopy. P2Y2 expression in atherosclerosis was measured by real-time polymerase chain reaction and immunohistochemistry and demonstrates an increased expression mainly caused by influx of P2Y2-expressing macrophages. To investigate the functional role of P2Y2 in atherogenesis, P2Y2-deficient low-density lipoprotein receptor(-/-) mice consumed high cholesterol diet. After 16 weeks, P2Y2-deficient mice showed significantly reduced atherosclerotic lesions with decreased macrophages compared with P2Y2-competent mice (n=11; aortic arch: control group, 0.25 mm(2); P2Y2-deficient, 0.14 mm2; P=0.04). Mechanistically, atherosclerotic lesions from P2Y2-deficient mice expressed less vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 RNA. CONCLUSIONS: We show that extracellular ATP induces vascular inflammation and atherosclerosis via activation of P2Y2.
Asunto(s)
Adenosina Trifosfato/toxicidad , Aorta/efectos de los fármacos , Enfermedades de la Aorta/inducido químicamente , Aterosclerosis/inducido químicamente , Inflamación/inducido químicamente , Receptores Purinérgicos P2Y2/efectos de los fármacos , Adenosina Trifosfato/administración & dosificación , Adenosina Trifosfato/sangre , Animales , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Genotipo , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Inyecciones Intraperitoneales , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Rodamiento de Leucocito/efectos de los fármacos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Peritonitis/genética , Peritonitis/metabolismo , Fenotipo , Placa Aterosclerótica , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores Purinérgicos P2Y2/deficiencia , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The aim of this study is to analyze the expression of purinergic receptors in the heart after ischemia-reperfusion, and their possible role in ischemia-reperfusion injury. Rat hearts were perfused according to the Langendorff technique and subjected to 30 min ischemia followed by 15 min reperfusion. Ischemia-reperfusion reduced the gene expression and protein content of purinergic receptors of the P2Y2 subtype, and increased the gene expression and protein content of the P2X7 subtype. Treatment with the agonist of the P2Y2 subtype 2-thio-UTP and with the antagonist of the P2X7 subtype Brilliant Blue improved myocardial function parameters, reduced cell death and increased the myocardial expression of antiapoptotic markers after ischemia-reperfusion. These results suggest that the myocardial expression of the protective P2Y2 subtype of purinergic receptors is reduced, whereas that of the harmful subtype P2X7 subtype is increased during coronary ischemia-reperfusion. This may contribute to myocardial injury in this condition.
Asunto(s)
Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Regulación hacia Abajo , Hemodinámica , Preparación de Corazón Aislado , Masculino , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Antagonistas del Receptor Purinérgico P2X/farmacología , Agonistas del Receptor Purinérgico P2Y/farmacología , Ratas Sprague-Dawley , Receptores Purinérgicos P2X7/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2Y2/efectos de los fármacos , Receptores Purinérgicos P2Y2/genética , Transducción de Señal , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Adenosine and uridine triphosphate (ATP and UTP) can act as extracellular signalling molecules, playing important roles in vascular biology and disease. ATP and UTP acting via the P2Y2-receptor have, for example, been shown to regulate endothelial dilatation, inflammation and angiogenesis. MicroRNAs (miRNAs), a class of regulatory, short, non-coding RNAs, have been shown to be important regulators of these biological processes. In this study, we used RNA deep-sequencing to explore changes in miRNA expression in the human microvascular endothelial cell line HMEC-1 upon UTP treatment. The expression of miR-22, which we have previously shown to target ICAM-1 mRNA in HMEC-1, increased significantly after stimulation. Up-regulation of miR-22 and down-regulation of cell surface ICAM-1 were confirmed with qRT-PCR and flow cytometry, respectively. siRNA-mediated knockdown of the P2Y2-receptor abolished the effect of UTP on miR-22 transcription. Leukocyte adhesion was significantly inhibited in HMEC-1 following miR-22 overexpression and treatment with UTP/ATP. In conclusion, extracellular UTP and ATP can attenuate ICAM-1 expression and leukocyte adhesion in endothelial cells through miR-22.
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Adenosina Trifosfato/farmacología , Antiinflamatorios/farmacología , Células Endoteliales/efectos de los fármacos , Inflamación/prevención & control , Molécula 1 de Adhesión Intercelular/metabolismo , MicroARNs/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Uridina Trifosfato/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/inmunología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/metabolismo , MicroARNs/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Interferencia de ARN , Receptores Purinérgicos P2Y2/efectos de los fármacos , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , TransfecciónRESUMEN
AIMS: Nucleotides are important paracrine regulators of vascular tone. We previously demonstrated that activation of P2Y2 receptors causes an acute, NO-independent decrease in blood pressure, indicating this signalling pathway requires an endothelial-derived hyperpolarization (EDH) response. To define the mechanisms by which activation of P2Y2 receptors initiates EDH and vasodilation, we studied intermediate-conductance (KCa3.1, expressed in endothelial cells) and big-conductance potassium channels (KCa1.1, expressed in smooth muscle cells) as well as components of the myoendothelial gap junction, connexins 37 and 40 (Cx37, Cx40), all hypothesized to be part of the EDH response. METHODS: We compared the effects of a P2Y2/4 receptor agonist in wild-type (WT) mice and in mice lacking KCa3.1, KCa1.1, Cx37 or Cx40 under anaesthesia, while monitoring intra-arterial blood pressure and heart rate. RESULTS: Acute activation of P2Y2/4 receptors (0.01-3 mg kg(-1) body weight i.v.) caused a biphasic blood pressure response characterized by a dose-dependent and rapid decrease in blood pressure in WT (maximal response % of baseline at 3 mg kg(-1) : -38 ± 1%) followed by a consecutive increase in blood pressure (+44 ± 11%). The maximal responses in KCa3.1(-/-) and Cx37(-/-) were impaired (-13 ± 5, +17 ± 7 and -27 ± 1, +13 ± 3% respectively), whereas the maximal blood pressure decrease in response to acetylcholine at 3 µg kg(-1) was not significantly different (WT: -53 ± 3%; KCa3.1(-/-) : -52 ± 3; Cx37(-/-) : -53 ± 3%). KCa1.1(-/-) and Cx40(-/-) showed an identical biphasic response to P2Y2/4 receptor activation compared to WT. CONCLUSIONS: The data suggest that the P2Y2/4 receptor activation elicits blood pressure responses via distinct mechanisms involving KCa3.1 and Cx37.
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Presión Sanguínea/efectos de los fármacos , Conexinas/metabolismo , Inosina/análogos & derivados , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Receptores Purinérgicos P2Y2/efectos de los fármacos , Uridina Trifosfato/análogos & derivados , Animales , Conexinas/deficiencia , Conexinas/genética , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Frecuencia Cardíaca/efectos de los fármacos , Inosina/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Agonistas del Receptor Purinérgico P2Y , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal/efectos de los fármacos , Uridina Trifosfato/farmacología , Vasodilatación/efectos de los fármacos , Proteína alfa-4 de Unión ComunicanteRESUMEN
Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2-/-) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24-72 h) in response to 70% PH were impaired in P2Y2-/- mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2-/- remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2-/- mice were treated with ATP or ATPγS for 5-120 min and 12-24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH.
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Hepatectomía , Hepatocitos/efectos de los fármacos , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclinas/farmacología , Proteína 1 de la Respuesta de Crecimiento Precoz/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Purinérgicos P2Y2/genéticaRESUMEN
Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5ß1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R(-/-) mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5ß1 integrin and the Rho GTPase Cdc42.
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Agregación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Glándula Parótida/efectos de los fármacos , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Glándula Submandibular/efectos de los fármacos , Uridina Trifosfato/farmacología , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Animales , Línea Celular , Células Epiteliales/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glándula Parótida/citología , Glándula Parótida/metabolismo , Fosforilación , Inhibidores de Proteasas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Receptores Purinérgicos P2Y2/deficiencia , Receptores Purinérgicos P2Y2/genética , Glándula Submandibular/citología , Glándula Submandibular/metabolismo , Transfección , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Extended N(4)-(3-arylpropyl)oxy derivatives of uridine-5'-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N(4)-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N(4)-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N(4)-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs.
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Iminas/química , Sondas Moleculares , Agonistas del Receptor Purinérgico P2/química , Receptores Purinérgicos P2Y2/efectos de los fármacos , Uridina Trifosfato/química , Colorantes Fluorescentes/química , Humanos , Microscopía Fluorescente , Agonistas del Receptor Purinérgico P2/farmacología , Receptores Purinérgicos P2Y2/química , Receptores Purinérgicos P2Y2/clasificaciónRESUMEN
Whole body knockout (KO) of the P2Y2 receptor (P2Y2R) results in enhanced vasopressin V2 receptor activity and increased renal Na⺠conservation. We hypothesized that P2Y2R KO mice would be less sensitive to lithium-induced natriuresis and kaliuresis due to attenuated downregulation of one or more of the major renal Na⺠or K⺠transporter/channel proteins. KO and wild-type (WT) mice were fed a control or lithium-added diet (40 mmol/kg food) for 14 days. Lithium-induced natriuresis and kaliuresis were significantly (~25%) attenuated in KO mice. The subunits of the epithelial Na⺠channel (ENaC) were variably affected by lithium and genotype, but, overall, medullary levels were decreased substantially by lithium (15-60%) in both genotypes. In contrast, cortical, ß-, and γ-ENaC were increased by lithium (~50%), but only in WT mice. Moreover, an assessment of ENaC activity by benzamil sensitivity suggested that lithium increased ENaC activity in WT mice but in not KO mice. In contrast, medullary levels of Naâº-Kâº-2Clâ» cotransporter 2 and cortical levels of the renal outer medullary K⺠channel were not downregulated by lithium and were significantly (15-76%) higher in KO mice under both dietary conditions. In addition, under control conditions, tissue osmolality of the inner medulla as well as furosemide sensitivity were significantly higher in KO mice versus WT mice. Therefore, we suggest that increased expression of these proteins, particularly in the control state, reduces Na⺠delivery to the distal nephron and provides a buffer to attenuate collecting duct-mediated natriuresis and kaliuresis. Additional studies are warranted to explore the potential therapeutic benefits of purinergic antagonism.
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Litio/farmacología , Natriuresis/efectos de los fármacos , Potasio/orina , Receptores Purinérgicos P2Y2/metabolismo , Aldosterona/metabolismo , Análisis de Varianza , Animales , Dieta , Diuréticos/farmacología , Canales Epiteliales de Sodio/metabolismo , Genotipo , Riñón/metabolismo , Médula Renal/efectos de los fármacos , Médula Renal/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Litio/toxicidad , Ratones , Ratones Noqueados , Nefronas/efectos de los fármacos , Nefronas/metabolismo , Concentración Osmolar , Receptores Purinérgicos P2Y2/efectos de los fármacos , Receptores Purinérgicos P2Y2/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12RESUMEN
PURPOSE: We characterized and identified the uroepithelial P2 receptor responsible for adenosine triphosphate mediated release of the cytokines interleukin-8 and 6. MATERIALS AND METHODS: The human renal epithelial cell line A498 (ATCC™) was cultured and stimulated with different purinergic agonists with or without prior inhibition with different antagonists or signaling pathway inhibitors. Supernatant was analyzed for interleukin-8 and 6 by enzyme-linked immunosorbent assay. P2 receptor mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction. The candidate receptor was knocked down with siRNA technology. Interleukin-8 and 6 responses were measured after purinergic stimulation of knocked down cells. RESULTS: ATP and ATP-γ-S (Roche Diagnostics, Mannheim, Germany) were equipotent as inducers of interleukin-8 and 6 release. Agonist profile experiments using different P2 receptor agonists indicated that P2Y(2) was the main contributor to this release, although P2Y(11) and P2X(7) activation could not be excluded. Signaling pathway experiments showed that interleukin-8 release involved phospholipase C and inositol trisphosphate mediated signaling, indicating a P2Y receptor subtype. Antagonist experiments indicated P2Y(2) as the responsible receptor. Gene expression analysis of P2 receptors showed that strong expression of P2Y(2) receptor and subsequent knockdown of P2Y(2) receptor mRNA for 72 and 96 hours abrogated interleukin-8 and 6 release after purinergic stimulation with adenosine triphosphate-γ-S. CONCLUSIONS: Interleukin-8 and 6 release after purinergic stimulation in uroepithelial A498 cells is mediated through P2Y(2) receptor activation.
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Adenosina Trifosfato/farmacología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Receptores Purinérgicos P2Y2/efectos de los fármacos , Urotelio/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , ARN Interferente Pequeño/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Transducción de Señal , Infecciones Urinarias/fisiopatología , Urotelio/citología , Urotelio/metabolismoRESUMEN
BACKGROUND & AIMS: During progression of liver disease, inflammation affects survival of hepatocytes. Endogenous release of adenosine triphosphate (ATP) in the liver activates purinergic P2 receptors (P2R), which regulate inflammatory responses, but little is known about the roles of these processes in the development of acute hepatitis. METHODS: We induced acute hepatitis in C57BL/6 mice by intravenous injection of concanavalin A and then analyzed liver concentrations of ATP and expression of P2R. We assessed P2Y(2)R(-/-) mice and C57BL/6 wild-type mice injected with suramin, a pharmacologic inhibitor of P2YR. Toxic liver failure was induced in mice by intraperitoneal injection of acetaminophen. Hepatocyte-specific functions of P2R signaling were analyzed in primary mouse hepatocytes. RESULTS: Induction of acute hepatitis in wild-type C57BL/6 mice released large amounts of ATP from livers and induced expression of P2Y(2)R. Liver damage and necrosis were greatly reduced in P2Y(2)R(-/-) mice and C57BL/6 mice given injections of suramin. Acetaminophen-induced liver damage was reduced in P2Y(2)R(-/-) mice. Analysis of liver-infiltrating immune cells during acute hepatitis revealed that expression of P2Y(2)R in bone marrow-derived cells was required for liver infiltration by neutrophils and subsequent liver damage. Hepatic expression of P2Y(2)R interfered with expression of genes that regulate cell survival, and promoted tumor necrosis factor-α-mediated cell death, in a cell-autonomous manner. CONCLUSIONS: Extracellular ATP and P2Y(2)R have cell-type specific, but synergistic functions during liver damage that regulate cellular immune responses and promote hepatocyte death. Reagents designed to target P2Y(2)R might be developed to treat inflammatory liver disease.
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Apoptosis/fisiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hepatocitos/patología , Infiltración Neutrófila/fisiología , Receptores Purinérgicos P2Y2/fisiología , Enfermedad Aguda , Adenosina Trifosfato/metabolismo , Animales , Movimiento Celular/fisiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Concanavalina A/efectos adversos , Modelos Animales de Enfermedad , Hepatocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Purinérgicos P2Y2/deficiencia , Receptores Purinérgicos P2Y2/efectos de los fármacos , Suramina/farmacologíaRESUMEN
Cardiac fibroblasts (CFs) play an essential role in remodeling of the cardiac extracellular matrix. Extracellular nucleotide signaling may provoke a profibrotic response in CFs. We tested the hypothesis that physical perturbations release ATP from CFs and that ATP participates in profibrotic signaling. ATP release was abolished by the channel inhibitor carbenoxolone and inhibited by knockdown of either connexin (Cx)43 or Cx45 (47 and 35%, respectively), implying that hypotonic stimulation induces ATP release via Cx43 and Cx45 hemichannels, although pannexin 1 may also play a role. ATP released by hypotonic stimulation rapidly (<10 min) increased phosphorylated ERK by 5-8 fold, an effect largely eliminated by P2Y(2) receptor knockdown or ATP hydrolysis with apyrase. ATP stimulation of P2Y(2) receptors increased α-smooth muscle actin (α-SMA) production, and in an ERK-dependent manner, ATP increased collagen accumulation by 60% and mRNA expression of profibrotic markers: plasminogen activator inhibitor-1 and monocyte chemotactic protein-1 by 4.5- and 4.0-fold, respectively. Apyrase treatment substantially reduced the basal profibrotic phenotype, decreasing collagen and α-SMA content and increasing matrix metalloproteinase expression. Thus, ATP release activates P2Y(2) receptors to mediate profibrotic responses in CFs, implying that nucleotide release under both basal and activated states is likely an important mechanism for fibroblast homeostasis.