RESUMEN
Intraerythrocytic stages of Plasmodium falciparum responsible for all clinical manifestations of malaria are regulated by array of signalling cascades that represent attractive targets for antimalarial therapy. G-protein coupled receptors (GPCRs) are druggable targets in the treatment of various pathological conditions, however, there is limited understanding about the role of GPCRs in malaria pathogenesis. In Plasmodium, serpentine receptors (PfSR1, PfSR10, PfSR12 and PfSR25) with GPCR-like membrane topology have been reported with the finite knowledge about their potential as antimalarial targets. We analyzed the localization of these receptors in malaria parasite by immunofluorescence assays. All four receptors were expressed in blood stages with PfSR12 expressing more in late intraerythrocytic stages. Further, we evaluated the druggability of PfSR12 using FDA-approved P2Y purinergic receptor antagonist, Prasugrel and its active metabolite R138727, which is proposed to be specific towards PfSR12. Interestingly, biophysical analysis indicated strong binding between PfSR12 and R138727 as compared to the prodrug Prasugrel. This binding interaction was further confirmed by thermal shift assay. Treatment of parasite with Prasugrel and R138727 resulted in growth inhibition of P. falciparum indicating an important role of purinergic signalling and PfSR12 in parasite survival. Next, progression studies indicated the inhibitory effect of Prasugrel begins in late erythrocyte stages corroborating with PfSR12 expression at these stages. Furthermore, Prasugrel also blocked in vivo growth of malaria parasite in a mouse experimental model. This study indicates the presence of P2Y type of purinergic signalling in growth and development of malaria parasite and suggests PfSR12, putative purinergic receptor druggability through Prasugrel.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Animales , Ratones , Plasmodium falciparum , Antimaláricos/metabolismo , Clorhidrato de Prasugrel/metabolismo , Clorhidrato de Prasugrel/farmacología , Clorhidrato de Prasugrel/uso terapéutico , Malaria Falciparum/tratamiento farmacológico , Malaria/tratamiento farmacológico , Receptores Purinérgicos/metabolismo , Receptores Purinérgicos/uso terapéutico , Receptores Acoplados a Proteínas G/metabolismo , Eritrocitos/metabolismo , Antagonistas Purinérgicos/metabolismo , Antagonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/uso terapéutico , Proteínas Protozoarias/metabolismoRESUMEN
G-protein-coupled receptors for extracellular nucleotides are known as P2Y receptors and are made up of eight members that are encoded by distinct genes and can be classified into two classes based on their affinity for specific G-proteins. P2Y receptor modulators have been studied extensively, but only a few small-molecule P2Y receptor antagonists have been discovered so far and approved by drug agencies. Derivatives of indole carboxamide have been identified as P2Y12 and P2X7 antagonist, as a result, we developed and tested a series of indole derivatives4a-lhaving thiourea moiety as P2Y receptor antagonist by using a fluorescence-based assay to measure the inhibition of intracellular calcium release in 1321N1 astrocytoma cells that had been stably transfected with the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Most of the compounds exhibited moderate to excellent inhibition activity against P2Y1 receptor subtype. The series most potent compound, 4h exhibited an IC50 value of 0.36 ± 0.01 µM selectivity against other subtypes of P2Y receptor. To investigate the ligand-receptor interactions, the molecular docking studies were carried out. Compound 4h is the most potent P2Y1 receptor antagonist due to interaction with an important amino acid residue Pro105, in addition to Ile108, Phe119, and Leu102.
Asunto(s)
Indometacina/farmacología , Antagonistas Purinérgicos/farmacología , Receptores Purinérgicos/metabolismo , Tiourea/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Indometacina/síntesis química , Indometacina/química , Estructura Molecular , Antagonistas Purinérgicos/síntesis química , Antagonistas Purinérgicos/química , Relación Estructura-Actividad , Tiourea/químicaRESUMEN
Cytokines are important neuroinflammatory modulators in neurodegenerative brain disorders including traumatic brain injury (TBI) and stroke. However, their temporal effects on the physiological properties of microglia and neurons during the recovery period have been unclear. Here, using an ATP-induced cortical injury model, we characterized selective effects of ATP injection compared to needle-control. In the damaged region, the fluorescent intensity of CX3CR1-GFP (+) cells, as well as the cell density, was increased and the maturation of newborn BrdU (+) cells continued until 28 day-post-injection (dpi) of ATP. The excitability and synaptic E/I balance of neurons and the inward and outward membrane currents of microglia were increased at 3 dpi, when expressions of tumor necrosis factor (TNF)-α/interleukin (IL)-1ß and IL-10/IL-4 were also enhanced. These changes of both cells at 3 dpi were mostly decayed at 7 dpi and were suppressed by any of IL-10, IL-4, suramin (P2 receptor inhibitor) and 4-AP (K+ channel blocker). Acute ATP application alone induced only small effects from both naïve neurons and microglial cells in brain slice. However, TNF-α alone effectively increased the excitability of naïve neurons, which was blocked by suramin or 4-AP. TNF-α and IL-1ß increased and decreased membrane currents of naïve microglia, respectively. Our results suggest that ATP and TNF-α dominantly induce the physiological activities of 3 dpi neurons and microglia, and IL-10 effectively suppresses such changes of both activated cells in K+ channel- and P2 receptor-dependent manner, while IL-4 suppresses neurons preferentially.
Asunto(s)
Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Potenciales de la Membrana , Microglía/fisiología , Adenosina Trifosfato/metabolismo , Animales , Lesiones Traumáticas del Encéfalo/diagnóstico , Lesiones Traumáticas del Encéfalo/etiología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Expresión Génica , Genes Reporteros , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Antagonistas Purinérgicos/farmacologíaRESUMEN
We sought to determine if a pannexin/purinergic-dependent intravascular communication pathway exists in skeletal muscle microvasculature that facilitates capillary communication with upstream arterioles that control their perfusion. Using the hamster cremaster muscle and intravital microscopy, we locally stimulated capillaries and observed the vasodilatory response in the associated upstream 4A arteriole. We stimulated capillaries with vasodilators relevant to muscle contraction: 10-6 M S-nitroso-N-acetyl-dl-penicillamine (SNAP; nitric oxide donor), 10-6 M adenosine, 10 mM potassium chloride, 10-5 M pinacidil, as well as a known initiator of gap-junction-dependent intravascular communication, acetylcholine (10-5 M), in the absence and the presence of the purinergic membrane receptor blocker suramin (10-5 M), pannexin blocker mefloquine (2 × 10-5 M), or probenecid (5 × 10-6 M) and gap-junction inhibitor halothane (0.07%) applied in the transmission pathway, between the capillary stimulation site and the upstream 4A observation site. Potassium chloride, SNAP, and adenosine-induced upstream vasodilations were significantly inhibited by suramin, mefloquine, and probenecid but not halothane, indicating the involvement of a pannexin/purinergic-dependent signaling pathway. Conversely, SNAP-induced upstream vasodilation was only inhibited by halothane indicating that communication was facilitated by gap junctions. Both pinacidil and acetylcholine were inhibited by suramin but only acetylcholine was inhibited by halothane. These data demonstrate the presence of a pannexin/purinergic-dependent communication pathway between capillaries and upstream arterioles controlling their perfusion. This pathway adds to the gap-junction-dependent pathway that exists at this vascular level as well. Given that vasodilators relevant to muscle contraction can use both of these pathways, our data implicate the involvement of both pathways in the coordination of skeletal muscle blood flow.NEW & NOTEWORTHY Blood flow control during increased metabolic demand in skeletal muscle is not fully understood. Capillaries have been implicated in controlling blood flow to active skeletal muscle, but how capillaries communicate to the arteriolar vascular network is not clear. Our study uncovers a novel pathway through which capillaries can communicate to upstream arterioles to cause vasodilation and therefore control perfusion. This work implicates a new vascular communication pathway in blood flow control in skeletal muscle.
Asunto(s)
Músculos Abdominales/irrigación sanguínea , Arteriolas/metabolismo , Capilares/metabolismo , Comunicación Celular , Conexinas/metabolismo , Purinas/metabolismo , Receptores Purinérgicos/metabolismo , Vasodilatación , Animales , Capilares/efectos de los fármacos , Conexinas/antagonistas & inhibidores , Uniones Comunicantes/metabolismo , Masculino , Mesocricetus , Contracción Muscular , Agonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/farmacología , Flujo Sanguíneo Regional , Transducción de Señal , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Neural progenitor cells (NPCs) are self-renewing and multipotent cells that persist in the postnatal and adult brain in the subventricular zone and the hippocampus. NPCs can be expanded in vitro to be used in cell therapy. However, expansion is limited, since the survival and proliferation of adult NPCs decrease with serial passages. Many signaling pathways control NPC survival and renewal. Among these, purinergic receptor activation exerts differential effects on the biology of adult NPCs depending on the cellular context. In this study, we sought to analyze the effect of a general blockade of purinergic receptors with suramin on the proliferation and survival of NPCs isolated from the subventricular zone of postnatal rats, which are cultured as neurospheres. Treatment of neurospheres with suramin induced a significant increase in neurosphere diameter and in NPC number attributed to a decrease in apoptosis. Proliferation and multipotency were not affected. Suramin also induced an increase in the gap junction protein connexin43 and in vascular endothelial growth factor, which might be involved in the anti-apoptotic effect. Our results offer a valuable tool for increasing NPC survival before implantation in the lesioned brain and open the possibility of using this drug as adjunctive therapy to NPC transplantation.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Antagonistas Purinérgicos/farmacología , Receptores Purinérgicos/metabolismo , Células Madre/efectos de los fármacos , Suramina/farmacología , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ventrículos Laterales/efectos de los fármacos , Ventrículos Laterales/metabolismo , Masculino , Células-Madre Neurales/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Células Madre/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Evolving information has identified disease mechanisms and dysregulation of host biology that might be targeted therapeutically in coronavirus disease 2019 (COVID-19). Thrombosis and coagulopathy, associated with pulmonary injury and inflammation, are emerging clinical features of COVID-19. We present a framework for mechanisms of thrombosis in COVID-19 that initially derive from interaction of SARS-CoV-2 with ACE2, resulting in dysregulation of angiotensin signaling and subsequent inflammation and tissue injury. These responses result in increased signaling by thrombin (proteinase-activated) and purinergic receptors, which promote platelet activation and exert pathological effects on other cell types (e.g., endothelial cells, epithelial cells, and fibroblasts), further enhancing inflammation and injury. Inhibitors of thrombin and purinergic receptors may, thus, have therapeutic effects by blunting platelet-mediated thromboinflammation and dysfunction in other cell types. Such inhibitors include agents (e.g., anti-platelet drugs) approved for other indications, and that could be repurposed to treat, and potentially improve the outcome of, COVID-19 patients. COVID-19, caused by the SARS-CoV-2 virus, drives dysregulation of angiotensin signaling, which, in turn, increases thrombin-mediated and purinergic-mediated activation of platelets and increase in inflammation. This thromboinflammation impacts the lungs and can also have systemic effects. Inhibitors of receptors that drive platelet activation or inhibitors of the coagulation cascade provide opportunities to treat COVID-19 thromboinflammation.
Asunto(s)
COVID-19/complicaciones , Inflamación/etiología , Receptores Proteinasa-Activados/metabolismo , Receptores Purinérgicos/metabolismo , SARS-CoV-2 , Trombosis/etiología , Humanos , Inflamación/tratamiento farmacológico , Antagonistas Purinérgicos/farmacología , Receptores Proteinasa-Activados/antagonistas & inhibidores , Receptores Proteinasa-Activados/genética , Receptores Purinérgicos/genética , Trombosis/prevención & controlRESUMEN
Patch clamp investigations of single ion channels give insight into the function of these proteins on the molecular level. Utilizing this technique, we performed detailed investigations of the human P2X7 receptor, which is a ligand gated ion channel opened by binding of ATP, like the other P2X receptor subtypes. P2X7 receptors become activated under pathological conditions of ATP release like hypoxia or cell destruction. They are involved in inflammatory and nociceptive reactions of the organism to these pathological events. Knowledge about the function of the P2X7 receptor might lead to a deeper insight into the signaling within these pathophysiological processes and to reveal targets of anti-inflammatory and anti-nociceptive therapies. We found that hP2X7 receptors become activated by ATP within a few milliseconds and are permeable only to cations. Their ion channel conductance remains constant across minutes of activation, which argues against dilation of the ion channel pore. Substitution of Na+ or Cl- ions not only influences the ion channel current amplitude but also the channel gating. Polar residues of the second transmembrane domains of the three protein subunits are important for ion conduction, with S342 constituting the ion selectivity filter and the gate of the channel. The specific long C-terminal domains are important for hP2X7 receptor ion channel function, as their loss strongly decreases ion channel currents.
Asunto(s)
Adenosina Trifosfato/metabolismo , Activación del Canal Iónico/fisiología , Receptores Purinérgicos P2X7/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Agonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
Recent studies have proven that the purinergic signaling pathway plays a key role in neurotransmission and neuromodulation, and is involved in various neurodegenerative diseases and psychiatric disorders. With the characterization of the subtypes of receptors in purinergic signaling, i.e. the P1 (adenosine), P2X (ion channel) and P2Y (G protein-coupled), more attention has been paid to the pathophysiology and therapeutic potential of purinergic signaling in the central nervous system disorders. Alzheimer's disease (AD) is a progressive and deadly neurodegenerative disease that is characterized by memory loss, cognitive impairment and dementia. However, as drug development aimed to prevent or control AD has series of failures in recent years, more researchers have focused on the neuroprotection-related mechanisms such as purinergic signaling in AD patients to find a potential cure. This article reviews the recent discoveries of purinergic signaling in AD, and summarizes the potential agents as modulators for the receptors of purinergic signaling in AD-related research and treatments. Thus, our paper provides an insight into purinergic signaling in the development of anti- AD therapies.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Agonistas Purinérgicos/uso terapéutico , Antagonistas Purinérgicos/uso terapéutico , Receptores Purinérgicos/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacología , Adenosina/uso terapéutico , Enfermedad de Alzheimer/patología , Cafeína/química , Cafeína/metabolismo , Cafeína/farmacología , Cafeína/uso terapéutico , Humanos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Agonistas Purinérgicos/química , Agonistas Purinérgicos/metabolismo , Agonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/química , Antagonistas Purinérgicos/metabolismo , Antagonistas Purinérgicos/farmacología , Receptores Purinérgicos/química , Transducción de Señal/efectos de los fármacosRESUMEN
Many G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C-C chemokine receptor type 5 (CCR5), which serves as a co-receptor to facilitate cellular entry of human immunodeficiency virus 1 (HIV-1), normally signals through the heterotrimeric G protein, Gi. However, CCR5 also exhibits G protein signaling bias and certain chemokine analogs can cause a switch to Gq pathways to induce Ca2+ signaling. We want to understand how much of the Ca2+ signaling from Gi-coupled receptors is due to G protein promiscuity and how much is due to transactivation and crosstalk with other receptors. We propose a possible mechanism underlying the apparent switching between different G protein signaling pathways. We show that chemokine-mediated Ca2+ flux in HEK293T cells expressing CCR5 can be primed and enhanced by ATP pretreatment. In addition, agonist-dependent lysosomal exocytosis results in the release of ATP to the extracellular milieu, which amplifies cellular signaling networks. ATP is quickly degraded via ADP and AMP to adenosine. ATP, ADP and adenosine activate different cell surface purinergic receptors. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signaling and allow for Gi- and Gq-coupled receptor signaling pathways to converge. Associated secretory release of GPCR ligands, such as chemokines, opioids, and monoamines, should also lead to concomitant release of ATP with a synergistic effect on Ca2+ signaling. Our results suggest that crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs is an important cooperative mechanism to amplify the intracellular Ca2+ signaling response.
Asunto(s)
Señalización del Calcio/fisiología , Receptor Cross-Talk/fisiología , Receptores CCR5/agonistas , Receptores CCR5/metabolismo , Receptores Purinérgicos/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Señalización del Calcio/efectos de los fármacos , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacología , Células HEK293 , Humanos , Agonistas Purinérgicos/metabolismo , Agonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/metabolismo , Antagonistas Purinérgicos/farmacología , Receptor Cross-Talk/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Suramina/metabolismo , Suramina/farmacologíaRESUMEN
Novel coronavirus disease 2019 (COVID-19) causes pulmonary and cardiovascular disorders and has become a worldwide emergency. Myocardial injury can be caused by direct or indirect damage, particularly mediated by a cytokine storm, a disordered immune response that can cause myocarditis, abnormal coagulation, arrhythmia, acute coronary syndrome, and myocardial infarction. The present review focuses on the mechanisms of this viral infection, cardiac biomarkers, consequences, and the possible therapeutic role of purinergic and adenosinergic signalling systems. In particular, we focus on the interaction of the extracellular nucleotide adenosine triphosphate (ATP) with its receptors P2X1, P2X4, P2X7, P2Y1, and P2Y2 and of adenosine (Ado) with A2A and A3 receptors, as well as their roles in host immune responses. We suggest that receptors of purinergic signalling could be ideal candidates for pharmacological targeting to protect against myocardial injury caused by a cytokine storm in COVID-19, in order to reduce systemic inflammatory damage to cells and tissues, preventing the progression of the disease by modulating the immune response and improving patient quality of life.
Asunto(s)
Adenosina Trifosfato/metabolismo , COVID-19/inmunología , Enfermedades Cardiovasculares/virología , Receptores Purinérgicos/metabolismo , SARS-CoV-2 , Agonistas del Receptor de Adenosina A2/farmacología , COVID-19/metabolismo , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/fisiopatología , Citocinas/metabolismo , Humanos , Isquemia Miocárdica/inmunología , Isquemia Miocárdica/fisiopatología , Isquemia Miocárdica/virología , Pandemias , Antagonistas Purinérgicos/farmacología , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Tratamiento Farmacológico de COVID-19RESUMEN
Brain cells continuously produce and release protons into the extracellular space, with the rate of acid production corresponding to the levels of neuronal activity and metabolism. Efficient buffering and removal of excess H+ is essential for brain function, not least because all the electrogenic and biochemical machinery of synaptic transmission is highly sensitive to changes in pH. Here, we describe an astroglial mechanism that contributes to the protection of the brain milieu from acidification. In vivo and in vitro experiments conducted in rodent models show that at least one third of all astrocytes release bicarbonate to buffer extracellular H+ loads associated with increases in neuronal activity. The underlying signalling mechanism involves activity-dependent release of ATP triggering bicarbonate secretion by astrocytes via activation of metabotropic P2Y1 receptors, recruitment of phospholipase C, release of Ca2+ from the internal stores, and facilitated outward HCO3- transport by the electrogenic sodium bicarbonate cotransporter 1, NBCe1. These results show that astrocytes maintain local brain extracellular pH homeostasis via a neuronal activity-dependent release of bicarbonate. The data provide evidence of another important metabolic housekeeping function of these glial cells.
Asunto(s)
Astrocitos/metabolismo , Bicarbonatos/metabolismo , Encéfalo/metabolismo , Espacio Extracelular/metabolismo , Acetazolamida/farmacología , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/efectos de los fármacos , Anhidrasas Carbónicas/metabolismo , Células Cultivadas , Estimulación Eléctrica , Fluorescencia , Hipocampo/metabolismo , Concentración de Iones de Hidrógeno , Ratones Endogámicos C57BL , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Antagonistas Purinérgicos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Receptores Purinérgicos/metabolismo , Transducción de Señal , Simportadores de Sodio-Bicarbonato/metabolismoRESUMEN
T cells must migrate to encounter antigen-presenting cells and perform their roles in host defense. Here, we found that autocrine stimulation of the purinergic receptor P2Y11 regulates the migration of human CD4 T cells. P2Y11 receptors redistributed from the front to the back of polarized cells where they triggered intracellular cAMP/PKA signals that attenuated mitochondrial metabolism at the back. The absence of P2Y11 receptors at the front of cells resulted in hotspots of mitochondrial metabolism and localized ATP production that stimulated P2X4 receptors, Ca2+ influx, and pseudopod protrusion at the front. This regulatory function of P2Y11 receptors depended on their subcellular redistribution and autocrine stimulation by cellular ATP release and was perturbed by indiscriminate global stimulation. We conclude that excessive extracellular ATP-such as in response to inflammation, sepsis, and cancer-disrupts this autocrine feedback mechanism, which results in defective T cell migration, impaired T cell function, and loss of host immune defense.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Mitocondrias/metabolismo , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Células Jurkat , Microscopía Fluorescente/métodos , Agonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/farmacología , Receptores Purinérgicos P2/metabolismoRESUMEN
OBJECTIVE: The P2Y6 receptor has been shown to be involved in many cardiovascular diseases, including hypertension and atherosclerosis. The study is aimed at exploring the role of the P2Y6 receptor in Ang II-induced abdominal aortic aneurysm (AAA) formation in apolipoprotein E-deficient (apoE-/-) mice by using its selective antagonist. METHODS: Male apoE-/- mice were fed with high-fat diet and infused with angiotensin (Ang) II (1000 ng/kg/min) for 4 weeks to induce AAA or saline as controls. Mice were divided into four groups: normal saline (NS, placebo control) group (n = 8), Ang II+vehicle (Ang II) group (n = 14), Ang II-low dose MRS2578 (Ang II+MRS-16 mg) group (n = 14), and Ang II-high dose MRS2578 (Ang II+MRS-32 mg) group (n = 14). Daily intraperitoneal injection with vehicle or MRS2578 was pretreated one week before Ang II infusion. On postoperative day 10, aorta imaging of each group was taken by ultrasonography. After 4 weeks of Ang II infusion, the excised aortas were processed for diameter measurement and quantification of aneurysm severity and tissue characteristics; the blood samples were collected for measurement of the lipid profile and levels of cytokines. Verhoeff's Van Gieson (EVG) staining and immunochemistry staining were performed to evaluate disruption of the extracellular matrix (ECM) and infiltration of macrophages. Expression and activity of matrix metalloproteinases (MMPs) was measured by gelatin zymography. RESULTS: Treatment with MRS2578 made no significant difference in AAA formation, and maximal aortic diameter yet caused higher AAA rupture-induced mortality from 7% (Ang II) to 21.4% (Ang II+MRS-16 mg) or 42.9% (Ang II+MRS-32 mg), respectively (p < 0.05). Consistently, the severity of aneurysm tended to be more deteriorated in MRS2578-treated groups, especially the high-dosage group. The ratios of type III and IV aneurysm were much higher in the MRS2578-coadministered groups (p < 0.05). Furthermore, histological analyses showed that administration of MRS2578 significantly increased infiltration of macrophages, expression of monocyte chemotactic protein 1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1), and activities of MMP-2 and MMP-9 followed by aggravating degradation elastin in vivo (p < 0.05). However, the multiple effects of MRS2578 on the development of AAA are independent of changes in systolic blood pressure and lipid profiles. CONCLUSIONS: The present study demonstrated that administration of MRS2578 exacerbated the progression and rupture of experimental AAA through promoting proinflammatory response and MMP expression and activity, which indicated a crucial role of the P2Y6 receptor in AAA development. Clinical Relevance. Purinergic P2Y receptors have attracted much attention since the P2Y12 receptor antagonist had been successfully applied in clinical practice. Elucidating the underlying mechanisms of AAA and exploring potential therapeutic strategies are essential to prevent its progression and reduce the mortality rate.
Asunto(s)
Angiotensina II , Aneurisma de la Aorta Abdominal , Isotiocianatos , Antagonistas Purinérgicos , Receptores Purinérgicos P2/metabolismo , Tiourea/análogos & derivados , Angiotensina II/efectos adversos , Angiotensina II/farmacología , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Modelos Animales de Enfermedad , Isotiocianatos/efectos adversos , Isotiocianatos/farmacología , Ratones , Ratones Noqueados para ApoE , Antagonistas Purinérgicos/efectos adversos , Antagonistas Purinérgicos/farmacología , Tiourea/efectos adversos , Tiourea/farmacologíaRESUMEN
The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.
Asunto(s)
Proliferación Celular/genética , Osteoblastos/metabolismo , Osteogénesis/genética , Antagonistas Purinérgicos/farmacología , Receptores Purinérgicos P2Y/metabolismo , Transducción de Señal/genética , Azúcares de Uridina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Densidad Ósea/genética , Sistemas CRISPR-Cas , Calcio/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteogénesis/efectos de los fármacos , Fosforilación , Antagonistas Purinérgicos/metabolismo , Receptores Purinérgicos P2Y/genética , Transducción de Señal/efectos de los fármacos , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Glucosa/farmacología , Azúcares de Uridina Difosfato/farmacologíaRESUMEN
INTRODUCTION: Purines finely modulate physiological motor, secretory, and sensory functions in the gastrointestinal tract. Their activity is mediated by the purinergic signaling machinery, including receptors and enzymes regulating their synthesis, release, and degradation. Several gastrointestinal dysfunctions are characterized by alterations affecting the purinergic system. AREAS COVERED: The authors provide an overview on the purinergic receptor signaling machinery, the molecules and proteins involved, and a summary of medicinal chemistry efforts aimed at developing novel compounds able to modulate the activity of each player involved in this machinery. The involvement of purinergic signaling in gastrointestinal motor, secretory, and sensory functions and dysfunctions, and the potential therapeutic applications of purinergic signaling modulators, are then described. EXPERT OPINION: A number of preclinical and clinical studies demonstrate that the pharmacological manipulation of purinergic signaling represents a viable way to counteract several gastrointestinal diseases. At present, the paucity of purinergic therapies is related to the lack of receptor-subtype-specific agonists and antagonists that are effective in vivo. In this regard, the development of novel therapeutic strategies should be focused to include tools able to control the P1 and P2 receptor expression as well as modulators of the breakdown or transport of purines.
Asunto(s)
Diseño de Fármacos , Enfermedades Gastrointestinales/tratamiento farmacológico , Receptores Purinérgicos/efectos de los fármacos , Animales , Descubrimiento de Drogas , Fármacos Gastrointestinales/farmacología , Enfermedades Gastrointestinales/fisiopatología , Humanos , Agonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/farmacología , Purinas/metabolismo , Receptores Purinérgicos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Adenosine triphosphate (ATP) is released into the extracellular environment during transplantation, and acts via purinergic receptors to amplify the alloimmune response. Here, using a well-established murine model of allogeneic corneal transplantation, we investigated the immunomodulatory mechanisms of the purinergic receptor antagonist oxidized ATP (oATP). Corneal transplantation was performed using C57BL/6 donors and BALB/c hosts. oATP or sterile saline was administered via intraperitoneal injection for 2 weeks postoperatively. Frequencies of CD45+ leukocytes, CD11b+MHCII+ antigen presenting cells (APCs), CD4+IFN-γ+ effector Th1 cells and CD4+Foxp3+ regulatory T cells (Tregs) were evaluated by flow cytometry. Slit-lamp microscopy was performed weekly for 8 weeks to evaluate graft opacity and determine transplant rejection. Treatment with oATP was shown to significantly reduce graft infiltration of CD45+ leukocytes, decrease APC maturation and suppress effector Th1 cell generation relative to saline-treated control. No difference in Treg frequencies or Foxp3 expression was observed between the oATP-treated and control groups. Finally, oATP treatment was shown to reduce graft opacity and increase graft survival. This report demonstrates that oATP limits the alloimmune response by regulating APC maturation and suppressing the generation of alloreactive Th1 immunity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Trasplante de Córnea , Rechazo de Injerto/inmunología , Antagonistas Purinérgicos/uso terapéutico , Receptores Purinérgicos/metabolismo , Aloinjertos/efectos de los fármacos , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Córnea/efectos de los fármacos , Córnea/patología , Leucocitos/efectos de los fármacos , Recuento de Linfocitos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oxidación-Reducción , Antagonistas Purinérgicos/farmacologíaRESUMEN
Immune-mediated inflammatory diseases (IMIDs) encompass a wide range of seemingly unrelated conditions, such as multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases, asthma, chronic obstructive pulmonary disease, and systemic lupus erythematosus. Despite differing etiologies, these diseases share common inflammatory pathways, which lead to damage in primary target organs and frequently to a plethora of systemic effects as well. The purinergic signaling complex comprising extracellular nucleotides and nucleosides and their receptors, the P2 and P1 purinergic receptors, respectively, as well as catabolic enzymes and nucleoside transporters is a major regulatory system in the body. The purinergic signaling complex can regulate the development and course of IMIDs. Here we provide a comprehensive review on the role of purinergic signaling in controlling immunity, inflammation, and organ function in IMIDs. In addition, we discuss the possible therapeutic applications of drugs acting on purinergic pathways, which have been entering clinical development, to manage patients suffering from IMIDs.
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Inflamación/tratamiento farmacológico , Inflamación/inmunología , Agonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/farmacología , Purinas/metabolismo , Receptores Purinérgicos/metabolismo , Animales , Humanos , Inflamación/metabolismo , Terapia Molecular Dirigida , Purinas/inmunología , Receptores Purinérgicos/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
Identifying novel small-molecule P2X1 and P2X4 ligands with sub-type specificity and high-affinity remains a pharmacological challenge. Here we use computational methods, electrophysiology and fluorescent microplate assays to screen for ligand candidates acting at these receptors. Modelling and docking identified 80 compounds for testing at P2X4 receptors, and 20 of these showed >50% inhibition in fluorescence-based assays, making them appealing for further SAR studies. Confirmation of activity by two-electrode voltage clamp, followed by their elaboration resulted in only minor improvements in potency, with the highest IC50 being 295⯵M. Testing on P2X1 receptors, resulted in a series of biguanide compounds that yielded a maximum IC50 of 100⯵M, but no consistent SAR could be found. Potencies of established antagonists gave expected results, although the measured potencies varied between techniques and no antagonism could be found for compounds such as paroxetine, carbamazepine, 9(10H)-acridanone, acridinol and phenoxazine-type heterocycles. This study highlights the challenge of identifying P2X4 and P2X1 ligands and suggests that a combination of complimentary approaches is needed if we are to be confident of ligand activities at these receptors.
Asunto(s)
Descubrimiento de Drogas/métodos , Ligandos , Antagonistas Purinérgicos/farmacología , Receptores Purinérgicos P2X1/efectos de los fármacos , Receptores Purinérgicos P2X4/efectos de los fármacos , Animales , Biguanidas/farmacología , Células Cultivadas , Simulación por Computador , Humanos , Simulación del Acoplamiento Molecular , Oocitos/fisiología , Técnicas de Placa-Clamp , Agonistas Purinérgicos/farmacología , Relación Estructura-Actividad , Xenopus laevisRESUMEN
AIM: To explore the role of P2Y6 receptors in the maintenance of neuropathic pain and progression of oxidative stress, we investigated the efficacy of the selective P2Y6 receptors antagonist MRS2578 on the antiallodynic effects and improvement of pathological neuropathic pain-induced oxidative stress, thereby finding a potential therapeutic target in neurological disease. MATERIALS AND METHODS: The mechanical allodynia in the ipsilateral spinal dorsal horn (SDH) of rats was observed in rats after chronic constriction injury (CCI). Meanwhile, the messenger RNA (mRNA) levels of biological parameters, including superoxide dismutase (SOD), glutathione (GSH), and heme oxygenase-1 (HO-1) in the SDH of rats were measured by real-time polymerase chain reaction (RT-PCR). In addition, the mRNA expression and protein levels of P2Y6 were measured by RT-PCR and Western blot assay, respectively. Next, the rats subjected to CCI were intrathecally infused with MRS2578 to block the expression of P2Y6 receptors. The positive expression of P2Y6 receptors was examined by immunohistochemistry. RESULTS: In the present study, the results revealed that the P2Y6 expression in the ipsilateral SDH of CCI rats was significantly upregulated. In addition, inhibition of the P2Y6 receptor in SDH increased CCI-induced tactile allodynia. Furthermore, the levels of SOD, GSH, and HO-1 which were correlated with oxidative stress produced by CCI were also decreased. CONCLUSION: The results demonstrated that inhibition of the P2Y6 receptor can generate antiallodynic effects and improved the pathological neuropathic pain-induced oxidative stress. Thus, this study provides a potential approach for the therapy of neurological disease.
Asunto(s)
Analgésicos/farmacología , Hiperalgesia/tratamiento farmacológico , Isotiocianatos/farmacología , Neuralgia/tratamiento farmacológico , Antagonistas Purinérgicos/farmacología , Receptores Purinérgicos P2/genética , Tiourea/análogos & derivados , Animales , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Inyecciones Espinales , Ligadura , Neuralgia/genética , Neuralgia/metabolismo , Neuralgia/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Tiourea/farmacologíaRESUMEN
KEY POINTS: In the epididymis, elaborate communication networks between epithelial cells are important with respect to establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa, which is essential for male fertility. Proton secretion by epididymal clear cells is achieved via the proton pumping V-ATPase located in their apical membrane. In the present study, we dissect the molecular mechanisms by which clear cells respond to luminal ATP and adenosine to modulate their acidifying activity via the adenosine receptor ADORA2B and the pH-sensitive ATP receptor P2X4. We demonstrate that the hydrolysis of ATP to produce adenosine by ectonucleotidases plays a key role in V-ATPase-dependent proton secretion, and is part of a feedback loop that ensures acidification of the luminal compartment These results help us better understand how professional proton-secreting cells respond to extracellular cues to modulate their functions, and how they communicate with neighbouring cells. ABSTRACT: Cell-cell cross-talk is crucial for the dynamic function of epithelia, although how epithelial cells detect and respond to variations in extracellular stimuli to modulate their environment remains incompletely understood. In the present study, we used the epididymis as a model system to investigate epithelial cell regulation by luminal factors. In the epididymis, elaborate communication networks between the different epithelial cell types are important for establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa. In particular, clear cells (CCs) secrete protons into the lumen via the proton pumping V-ATPase located in their apical membrane, a process that is activated by luminal alkalinization. However, how CCs detect luminal pH variations to modulate their function remains uncharacterized. Purinergic regulation of epithelial transport is modulated by extracellular pH in other tissues. In the present study, functional analysis of the mouse cauda epididymis perfused in vivo showed that luminal ATP and adenosine modulate the acidifying activity of CCs via the purinergic ADORA2B and P2X4 receptors, and that luminal adenosine content is itself regulated by luminal pH. Altogether, our observations illustrate mechanisms by which CCs are activated by pH sensitive P2X4 receptor and ectonucleotidases, providing a feedback mechanism for the maintenance of luminal pH. These novel mechanisms by which professional proton-secreting cells respond to extracellular cues to modulate their functions, as well as how they communicate with neighbouring cells, might be translatable to other acidifying epithelia.