RESUMEN
Antagonists of the P2X7 receptor (P2X7R) have the potential to treat diseases where neuroinflammation is present such as depression, chronic pain and Alzheimer's disease. We recently developed a structural hybrid (C1; 1-((adamantan-1-yl)methyl)-2-cyano-3-(quinolin-5-yl)guanidine) of a purported competitive P2X7R antagonist (C2; 2-cyano-1-((1S)-1-phenylethyl)-3-(quinolin-5-yl)guanidine) and a likely negative allosteric modulator (NAM) of the P2X7R (C3; N-((adamantan-1-yl)methyl)-2-chloro-5-methoxybenzamide). Here we aimed to pharmacologically characterize C1, to gain insights into how select structural components impact antagonist interaction with the P2X7R. A second aim was to examine the role of the peptide LL-37, an apparent activator of the P2X7R, and compare the ability of multiple P2X7R antagonists to block its effects. Compounds 1, 2 and 3 were characterised using washout, Schild and receptor protection studies, all using dye uptake assays in HEK293 cells expressing the P2X7R. LL-37 was examined in the same HEK293 cells and THP-1 monocytes. Compounds 2 and 3 acted as a BzATP-competitive antagonist and NAM of the P2X7R respectively. Compound 1 was a slowly reversible NAM of the P2X7R suggesting the incorporation of an appropriately positioned adamantane promotes binding to the allosteric site of the P2X7R. LL-37 was shown to potentiate the ability of ATP to induce dye uptake at low concentrations (1-3 µg mL-1) or induce dye uptake alone at higher concentrations (10-20 µg mL-1). None of the P2X7R antagonists studied were able to block LL-37-induced dye uptake bringing in to question the ability of current P2X7R antagonists to inhibit the inflammatory action of LL-37 in vivo.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Enfermedades Neuroinflamatorias , Antagonistas del Receptor Purinérgico P2X , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Desarrollo de Medicamentos , Células HEK293 , Humanos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Porinas/metabolismo , Agonistas Purinérgicos/farmacología , Antagonistas del Receptor Purinérgico P2X/clasificación , Antagonistas del Receptor Purinérgico P2X/farmacología , Proteínas Modificadoras de la Actividad de Receptores/metabolismo , Células THP-1 , CatelicidinasRESUMEN
P2Y13 is an ADP-stimulated G-protein coupled receptor implicated in many physiological processes, including neurotransmission, metabolism, pain, and bone homeostasis. Quantitative understanding of P2Y13 activation dynamics is important for translational studies. We systematically identified PubMed annotated studies that characterized concentration-dependence of P2Y13 responses to natural and synthetic agonists. Since the comparison of the efficacy (maximum response) is difficult for studies performed in different systems, we normalized the data and conducted a meta-analysis of EC50 (concentration at half-maximum response) and Hill coefficient (slope) of P2Y13-mediated responses to different agonists. For signaling events induced by heterologously expressed P2Y13, EC50 of ADP-like agonists was 17.2 nM (95% CI: 7.7-38.5), with Hills coefficient of 4.4 (95% CI: 3.3-5.4), while ATP-like agonists had EC50 of 0.45 µM (95% CI: 0.06-3.15). For functional responses of endogenously expressed P2Y13, EC50 of ADP-like agonists was 1.76 µM (95% CI: 0.3-10.06). The EC50 of ADP-like agonists was lower for the brain P2Y13 than the blood P2Y13. ADP-like agonists were also more potent for human P2Y13 compared to rodent P2Y13. Thus, P2Y13 appears to be the most ADP-sensitive receptor characterized to date. The detailed understanding of tissue- and species-related differences in the P2Y13 response to ADP will improve the selectivity and specificity of future pharmacological compounds.
Asunto(s)
Agonistas Purinérgicos/farmacología , Receptores Purinérgicos P2/metabolismo , Transducción de Señal/efectos de los fármacos , Adenosina Difosfato/química , Animales , Encéfalo/efectos de los fármacos , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Ratones , Ratas , Programas Informáticos , Resultado del TratamientoRESUMEN
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
BACKGROUND: The aim of the present study was to investigate the effects of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) on bladder function and pathophysiology. METHODS: To create a model for CPPS, rats were intraprostatically injected with zymosan or saline, serving as control. Metabolic cage experiments were performed 7, 14, or 21 days after zymosan injection and after 14 days in the control group. Thereafter, cystometry was performed in which simulated micturition cycles were induced by saline infusion and contractile responses to the cholinergic agonist methacholine and the purinergic agonist ATP were measured. Following cystometry, the prostate and urinary bladder were excised and assessed histopathologically for possible inflammatory changes. RESULTS: Metabolic cage data revealed a significantly increased urinary frequency in zymosan treated rats. Likewise, the volume per micturition was significantly lower in all CPPS groups compared to controls. Cystometry showed a significant increase in the number of nonvoiding contractions, longer voiding time, and a trend towards lower compliance in CPPS rats compared to controls. Induction of CPPS led to significantly reduced cholinergic and purinergic contractile responses. Histopathological analysis demonstrated prostatic inflammation in all CPPS groups, in particular in later stage groups. Both the extent and grade of bladder inflammation were significantly higher in CPPS groups compared to controls. CONCLUSIONS: The current findings demonstrate a potential prostate-to-bladder cross-sensitization leading to symptoms of bladder overactivity and signs of bladder inflammation. Future clinical studies are required to verify the outcomes of the current study and enable advancement of patient care.
Asunto(s)
Síntomas del Sistema Urinario Inferior , Dolor Pélvico , Próstata , Prostatitis , Vejiga Urinaria Hiperactiva , Vejiga Urinaria , Animales , Agonistas Colinérgicos/farmacología , Dolor Crónico , Inflamación/metabolismo , Síntomas del Sistema Urinario Inferior/metabolismo , Síntomas del Sistema Urinario Inferior/fisiopatología , Masculino , Cloruro de Metacolina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiopatología , Dolor Pélvico/etiología , Dolor Pélvico/fisiopatología , Próstata/efectos de los fármacos , Próstata/metabolismo , Próstata/patología , Prostatitis/complicaciones , Prostatitis/fisiopatología , Agonistas Purinérgicos/farmacología , Ratas , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Vejiga Urinaria/fisiopatología , Vejiga Urinaria Hiperactiva/metabolismo , Vejiga Urinaria Hiperactiva/fisiopatología , Micción/efectos de los fármacos , Micción/fisiología , Zimosan/farmacologíaRESUMEN
Non-adrenergic prostate smooth muscle contractions may account for the limited effectiveness of α1-adrenoceptor antagonists, which are the first-line option for medical treatment of voiding symptoms suggestive of benign prostatic hyperplasia. In non-human prostates, purinergic agonists induce contractions reaching similar magnitudes as α1-adrenergic contractions. However, evidence for the human prostate is highly limited, and pointed to much weaker purinergic contractions. Here, we examined contractions of different purinergic agonists in human prostate tissues. Tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, and expression of purinergic receptors was studied by RT-PCR. Electric field stimulation (EFS)-induced contractions amounted to 104% of KCl-induced contractions (95% CI: 84-124%). From all tested agonists, only ATP induced concentration-dependent contractions, reaching an average maximum of 18% (12-24%) of KCl. Maximum tensions following application of other agonists averaged to 7.1% of KCl for α,ß-methylene-ATP (1.8-12.4%), 3.9% for ß,γ-methylene-ATP (2.0-5.4%), 3.1% for 2-methylthio-ATP (- 0.1-6.3%), and 5.1% for ATPγS (1.0-9.2%). Responses were not affected by the P2X antagonist NF023 or the P2Y antagonist PPADS. mRNA expression of P2X1-4 correlated with expression of a marker for catecholaminergic nerves, although neither ATP, NF023, nor PPADS changed EFS-induced contractions. Correlation between expression of receptors and the smooth muscle marker calponin was not observed. Our findings point to a low relevance of purinergic contractions in the human prostate, compared to other contractile stimuli in the human prostate and compared to purinergic contractions in non-human prostates. Purinergic contractions in the human prostate are not sensitive to NF023 or PPADS.
Asunto(s)
Músculo Liso/efectos de los fármacos , Próstata/efectos de los fármacos , Agonistas Purinérgicos/farmacología , Receptores Purinérgicos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Humanos , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/metabolismo , Próstata/metabolismo , Agonistas Purinérgicos/administración & dosificación , Receptores Purinérgicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , PorcinosRESUMEN
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
AIMS: To investigates the effects of water avoidance stress on voiding behaviour and functional bladder responses in mice. MAIN METHODS: Mice in the Stress group were exposed to water avoidance stress (WAS) for 1 h/day for 10 days, Controls were age-matched and housed normally. Voiding behaviour was measured periodically throughout the stress protocol and bladders were isolated 24-h after final stress exposure to measure bladder compliance, spontaneous phasic activity, contractile responses, and release of urothelial mediators. KEY FINDINGS: Repeated stress exposure induced a significant increase in plasma corticosterone levels in the WAS group compared to control. An overactive bladder phenotype was observed in WAS mice, causing a significant increase in the number of voiding events observed from as early as day-3, and a 7-fold increase following 10-days' stress. This increase in voiding frequency was associated with a significant decrease in void size, an increase in the number of small voids, but no change in total voided volume. Bladders from stressed mice showed a significant increase in the maximum responses to the muscarinic agonist carbachol (p < 0.01), in addition to enhanced pressure responses to the purinergic agonists ATP (p < 0.05) and αß-mATP (p < 0.05), and non-receptor mediated contractions to KCl (p < 0.05) compared to controls. Nerve-mediated bladder contractions to electric field stimulation were not significantly affected by stress, nor were spontaneous phasic contractions or release of urothelial ATP and acetylcholine. SIGNIFICANCE: Repeated exposure to water avoidance stress produced an overactive bladder phenotype, confirmed by increased voiding frequency, and associated with enhanced bladder contractile responses.
Asunto(s)
Contracción Muscular/fisiología , Estrés Psicológico/metabolismo , Vejiga Urinaria Hiperactiva/fisiopatología , Acetilcolina , Animales , Carbacol/farmacología , Corticosterona , Femenino , Ratones , Ratones Endogámicos C57BL , Agonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Cloruro de Potasio/farmacología , Agonistas Purinérgicos/farmacología , Estrés Psicológico/fisiopatología , Vejiga Urinaria/patología , Vejiga Urinaria Hiperactiva/metabolismo , Micción/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
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
INTRODUCTION: Spinal cord injury (SCI) causes most severe motor and sensory dysfunctions. In Chinese traditional medicine, the agonist of a purinergic receptor is believed to have a positive effect on SCIs, and 2-Methylthio-adenosine-5'-diphosphate (2-MesADP) is a selective agonist of the P2Y purinergic receptor. METHODS: To investigate its therapeutic function and molecular mechanism in SCI, transcriptome analysis associated with weighted gene co-expression network analysis (WGCNA) was carried out at various time points after T9 crush injury. RESULTS: 2-MesADP demonstrated recovery of limb motor function at the 6 weeks after injury, accompanied by neuronal regeneration and axon remyelination at 2 and 6 weeks. Furthermore, gene profiling revealed alternated gene expression with the treatment of 2-MesADP. These genes were assigned to a total of 38 modules, followed by gene ontology analysis; of these, 18 represented neuronal apoptosis and regeneration, immune response, synaptic transmission, cell cycle, and angiogenesis. In the neuronal apoptosis and regeneration module, Nefh, NeuroD6, and Dcx in the 2-MesADP group were noticed due to their interesting expression pattern. The gene expression patterns of Mag, Mog, and Cnp, which played key roles in myelination, were significantly changed with the treatment of 2-MesADP. Wnt signal pathway was the most important pathway in 2-MesADP treatment for acute SCI. CONCLUSION: 2-MesADP enhanced locomotor recovery in mouse SCI by altering the expression of neuronal apoptosis and remyelination-related genes and Wnt signaling pathways.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Locomoción/fisiología , Agonistas Purinérgicos/farmacología , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal , Tionucleótidos/farmacología , Adenosina Difosfato/farmacología , Animales , Proteína Doblecortina , Humanos , Ratones , Regeneración Nerviosa/efectos de los fármacos , Recuperación de la Función/fisiología , Remielinización/efectos de los fármacos , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
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
Purpose: Osteopontin (OPN) is a neuroprotective factor in the retina that improves photoreceptor survival. The aim of the present study was to investigate whether human RPE cells express and respond to OPN. Methods: Hypoxia and chemical hypoxia were induced by cell culture in 0.25% O2 and the addition of CoCl2, respectively. Hyperosmolarity was produced by the addition of 100 mM NaCl or 200 mM sucrose. Gene expression was quantified with real-time reverse transcription (RT)-PCR, and protein secretion was investigated with enzyme-linked immunosorbent assay (ELISA). Nuclear factor of activated T cell 5 (NFAT5) was depleted with siRNA. Results: The acutely isolated RPE cells and the cultured RPE cells expressed OPN. OPN gene expression was induced by hypoxia and hyperosmotic media, as well as by exogenous bFGF. High extracellular NaCl and hypoxia induced secretion of OPN. Hyperosmotic expression of the OPN gene was mediated by the p38 MAPK and ERK1/2 signal transduction pathways, and the transcriptional activities of CREB and NFAT5. The hypoxic expression of the OPN gene was mediated by the PI3K signal transduction pathway and caspase-mediated, necrosis-related pathways. Phospholipases A2 were involved in mediating hyperosmotic and hypoxic OPN gene expression. Autocrine or paracrine P2Y2 receptor signaling induced by extracellular ATP contributed to hyperosmotic expression of the OPN gene whereas activation of A1 receptors by extracellularly formed adenosine contributed to thypoxic OPN gene expression. Autocrine or paracrine VEGF signaling exerted an inhibitory effect on expression of the OPN gene. Exogenous OPN induced expression and secretion of bFGF, but not of VEGF. Conclusions: The data indicated that RPE cells produce and respond to OPN; OPN expression is, in part, induced by the cellular danger signal ATP. RPE-derived neuroprotective factors such as bFGF may contribute to the prosurvival effect of OPN on photoreceptor cells.
Asunto(s)
Hipoxia de la Célula/efectos de los fármacos , Células Epiteliales/metabolismo , Presión Osmótica/efectos de los fármacos , Osteopontina/metabolismo , Agonistas Purinérgicos/farmacología , Receptores Purinérgicos/metabolismo , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Adenosina Trifosfato/farmacología , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Hipoxia de la Célula/genética , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Osteopontina/genética , Osteopontina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasas A2/metabolismo , ARN Interferente Pequeño , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Cloruro de Sodio/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.SIGNIFICANCE STATEMENT Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.
Asunto(s)
Epilepsia del Lóbulo Temporal/fisiopatología , Glioma/fisiopatología , Microglía/fisiología , Receptores Purinérgicos P2Y12/fisiología , Receptores Purinérgicos P2Y1/fisiología , Receptores Purinérgicos P2/fisiología , Neoplasias Supratentoriales/fisiopatología , Adenosina Difosfato/farmacología , Adulto , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Forma de la Célula/efectos de los fármacos , Extensiones de la Superficie Celular/efectos de los fármacos , Extensiones de la Superficie Celular/fisiología , Extensiones de la Superficie Celular/ultraestructura , Epilepsia del Lóbulo Temporal/etiología , Epilepsia del Lóbulo Temporal/patología , Femenino , Glioma/patología , Humanos , Microscopía Intravital , Masculino , Microglía/efectos de los fármacos , Microglía/ultraestructura , Persona de Mediana Edad , Lectinas de Plantas , Agonistas Purinérgicos/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Neoplasias Supratentoriales/patología , Esclerosis Tuberosa/complicacionesRESUMEN
Oxalate crystal-induced renal inflammation is associated with progressive kidney failure due to activation of the NLRP3/CASP-1 inflammasome. It has been suggested previously that purinergic P2X7 receptor signaling is critical for crystal-induced inflammasome activation and renal injury. Therefore, we investigated the role of the P2X7 receptor in response to crystal-induced cytokine release, inflammation, and kidney failure using in vitro and in vivo models. Dendritic cells and macrophages derived from murine bone marrow and human peripheral blood mononucleated cells stimulated with calcium-oxalate crystals, monosodium urate crystals, or ATP lead to the robust release of interleukin-1beta (IL-1ß). Treatment with the P2X7 inhibitor A740003 or the depletion of ATP by apyrase selectively abrogated ATP-induced, but not oxalate and urate crystal-induced IL-1ß release. In line with this finding, dendritic cells derived from bone marrow (BMDCs) from P2X7-/- mice released reduced amounts of IL-1ß following stimulation with ATP, while oxalate and urate crystal-induced IL-1ß release was unaffected. In sharp contrast, BMDCs from Casp1-/- mice exhibited reduced IL-1ß release following either of the three stimulants. In addition, P2X7-/- mice demonstrated similar degrees of crystal deposition, tubular damage and inflammation when compared with WT mice. In line with these findings, increases in plasma creatinine were no different between WT and P2X7-/- mice. In contrast to previous reports, our results indicate that P2X7 receptor is not required for crystal-induced CKD and it is unlikely to be a suitable therapeutic target for crystal-induced progressive kidney disease.
Asunto(s)
Cálculos Renales/inducido químicamente , Oxalatos/toxicidad , Agonistas Purinérgicos/farmacología , Receptores Purinérgicos P2X7/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Caspasa 1/genética , Humanos , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores Purinérgicos P2X7/genética , Ácido Úrico/toxicidadRESUMEN
Localisation of platelet-derived growth factor receptor-α (PDGFRα) (+) cells expressing small-conductance Ca2+-activated K+ (SK3) channels in the urinary bladder was investigated, while putative roles of SK3 (+) PDGFRα (+) cells in suppressing detrusor smooth muscle (DSM) spontaneous activity were explored. In guinea-pig bladder, immunohistochemistry for SK3 channels, PDGFRα or vimentin was examined, as were the effects of purinergic agonists on spontaneous phasic contractions (SPCs). In bladder of PDGFRα-GFP mice, the effects of purinergic agonists on intracellular Ca2+ signaling in PDGFRα (+) cells or DSM cells in situ and SPCs were investigated. SK3 (+) cells co-expressing PDGFRα or vimentin were distributed in DSM bundles but not inter-bundle spaces or lamina propria. SK3 (+) cells had a stellate- or spindle-shape cell body extending processes. MRS2365 (100 nM or 1 µM), a P2Y1 agonist, caused a transient contraction without inhibiting SPCs in both DSM and lamina propria. In PDGFRα-GFP mice bladder, MRS2365, (100â¯nM), ADP (100⯵M) or ATP (100⯵M) increased the Ca2+ level of PDGFRα (+) cells without suppressing spontaneous Ca2+ transients in neighboring DSM cells, and also failed to suppress SPCs. Preferential localisation of SK3 positive PDGFRα (+) cells in DSM bundles appears to indicate their functional interaction with DSM cells. However, increases in Ca2+ level of PDGFRα (+) cells upon purinergic stimulation are not associated with the inhibition of Ca2+ or contractile activity in DSM cells. Thus, it is unlikely that the SK3-dependent hyperpolarisation generated in SK3 expressing PDGFRα (+) cells is transmitted to DSMs to suppress their excitability.
Asunto(s)
Regulación de la Expresión Génica , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Vejiga Urinaria/citología , Animales , Señalización del Calcio/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Cobayas , Masculino , Ratones , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Agonistas Purinérgicos/farmacología , Vejiga Urinaria/fisiologíaRESUMEN
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.
Asunto(s)
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
BACKGROUND: The arenavirus Junin virus (JUNV), causative agent of the argentine hemorrhagic fever, is able to modulate several signaling pathways involved in cell survival and multiplication. OBJECTIVES: We aimed to characterize the infection of rat osteoblasts (OBCs) with JUNV and its consequence on the modulation of osteogenic genes expression, thus studying the ability of this virus to induce cell differentiation. In addition, we evaluated the effect of purinergic agonists on viral replication. METHOD: Quantification of infectivity by plaque forming unit (PFU) assay, synthesis of viral proteins by western blot and immunofluorescence, and expression of osteogenic differentiation markers (ODM) by quantitative real-time polymerase chain reaction were employed. RESULTS: Infection of OBCs with JUNV (MOI 0.01 PFU/cell) showed a peak of infectivity, reaching 1.5 × 105 PFU/mL at the second day post-infection (p.i.). A marked restriction in multiplication was detected at day 7 p.i. that did not impair the establishment of a persistent stage of infection in OBCs. Analysis of mRNAs corresponding to ODM such as alkaline phosphatase, bone sialo-protein, and bone morphogenetic proteins (BMPs) 4 and 6 revealed that only the levels of BMP-6 were significantly higher in infected cells. Treatment with the purinergic agonists ATPγS, UTP, ADP, or UDP diminished viral titer and reduced the expression of the viral nucleoprotein. Also, treatment with 10 µM ATPγS reduced the stimulation of BMP-6 expression induced by the infection. CONCLUSIONS: These data demonstrate that JUNV is capable of infecting OBCs and point out BMP-6 as a key factor during this process.
Asunto(s)
Proteína Morfogenética Ósea 6/genética , Virus Junin/fisiología , Osteoblastos/virología , Osteogénesis/genética , Animales , Diferenciación Celular , Células Cultivadas , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Agonistas Purinérgicos/farmacología , Ratas , Transducción de Señal , Replicación Viral/efectos de los fármacosRESUMEN
In this study the nitric oxide (NO)-soluble guanylate cyclase (sGC) and phosphodiesterase-5 (PDE-5) pathway was characterized in tortoise Chelonoidis carbonaria aorta. Concentration response curves (CCR) to ATP, ADP, AMP, adenosine and histamine were performed in the presence and absence of L-NAME in aorta pre-contracted with ACh (3⯵M). CCR to SNP, BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator) and tadalafil (PDE-5 inhibitor) were constructed in the presence and absence of ODQ (10⯵M). ATP (pEC50 6.1⯱â¯0.1), ADP (pEC50 6.0⯱â¯0.2), AMP (pEC50 6.8⯱â¯0.1) and histamine (pEC50 6.8⯱â¯0.12) relaxed Chelonoidis aorta and the addition of L-NAME reduced their efficacy (pâ¯<â¯.05). Adenosine effects (pEC50 6.6⯱â¯0.1) were not changed in the presence of L-NAME. SNP (pEC50 7.5⯱â¯0.7; Emax 102.2⯱â¯2.5%), BAY 41-2272 (pEC50 7.3⯱â¯0.2; Emax 130.3⯱â¯10.2%), BAY 60-2770 (pEC50 11.4⯱â¯0.1; Emax 130.3⯱â¯6.5%) and tadalafil (pEC50 6.7⯱â¯0.3; Emax 121.3⯱â¯15.3%) relaxed Chelonoidis aorta. The addition of ODQ reduced the SNP and tadalafil maximum response (pâ¯<â¯.05) and promoted 63 fold right shift on BAY 41-2272 curve. In contrast, no alteration was observed on BAY 60-2770 response. Transcriptomic analysis for eNOS and sGC were found in aorta and brain libraries with high homology when compared with human transcripts. The NO-sGC-PDE-5 is functionally present in Chelonoidis aorta with a functional and genomic similarity to mammalian vessels. Unlike most of mammalian vessels, ACh did not cause endothelium-dependent relaxation in Chelonoidis carbonaria aortic rings.