RESUMEN
The role of adenosine receptors in fascial manipulation-induced analgesia has not yet been investigated. The purpose of this study was to evaluate the involvement of the adenosine A1 receptor (A1R) in the antihyperalgesic effect of plantar fascia manipulation (PFM), specifically in mice with peripheral inflammation. Mice injected with Complete Freund's Adjuvant (CFA) underwent behavioral, i.e. mechanical hyperalgesia and edema. The mice underwent PFM for either 3, 9 or 15 min. Response frequency to mechanical stimuli was then assessed at 24 and 96 h after plantar CFA injection. The adenosinergic receptors were assessed by systemic (intraperitoneal, i.p.), central (intrathecal, i.t.), and peripheral (intraplantar, i.pl.) administration of caffeine. The participation of the A1R was investigated using the 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective A1R subtype antagonist. PFM inhibited mechanical hyperalgesia induced by CFA injection and did not reduce paw edema. Furthermore, the antihyperalgesic effect of PFM was prevented by pretreatment of the animals with caffeine given by i.p., i.pl., and i.t. routes. In addition, i.pl. and i.t. administrations of DPCPX blocked the antihyperalgesia caused by PFM. These observations indicate that adenosine receptors mediate the antihyperalgesic effect of PFM. Caffeine's inhibition of PFM-induced antihyperalgesia suggests that a more precise understanding of how fascia-manipulation and caffeine interact is warranted.
Asunto(s)
Modelos Animales de Enfermedad , Adyuvante de Freund , Hiperalgesia , Inflamación , Receptor de Adenosina A1 , Xantinas , Animales , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A1/efectos de los fármacos , Ratones , Masculino , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Xantinas/farmacología , Fascia/efectos de los fármacos , Cafeína/farmacología , Cafeína/administración & dosificación , Analgesia/métodos , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Antagonistas del Receptor de Adenosina A1/farmacologíaRESUMEN
The mechanisms responsible for glomerular hemodynamic regulation with sodium-glucose co-transporter 2 (SGLT2) inhibitors in kidney disease due to type 2 diabetes remain unclear. Therefore, we investigated changes in glomerular hemodynamic function using an animal model of type 2 diabetes, treated with an SGLT2 inhibitor alone or in combination with a renin-angiotensin-aldosterone system inhibitor using male Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats. Afferent and efferent arteriolar diameter and single-nephron glomerular filtration rate (SNGFR) were evaluated in ZDF rats measured at 0, 30, 60, 90, and 120 minutes after the administration of a SGLT2 inhibitor (luseogliflozin). Additionally, we assessed these changes under the administration of the adenosine A1 receptor (A1aR) antagonist (8-cyclopentyl-1,3-dipropylxanthine), along with coadministration of luseogliflozin and an angiotensin II receptor blocker (ARB), telmisartan. ZDF rats had significantly increased SNGFR, and afferent and efferent arteriolar diameters compared to ZL rats, indicating glomerular hyperfiltration. Administration of luseogliflozin significantly reduced afferent vasodilatation and glomerular hyperfiltration, with no impact on efferent arteriolar diameter. Urinary adenosine levels were increased significantly in the SGLT2 inhibitor group compared to the vehicle group. A1aR antagonism blocked the effect of luseogliflozin on kidney function. Co-administration of the SGLT2 inhibitor and ARB decreased the abnormal expansion of glomerular afferent arterioles, whereas the efferent arteriolar diameter was not affected. Thus, regulation of afferent arteriolar vascular tone via the A1aR pathway is associated with glomerular hyperfiltration in type 2 diabetic kidney disease.
Asunto(s)
Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Tasa de Filtración Glomerular , Glomérulos Renales , Ratas Zucker , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Masculino , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/fisiopatología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/fisiopatología , Glomérulos Renales/patología , Glomérulos Renales/irrigación sanguínea , Tasa de Filtración Glomerular/efectos de los fármacos , Ratas , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/fisiopatología , Hemodinámica/efectos de los fármacos , Xantinas/farmacología , Arteriolas/efectos de los fármacos , Arteriolas/fisiopatología , Antagonistas del Receptor de Adenosina A1/farmacología , Sistema Renina-Angiotensina/efectos de los fármacos , Transportador 2 de Sodio-Glucosa/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Sorbitol/análogos & derivadosRESUMEN
Peripheral A1 adenosine receptor signaling has been shown to have analgesic effects in a variety of pain conditions. However, it is not yet fully elucidated for the precise molecular mechanisms. Acid sensing ion channels (ASICs) are expressed predominantly in nociceptive sensory neurons responding to protons. Given that both A1 adenosine receptors and ASICs are present in dorsal root ganglia (DRG) neurons, we therefore investigated whether there was a cross-talk between the two types of receptors. Herein, electrophysiological recordings showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) suppressed acid-induced currents and action potentials, which were mediated by ASICs, in rat DRG neurons. CPA inhibited the maximum response to protons, as shown a downward shift of concentration-response curve for protons. The CPA-induced suppression of ASIC currents was blocked by the A1 adenosine receptor antagonist KW-3902 and also prevented by intracellular application of the Gi/o-protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. Finally, intraplantar pretreatment of CPA dose-dependently relieved acid-induced nociceptive responses in rats through peripheral A1 adenosine receptors. These results suggested that CPA suppressed ASICs via A1 adenosine receptors and intracellular Gi/o-proteins and cAMP signaling cascades in rat DRG neurons, which was a novel potential mechanism underlying analgesia of peripheral A1 adenosine receptors.
Asunto(s)
Canales Iónicos Sensibles al Ácido/efectos de los fármacos , Agonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Analgesia , Fenómenos Electrofisiológicos/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Nocicepción/efectos de los fármacos , Nociceptores/efectos de los fármacos , Receptor de Adenosina A1/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , RatasRESUMEN
A long evolution of knowledge of the psychostimulant caffeine led in the 1960s to another purine natural product, adenosine and its A2A receptor. Adenosine is a short-lived autocrine/paracrine mediator that acts pharmacologically at four different adenosine receptors in a manner opposite to the pan-antagonist caffeine and serves as an endogenous allostatic regulator. Although detrimental in the developing brain, caffeine appears to be cerebroprotective in aging. Moderate caffeine consumption in adults, except in pregnancy, may also provide benefit in pain, diabetes, and kidney and liver disorders. Inhibition of A2A receptors is one of caffeine's principal effects and we now understand this interaction at the atomic level. The A2A receptor has become a prototypical example of utilizing high-resolution structures of GPCRs for the rational design of chemically diverse drug molecules. The previous focus on discovery of selective A2A receptor antagonists for neurodegenerative diseases has expanded to include immunotherapy for cancer, and clinical trials have ensued. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.
Asunto(s)
Cafeína , Receptor de Adenosina A2A , Adenosina/metabolismo , Antagonistas del Receptor de Adenosina A1/farmacología , Cafeína/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismoRESUMEN
In previous studies using isolated, paced guinea pig left atria, we observed that FSCPX, known as a selective A1 adenosine receptor antagonist, paradoxically increased the direct negative inotropic response to A1 adenosine receptor agonists (determined using concentration/effect (E/c) curves) if NBTI, a nucleoside transport inhibitor, was present. Based on mathematical modeling, we hypothesized that FSCPX blunted the cardiac interstitial adenosine accumulation in response to nucleoside transport blockade, probably by inhibiting CD39 and/or CD73, which are the two main enzymes of the interstitial adenosine production in the heart. The goal of the present study was to test this hypothesis. In vitro CD39 and CD73 inhibitor assays were carried out; furthermore, E/c curves were constructed in isolated, paced rat and guinea pig left atria using adenosine, CHA and CPA (two A1 adenosine receptor agonists), FSCPX, NBTI and NBMPR (two nucleoside transport inhibitors), and PSB-12379 (a CD73 inhibitor), measuring the contractile force. We found that FSCPX did not show any inhibitory effect during the in vitro enzyme assays. However, we successfully reproduced the paradox effect of FSCPX in the rat model, mimicked the "paradox" effect of FSCPX with PSB-12379, and demonstrated the lipophilia of FSCPX, which could explain the negative outcome of inhibitor assays with CD39 and CD73 dissolved in a water-based solution. Taken together, these three pieces of indirect evidence are strong enough to indicate that FSCPX possesses an additional action besides the A1 adenosine receptor antagonism, which action may be the inhibition of an ectonucleotidase. Incidentally, we found that POM-1 inhibited CD73, in addition to CD39.
Asunto(s)
5'-Nucleotidasa/antagonistas & inhibidores , Antagonistas del Receptor de Adenosina A1/farmacología , Apirasa/antagonistas & inhibidores , Receptor de Adenosina A1/metabolismo , Xantinas/farmacología , 5'-Nucleotidasa/metabolismo , Animales , Antígenos CD/metabolismo , Apirasa/metabolismo , Cobayas , Masculino , Ratas , Ratas WistarRESUMEN
Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A3/farmacología , Sondas Moleculares/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A3/metabolismo , Xantinas/farmacología , Antagonistas del Receptor de Adenosina A1/síntesis química , Antagonistas del Receptor de Adenosina A3/síntesis química , Alquinos/química , Animales , Azidas/química , Células CHO , Química Clic , Cricetulus , Diseño de Fármacos , Colorantes Fluorescentes/química , Humanos , Ligandos , Sondas Moleculares/síntesis química , Receptor de Adenosina A1/química , Receptor de Adenosina A3/química , Xantinas/síntesis químicaRESUMEN
The role of peripheral adenosine receptors in pain is a controversial issue and seems to be quite different from the roles of spinal and central adenosine receptors. The present study is aimed at clarifying the role of these receptors in peripheral nociception. To clarify this, studies were done on Swiss mice with adenosine receptor agonists and antagonists. Nociceptive behavior was induced by subcutaneous injection of glutamate (10 µmol) into the ventral surface of the hind paw of mice. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Results showed that intraplantar (i.pl.) administration of N6-cyclohexyl-adenosine (CHA), an adenosine A1 receptor agonist, at 1 or 10 µg/paw significantly reduced glutamate-induced nociception (p<0.01 and p<0.001 vs. vehicle, respectively, n=8-10). In contrast, i.pl. injection of hydrochloride hydrate (CGS21680, an adenosine A2A receptor agonist) (1 µg/paw) induced a significant increase in glutamate-induced nociception compared to the vehicle (p<0.05, n=8), while 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, an adenosine A2A receptor antagonist) (20 µg/paw) caused a significant reduction (p<0.05, n=7-8). There were no significant effects on i.pl. administration of four additional adenosine receptor drugs-8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 antagonist, 1-10 µg/paw), N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA, an A2B agonist, 1-100 µg/paw), alloxazine (an A2B antagonist, 0.1-3 µg/paw), and 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) (an A3 agonist, 1-100 µg/paw) (p>0.05 vs. vehicle for all tests). We also found that prior administration of DPCPX (3 µg/paw) significantly blocked the anti-nociceptive effect of CHA (1 µg/paw) (p<0.05, n=7-9). Similarly, ZM241385 (20 µg/paw) administered prior to CGS21680 (1 µg/paw) significantly blocked CGS21680-induced exacerbation of nociception (p<0.05, n=8). Finally, inosine (10 and 100 µg/paw), a novel endogenous adenosine A1 receptor agonist recently reported by our research group, was also able to reduce glutamate-induced nociception (p<0.001 vs. vehicle, n=7-8). Interestingly, as an A1 adenosine receptor agonist, the inosine effect was significantly blocked by the A1 antagonist DPCPX (3 µg/paw) (p<0.05, n=7-9) but not by the A2A antagonist ZM241385 (10 µg/paw, p>0.05). In summary, these results demonstrate for the first time that i.pl administration of inosine induces an anti-nociceptive effect, similar to that elicited by CHA and possibly mediated by peripheral adenosine A1 receptor activation. Moreover, our results suggest that peripheral adenosine A2A receptor activation presents a pro-nociceptive effect, exacerbating glutamate-induced nociception independent of inosine-induced anti-nociceptive effects.
Asunto(s)
Glutamatos , Nocicepción/efectos de los fármacos , Dolor/inducido químicamente , Dolor/psicología , Sistema Nervioso Periférico/efectos de los fármacos , Receptores Purinérgicos P1/efectos de los fármacos , Agonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Agonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Femenino , Pie , Glutamatos/administración & dosificación , Inyecciones , Inosina/farmacología , Masculino , Ratones , Dimensión del Dolor/efectos de los fármacos , Receptor de Adenosina A2A/efectos de los fármacosRESUMEN
N-methyl D-aspartate (NMDA) administered at subtoxic dose plays a protective role against neuronal excitotoxicity, a mechanism described as preconditioning. Since the activation of adenosinergic receptors influences the achievement of NMDA preconditioning in the hippocampus, we evaluated the potential functional interplay between adenosine A1 and A2A receptors (A1R and A2AR) activities and NMDA preconditioning. Adult male Swiss mice received saline (NaCl 0.9 g%, i.p.) or a nonconvulsant dose of NMDA (75 mg/kg, i.p.) and 24 h later they were treated with the one of the ligands: A1R agonist (CCPA, 0.2 mg/kg, i.p.) or antagonist (DPCPX, 3 mg/kg, i.p.), A2AR agonist (CGS21680, 0.05 mg/kg, i.p.) or antagonist (ZM241385, 0.1 mg/kg, i.p.) and subjected to contextual fear conditioning task. Binding properties and content of A2AR and glutamate uptake were assessed in the hippocampus of mice subjected to NMDA preconditioning. Treatment with CGS21680 increased the time of freezing during the exposure of animals to the new environment. NMDA preconditioning did not affect the freezing time of mice per se, but it prevented the response observed after the activation of A2AR. Furthermore, the activation of A2AR by CGS21680 after the preconditioning blocked the increase of glutamate uptake induced by NMDA preconditioning. The immunodetection of A2AR in total hippocampal homogenates showed no significant differences evoked by NMDA preconditioning and did not alter A2AR maximum binding for the selective ligand [3H]CGS21680. These results demonstrate changes in A2AR functionality in mice following NMDA preconditioning.
Asunto(s)
Condicionamiento Clásico/fisiología , Miedo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Memoria/fisiología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Agonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Agonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Condicionamiento Clásico/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Memoria/efectos de los fármacos , Ratones , N-Metilaspartato/farmacologíaRESUMEN
The purpose of the study was to investigate whether the co-administration of Mg2+ and Zn2+ with selective A1 and A2A receptor antagonists might be an interesting antidepressant strategy. Forced swim, tail suspension, and spontaneous locomotor motility tests in mice were performed. Further, biochemical and molecular studies were conducted. The obtained results indicate the interaction of DPCPX and istradefylline with Mg2+ and Zn2+ manifested in an antidepressant-like effect. The reduction of the BDNF serum level after co-administration of DPCPX and istradefylline with Mg2+ and Zn2+ was noted. Additionally, Mg2+ or Zn2+, both alone and in combination with DPCPX or istradefylline, causes changes in Adora1 expression, DPCPX or istradefylline co-administered with Zn2+ increases Slc6a15 expression as compared to a single-drug treatment, co-administration of tested agents does not have a more favourable effect on Comt expression. Moreover, the changes obtained in Ogg1, MsrA, Nrf2 expression show that DPCPX-Mg2+, DPCPX-Zn2+, istradefylline-Mg2+ and istradefylline-Zn2+ co-treatment may have greater antioxidant capacity benefits than administration of DPCPX and istradefylline alone. It seems plausible that a combination of selective A1 as well as an A2A receptor antagonist and magnesium or zinc may be a new antidepressant therapeutic strategy.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Conducta Animal/efectos de los fármacos , Magnesio/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Xantinas/farmacología , Zinc/farmacología , Animales , Masculino , RatonesRESUMEN
6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson's disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 µM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A1 (A1R) and/or A2A receptors (A2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A1R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A1R agonist, did not alter GUO effects. Regarding A2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A1R antagonist DPCPX, and the A2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A1R-A2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.
Asunto(s)
Guanosina/farmacología , Enfermedades Mitocondriales/prevención & control , Neostriado/metabolismo , Fármacos Neuroprotectores/farmacología , Oxidopamina/toxicidad , Receptor de Adenosina A1/efectos de los fármacos , Receptor de Adenosina A2A/efectos de los fármacos , Estallido Respiratorio/efectos de los fármacos , Antagonistas del Receptor de Adenosina A1/farmacología , Animales , Evaluación Preclínica de Medicamentos , Técnicas In Vitro , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neostriado/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Xantinas/uso terapéuticoRESUMEN
Adenosine A1 receptors (A1ARs) are promising imaging biomarkers and targets for the treatment of stroke. Nevertheless, the role of A1ARs on ischemic damage and its subsequent neuroinflammatory response has been scarcely explored so far. Methods: In this study, the expression of A1ARs after transient middle cerebral artery occlusion (MCAO) was evaluated by positron emission tomography (PET) with [18F]CPFPX and immunohistochemistry (IHC). In addition, the role of A1ARs on stroke inflammation using pharmacological modulation was assessed with magnetic resonance imaging (MRI), PET imaging with [18F]DPA-714 (TSPO) and [18F]FLT (cellular proliferation), as well as IHC and neurofunctional studies. Results: In the ischemic territory, [18F]CPFPX signal and IHC showed the overexpression of A1ARs in microglia and infiltrated leukocytes after cerebral ischemia. Ischemic rats treated with the A1AR agonist ENBA showed a significant decrease in both [18F]DPA-714 and [18F]FLT signal intensities at day 7 after cerebral ischemia, a feature that was confirmed by IHC results. Besides, the activation of A1ARs promoted the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome including motor, sensory and reflex responses. These results show for the first time the in vivo PET imaging of A1ARs expression after cerebral ischemia in rats and the application of [18F]FLT to evaluate glial proliferation in response to treatment. Conclusion: Notably, these data provide evidence for A1ARs playing a key role in the control of both the activation of resident glia and the de novo proliferation of microglia and macrophages after experimental stroke in rats.
Asunto(s)
Encéfalo/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Inflamación/metabolismo , Receptor de Adenosina A1/metabolismo , Antagonistas del Receptor de Adenosina A1/farmacología , Animales , Encéfalo/diagnóstico por imagen , Didesoxinucleósidos , Inmunohistoquímica , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/fisiopatología , Inflamación/diagnóstico por imagen , Inflamación/fisiopatología , Leucocitos/metabolismo , Activación de Macrófagos/efectos de los fármacos , Imagen por Resonancia Magnética , Microglía/metabolismo , Imagen Multimodal , Tomografía de Emisión de Positrones , Pirazoles , Pirimidinas , Radiofármacos , Ratas , Xantinas/farmacologíaRESUMEN
The dual inhibition of adenosine receptors A1 (A1 AR) and A2 (A2A AR) has been considered as an efficient strategy in the treatment of Parkinson's disease (PD). This led to the recent development of a series of methoxy-substituted benzofuran derivatives among which compound 3j exhibited dual-inhibitory potencies in the micromolar range. Therefore, in this study, we seek to resolve the mechanisms by which this novel compound elicits its selective dual targeting against A1 AR and A2A AR. Unique to the binding of 3j in both proteins, from our findings, is the ring-ring interaction elicited by A1Phe275 (â A2Phe170) with the benzofuran ring of the compound. As observed, this π-stacking interaction contributes notably to the stability of 3j at the active sites of A1 and A2A AR. Besides, conserved active site residues in the proteins such as A1Ala170 (â A2Ala65), A1Ile173 (â A2Ile68), A1Val191 (â A2Val86), A1Leu192 (â A2Leu87), A1Ala195 (â A2Ala90), A1Met284 (â A2Met179), A1Tyr375 (â A2Tyr369), A1Ile378 (â A2Ile372), and A1His382 (â A2His376) were commonly involved with other ring substituents which further complement the dual binding and stability of 3j. This reflects a similar interaction mechanism that involved aromatic (π) interactions. Consequentially, vdW energies contributed immensely to the dual binding of the compound, which culminated in high ΔGbinds that were homogenous in both proteins. Furthermore, 3j commonly disrupted the stable and compact conformation of A1 and A2A AR, coupled with their active sites where Cα deviations were relatively high. Ligand mobility analysis also revealed that both compounds exhibited a similar motion pattern at the active site of the proteins relative to their optimal dual binding. We believe that findings from this study with significantly aid the structure-based design of highly selective dual-inhibitors of A1 and A2A AR.
Asunto(s)
Antiparkinsonianos/síntesis química , Benzofuranos/síntesis química , Diseño de Fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 2/genética , Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Benzofuranos/química , Dominio Catalítico , Simulación por Computador , Humanos , Ligandos , Conformación Molecular , Análisis de Componente Principal , Unión Proteica , Receptores Purinérgicos P1 , Relación Estructura-Actividad , TermodinámicaRESUMEN
Here, we tested the hypothesis that TNAP (tissue nonspecific alkaline phosphatase) modulates vascular responsiveness to norepinephrine. In the isolated, Tyrode's-perfused rat mesentery, 50 µmol/L of L-p-bromotetramisole (L-p-BT; selective TNAP inhibitor, Ki=56 µmol/L) significantly reduced TNAP activity and caused a significant 9.0-fold rightward-shift in the norepinephrine concentration versus vasoconstriction relationship. At 100 µmol/L, L-p-BT further reduced mesenteric TNAP activity and caused an additional significant right-shift of the norepinephrine concentration versus vasoconstriction relationship. A higher concentration (200 µmol/L) of L-p-BT had no further effect on either mesenteric TNAP activity or norepinephrine-induced vasoconstriction. L-p-BT did not alter vascular responses to vasopressin, thus ruling-out nonspecific suppression of vascular reactivity. Since in the rat mesenteric vasculature α1-adrenoceptors mediate norepinephrine-induced vasoconstriction, these finding indicate that TNAP inhibition selectively interferes with α1-adrenoceptor signaling. Additional experiments showed that the effects of TNAP inhibition on norepinephrine-induced vasoconstriction were not mediated by accumulation of pyrophosphate or ATP (TNAP substrates) nor by reduced adenosine levels (TNAP product). TNAP inhibition significantly reduced the Hillslope of the norepinephrine concentration versus vasoconstriction relationship from 1.8±0.2 (consistent with positive cooperativity of α1-adrenoceptor signaling) to 1.0±0.1 (no cooperativity). Selective activation of A1-adenosine receptors, which are known to participate in coincident signaling with α1-adrenoceptors, reversed the suppressive effects of L-p-BT on norepinephrine-induced vasoconstriction. In vivo, L-p-BT administration achieved plasma levels of ≈60 µmol/L and inhibited mesenteric vascular responses to exogenous norepinephrine and sympathetic nerve stimulation. TNAP modulates vascular responses to norepinephrine likely by affecting positive cooperativity of α1-adrenoceptor signaling via a mechanism involving A1 receptor signaling.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Proteínas de la Membrana/metabolismo , Mesenterio/efectos de los fármacos , Norepinefrina/farmacología , Tetramisol/análogos & derivados , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/genética , Animales , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Mesenterio/metabolismo , Ratas , Tetramisol/farmacología , Xantinas/farmacologíaRESUMEN
Parkinson's disease (PD) signs and symptoms regularly include tremor. Interestingly, the nucleoside guanosine (GUO) has already proven to be effective in reducing reserpine-induced tremulous jaw movements (TJMs) in rodent models, thus becoming a promising antiparkinsonian drug. Here, we aimed at revealing the mechanism behind GUO antiparkinsonian efficacy by assessing the role of adenosine A1 and A2A receptors (A1R and A2AR) on GUO-mediated anti-tremor effects in the reserpinized mouse model of PD. Reserpinized mice showed elevated reactive oxygen species (ROS) production and cellular membrane damage in striatal slices assessed ex vivo and GUO treatment reversed ROS production. Interestingly, while the simultaneous administration of sub-effective doses of GUO (5 mg/kg) and SCH58261 (0.01 mg/kg), an A2AR antagonist, precluded reserpine-induced TJMs, these were ineffective on reverting ROS production in ex vivo experiments. Importantly, GUO was able to reduce TJM and ROS production in reserpinized mouse lacking the A2AR, thus suggesting an A2AR-independent mechanism of GUO-mediated effects. Conversely, the administration of DPCPX (0.75 mg/kg), an A1R antagonist, completely abolished both GUO-mediated anti-tremor effects and blockade of ROS production. Overall, these results indicated that GUO anti-tremor and antioxidant effects in reserpinized mice were A1R dependent but A2AR independent, thus suggesting a differential participation of adenosine receptors in GUO-mediated effects.
Asunto(s)
Guanosina/uso terapéutico , Enfermedad de Parkinson Secundaria/metabolismo , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Temblor/metabolismo , Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2 , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Guanosina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Temblor/inducido químicamente , Temblor/tratamiento farmacológico , Xantinas/farmacologíaRESUMEN
Antagonists of the adenosine receptors (A1 and A2A subtypes) are widely researched as potential drug candidates for their role in Parkinson's disease-related cognitive deficits (A1 subtype), motor dysfunction (A2A subtype) and to exhibit neuroprotective properties (A2A subtype). Previously the benzo-α-pyrone based derivative, 3-phenyl-1H-2-benzopyran-1-one, was found to display both A1 and A2A adenosine receptor affinity in the low micromolar range. Prompted by this, the α-pyrone core was structurally modified to explore related benzoxazinone and quinazolinone homologues previously unknown as adenosine receptor antagonists. Overall, the C2-substituted quinazolinone analogues displayed superior A1 and A2A adenosine receptor affinity over their C2-substituted benzoxazinone homologues. The benzoxazinones were devoid of A2A adenosine receptor binding, with only two compounds displaying A1 adenosine receptor affinity. In turn, the quinazolinones displayed varying degrees of affinity (low micromolar range) towards the A1 and A2A adenosine receptor subtypes. The highest A1 adenosine receptor affinity and selectivity were favoured by methyl para-substitution of phenyl ring B (A1Ki = 2.50 µM). On the other hand, 3,4-dimethoxy substitution of phenyl ring B afforded the best A2A adenosine receptor binding (A2AKi = 2.81 µM) among the quinazolinones investigated. In conclusion, the quinazolinones are ideal lead compounds for further structural optimization to gain improved adenosine receptor affinity, which may find therapeutic relevance in Parkinson's disease-associated cognitive deficits and motor dysfunctions as well as exerting neuroprotective properties.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Quinazolinonas/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Antagonistas del Receptor de Adenosina A1/síntesis química , Antagonistas del Receptor de Adenosina A1/química , Antagonistas del Receptor de Adenosina A2/síntesis química , Antagonistas del Receptor de Adenosina A2/química , Animales , Relación Dosis-Respuesta a Droga , Estructura Molecular , Quinazolinonas/síntesis química , Quinazolinonas/química , Ratas , Relación Estructura-ActividadRESUMEN
The α2-adrenergic receptor (α2-AR) agonist dexmedetomidine increases baroreflex sensitivity (BRS). In the current study, we examined the potential role of adenosine A1 receptor (A1R) within the nucleus tractus solitaries (NTS) in such a response. Briefly, adult male Sprague-Dawley rats were anesthetized and randomly received microinjection of selective A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.1 pmol/1 µl) or saline vehicle into the right NTS. Ten min after the microinjection, dexmedetomidine infusion started at a rate of 30 µg/kg over 15 min followed by infusion at 15 µg·kg-1·h-1 for 105 min, or 100 µg/kg over 15 min followed by infusion at 50 µg·kg-1·h-1 for 105 min. BRS was examined using a standard phenylephrine method prior to infusion (T0), 60 min (T1) and 120 min (T2) after dexmedetomidine infusion started. Adenosine concentration in plasma and brainstem was measured with high-performance liquid chromatography with vs. without α2-AR antagonist atipamezole pretreatment (0.5 mg/kg, i.p.). Dexmedetomidine increased BRS at both 30 (T0: 0.55 ± 0.25 vs. T1: 2.45 ± 0.37, T2: 2.26 ± 0.56 ms/mmHg, P < 0.05) and 100 µg/kg (T0: 0.63 ± 0.24 vs. T1: 6.21 ± 1.87, T2: 6.30 ± 2.12 ms/mmHg, P < 0.05). DPCPX pretreatment obliterated BRS response to 100-µg/kg dexmedetomidine. At 100 µg/kg, dexmedetomidine increased adenosine concentration in plasma (0.23 ± 0.11 to 0.45 ± 0.07 µg/ml, P < 0.05) and brainstem (1.46 ± 0.30 to 2.52 ± 0.22 µg/ml, P < 0.05); such effect was blocked by atipamezole pretreatment. Western blot analysis showed α2-AR up-regulation by 100-µg/kg dexmedetomidine, which can be prevented by DPCPX. Double-labeling with glial fibrillary acidic protein showed α2-AR up-regulation in astrocytes in the NTS. These results suggest that dexmedetomidine enhances baroreflex sensitivity, possibly by increasing adenosine in NTS and α2-AR expression in astrocytes.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/farmacología , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Barorreflejo/efectos de los fármacos , Dexmedetomidina/farmacología , Receptor de Adenosina A1/metabolismo , Núcleo Solitario/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-Dawley , Núcleo Solitario/efectos de los fármacosRESUMEN
Glutamate cytotoxicity is implicated in neuronal death in different neurological disorders including stroke, traumatic brain injury, and neurodegenerative diseases. Adenosine is a nucleoside that plays an important role in modulating neuronal activity and its receptors have been identified as promising therapeutic targets for glutamate cytotoxicity. The purpose of this study is to elucidate the role of adenosine and its receptors on glutamate-induced injury in PC12 cells and to verify the protective effect of the novel A1 adenosine receptor positive allosteric modulator, TRR469. Flow cytometry experiments to detect apoptosis revealed that adenosine has a dual role in glutamate cytotoxicity, with A2A and A2B adenosine receptor (AR) activation exacerbating and A1 AR activation improving glutamate-induced cell injury. The overall effect of endogenous adenosine in PC12 cells resulted in a facilitating action on glutamate cytotoxicity, as demonstrated by the use of adenosine deaminase and selective antagonists. However, enhancing the action of endogenous adenosine on A1ARs by TRR469 completely abrogated glutamate-mediated cell death, caspase 3/7 activation, ROS production, and mitochondrial membrane potential loss. Our results indicate a novel potential therapeutic strategy against glutamate cytotoxicity based on the positive allosteric modulation of A1ARs.
Asunto(s)
Adenosina/farmacología , Ácido Glutámico/toxicidad , Neuroprotección/efectos de los fármacos , Receptor de Adenosina A1/metabolismo , Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Carbazoles/farmacología , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Colforsina/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Células PC12 , Piperazinas/farmacología , Pirroles/farmacología , Quinazolinas/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Receptores de Adenosina A2/metabolismo , Tiofenos/farmacología , Triazoles/farmacologíaRESUMEN
Adenosine receptors (ARs), like many otherGprotein-coupledreceptors (GPCRs), are targets of primary interest indrug design. However, one of the main limits for the development of drugs for this class of GPCRs is the complex selectivity profile usually displayed by ligands. Numerous efforts have been madefor clarifying the selectivity of ARs, leading to the development of many ligand-based models. The structure of the AR subtype A1 (A1AR) has been recently solved,providing important structural insights. In the present work, we rationalized the selectivity profile of two selective A1AR and A2AAR antagonists, investigating their recognition trajectories obtained by Supervised Molecular Dynamics from an unbound state and monitoring the role of the water molecules in the binding site.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/química , Simulación de Dinámica Molecular , Receptor de Adenosina A1/química , Antagonistas del Receptor de Adenosina A1/farmacología , Sitios de Unión , Humanos , Simulación del Acoplamiento Molecular/métodos , Unión Proteica , Receptor de Adenosina A1/metabolismo , Aprendizaje Automático SupervisadoRESUMEN
Adenosine A1 and/or A2A receptor antagonists hold promise for the potential treatment of neurological conditions, such as Parkinson's disease. Herein, a total of seventeen benzocycloalkanone derivatives were synthesised and evaluated for affinity towards adenosine receptors (A1 and A2A AR). The obtained results allowed for the conclusion that affinity and/or selectivity of the 2-benzylidene-1-indanone and -tetralone derivatives toward A1 and/or A2A ARs may be modulated by the nature of the substituents (either -OH, -OCH3 or morpholine) attached at position C4 of the 1-indanone core and C5 of the 1-tetralone core as well as the meta (C3') and/or para (C4') position(s) on ring B. Several compounds (2A: -B: , 3B: -C: and 4A: -B: ) possessed affinity for the A1 and/or A2A AR below 10 µM. Additionally, compounds 2A: , 3B: and 4A: were A1 AR antagonists. These results, once again, confirmed the importance of C4 methoxy-group substitution on ring A in combination with meta (C3') and/or para (C4') hydroxyl-group substitution ring B of the 2-benzylidene-1-indanone scaffold leading to drug-like compounds 1H: and 1J: with affinity in the nanomolar-range.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Técnicas de Química Sintética/métodos , Enfermedad de Parkinson/tratamiento farmacológico , Antagonistas del Receptor de Adenosina A1/síntesis química , Antagonistas del Receptor de Adenosina A2/síntesis química , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Chalconas/química , Química Farmacéutica/métodos , Simulación por Computador , Humanos , Estructura Molecular , Receptor de Adenosina A1/metabolismo , Receptores de Adenosina A2/metabolismo , Relación Estructura-ActividadRESUMEN
Here, we show that tumor ADORA1 deletion suppresses cell growth in human melanoma cell lines in vitro and tumor development in vivo in immune-deficient xenografts. However, this deletion induces the upregulation of PD-L1 levels, which inactivates cocultured T cells in vitro, compromises anti-tumor immunity in vivo, and reduces anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of ADORA1-deficient or ADORA1 antagonist-treated melanoma and NSCLC immune-competent mouse models. Mechanistically, we identify ATF3 as the factor transcriptionally upregulating PD-L1 expression. Tumor ATF3 deletion improves the effect of ADORA1 antagonist treatment of melanoma and NSCLC xenografts. We observe higher ADORA1, lower ATF3, and lower PD-L1 expression levels in tumor tissues from nonresponders among PD-1 mAb-treated NSCLC patients.