Purinergic Signalling最新文献

Adenosine receptor (AR) suppresses inflammation and fibrosis by activating cyclic adenosine monophosphate (cAMP) signaling. We investigated whether altered AR expression contributes to the development of fibrotic diseases and whether A_2AAR and A_2BAR upregulation inhibits fibrotic responses. Primary human lung fibroblasts (HLFs) from normal (NHLFs) or patients with idiopathic pulmonary fibrosis (DHLF) were used for in vitro testing. Murine models of fibrotic liver or pulmonary disease were developed by injecting thioacetamide intraperitoneally, by feeding a high-fat diet, or by intratracheal instillation of bleomycin. Modafinil, which activates cAMP signaling via A_2AAR and A_2BAR, was administered orally. The protein amounts of A_2AAR, A_2BAR, and exchange protein directly activated by cAMP (Epac) were reduced, while collagen and α-smooth muscle actin (α-SMA) were elevated in DHLFs compared to NHLFs. In liver or lung tissue from murine models of fibrotic diseases, A_2AAR and A_2BAR were downregulated, but A₁AR and A₃AR were not. Epac amounts decreased, and amounts of collagen, α-SMA, K_Ca2.3, and K_Ca3.1 increased compared to the control. Modafinil restored the amounts of A_2AAR, A_2BAR, and Epac, and reduced collagen, α-SMA, K_Ca2.3, and K_Ca3.1 in murine models of fibrotic diseases. Transforming growth factor-β reduced the amounts of A_2AAR, A_2BAR, and Epac, and elevated collagen, α-SMA, K_Ca2.3, and K_Ca3.1 in NHLFs; however, these alterations were inhibited by modafinil. Our investigation revealed that A_2AAR and A_2BAR downregulation induced liver and lung fibrotic diseases while upregulation attenuated fibrotic responses, suggesting that A_2AAR and A_2BAR-upregulating agents, such as modafinil, may serve as novel therapies for fibrotic diseases.

This study was designed to investigate the potential of extracellular adenosine 5'-triphosphate (ATP) via the P2 X 7 receptor to activate the renal fibrotic processes in rats. The present study demonstrates that administration of ATP rapidly activated transforming growth factor-β (TGF-β) to induce phosphorylation of Smad-2/3. Renal connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA and protein expressions were also increased following ATP administration. A decrease in TGF-β amount in serum as well as renal Smad-2/3 phosphorylation was noticed in animals pre-treated with the specific antagonist of P2 X 7 receptor, A 438,079. In addition, a significant reduction in mRNA and protein expression of CTGF and TIMP-1were also observed in the kidneys of those animals. Collectively, the current findings demonstrate that ATP has the ability to augment TGF-β-mediated Smad-2/3 phosphorylation and enhance the expression of the pro-fibrotic genes, CTGF and TIMP-1, an effect that is largely mediated via P2 X 7 receptor.

Head and neck squamous cell carcinomas (HNSCCs) are a heterogenous group of tumors and among the top 10 most common cancers and they arise from the epithelial tissues of the mucosal surfaces of the oral cavity, oropharynx, and larynx. Aberrant purinergic signaling has been associated with various cancer types. Here, we studied the role of the P2Y₂ purinergic receptor (P2Y₂R) in the context of oral cancer. We utilized bioinformatics analysis of deposited datasets to examine purinome gene expression in HNSCC tumors and cells lines and functionally characterized nucleotide-induced P2 receptor signaling in human FaDu and Cal27 and murine MOC2 oral cancer cell lines. Utilizing tumorigenesis assays with wild-type or P2ry2 knockout MOC2 cells we evaluated the role of P2Y₂Rs in tumor growth and the host anti-tumor immune responses. Our data demonstrate that human and murine oral cancer cell lines express numerous P2 receptors, with the P2Y₂R being highly expressed. Using syngeneic tumor grafts in wild-type mice, we observed that MOC2 tumors expressing P2Y₂R were larger than P2Y₂R^-/- tumors. Wild-type MOC2 tumors contained a lower population of tumor-infiltrating CD11b⁺F4/80⁺ macrophages and CD3⁺ cells, which were revealed to be CD3⁺CD4⁺IFNγ⁺ T cells, compared to P2Y₂R^-/- tumors. These results were mirrored when utilizing P2Y₂R^-/- mice, indicating that the changes in MOC2 tumor growth and to the host anti-tumor immune response were independent of host derived P2Y₂Rs. Results suggest that targeted suppression of the P2Y₂R in HNSCC cells in vivo, rather than systemic P2Y₂R antagonism, may be a more effective treatment strategy for HNSCCs.

We aimed to evaluate the effect of caffeine on viability, apoptosis, migration, redox profile and modulatory effect of the purinergic system of cutaneous melanoma cells. The melanoma cells SK-MEL-28 and non-tumoural CCD-1059sk cells were treated for 24 h with different concentrations of caffeine. Cell viability was evaluated by a biochemical assay and fluorescence microscopy, and flow cytometry assessed apoptosis induction. A wound-healing assay assessed cell migration. The redox profile was evaluated by the levels of markers of reactive oxygen species (ROS), nitric oxide (NOx), total thiols (PSH) and non-protein thiols (NPSH). RT-qPCR and flow cytometry assessed the expression of CD39 and CD73. ATPase/ADPase and AMPase enzyme activities were evaluated by hydrolysis of ATP, ADP and AMP nucleotides. A bioluminescent assay assessed extracellular ATP levels. Caffeine significantly reduced melanoma cell viability and migration and did not affect non-tumoural cells. Caffeine increased ROS levels and improved PSH levels in melanoma cells. Furthermore, caffeine reduced CD39 and CD73 expression, decreased ATP, ADP and AMP nucleotide hydrolysis and increased extracellular ATP levels. We have shown that caffeine reduces metastatic cutaneous melanoma cell viability and migration, induces ROS generation and improves PSH levels. In an unprecedented manner, we also showed that caffeine reduces the expression of CD39 and CD73 and, consequently, ATPase/ADPase/AMPase hydrolytic activity of ectonucleotidases, thus displacing the CD39/CD73 axis and increasing extracellular ATP levels. Therefore, caffeine may be an interesting compound for clinical trials with the CD39/CD73 axis as a therapeutic target.

Nucleosides and purine nucleotides serve as transmitter and modulator agents that extend their functions beyond the cell. In this context, purinergic signaling plays a crucial role in regulating energy homeostasis and modulating metabolic alterations in tumor cells. Therefore, it is essential to consider the pharmacological targeting of purinergic receptors (PUR), which encompass the expression and inhibition of P1 receptors (metabotropic adenosine receptors) as well as P2 receptors (extracellular ATP/ADP) comprising P2X and P2Y receptors. Thus, the pharmacological interaction between inhibitors (such as RNA, monoclonal antibodies, and small molecules) and PUR represents a key aspect in facilitating the development of therapeutic interventions. Moreover, this review explores recent advancements in pharmacological inhibitors and the regulation of innate and adaptive immunity of PUR, specifically in relation to immunological and inflammatory responses. These responses encompass the release of pro-inflammatory cytokines (PIC), the production of reactive oxygen and nitrogen species (ROS and RNS), the regulation of T cells, and the activation of inflammasomes in all human leukocytes.

Inflammatory pain, sustained by a complex network of inflammatory mediators, is a severe and persistent illness affecting many of the general population. We explore possible anti-inflammatory pathways of Polyphyllin VI (PPVI) based on our prior study, which showed that PPVI reduces inflammation in mice to reduce pain. Network pharmacology and RNA-Seq identified the contribution of the MAPK signaling pathway to inflammatory pain. In the LPS/ATP-induced RAW264.7 cell model, pretreatment with PPVI for 1 h inhibited the release of IL-6 and IL-8, down-regulated expression of the P2X₇ receptor(P2X₇R), and decreased phosphorylation of p38 and ERK1/2 components of the MAPK pathway. Moreover, PPVI decreased expression of IL-6 and IL-8 was observed in the serum of the inflammatory pain mice model and reduced phosphorylation of p38 and ERK1/2 in the dorsal root ganglia while the reductions of expression of IL-6 and phosphorylation of ERK1/2 were not observed after the pre-treatment with A740003 (an antagonist of the P2X₇R). These results suggest that PPVI may inhibit the release of IL-8 by regulating P2X₇R to reduce the phosphorylation of p38. However, the modulation of PPVI on the release of IL-6 and phosphorylation of ERK1/2 may mediated by other P2X₇R-independent signals.

Purine nucleotide adenosine triphosphate (ATP) is a source of intracellular energy maintained by mitochondrial oxidative phosphorylation. However, when released from ischemic cells into the extracellular space, they act as death-signaling molecules (eATP). Despite there being potential benefit in using pyruvate to enhance mitochondria by inducing a highly oxidative metabolic state, its association with eATP levels is still poorly understood. Therefore, while we hypothesized that pyruvate could beneficially increase intracellular ATP with the enhancement of mitochondrial function after cardiac arrest (CA), our main focus was whether a proportion of the raised intracellular ATP would detrimentally leak out into the extracellular space. As indicated by the increased levels in systemic oxygen consumption, intravenous administrations of bolus (500 mg/kg) and continuous infusion (1000 mg/kg/h) of pyruvate successfully increased oxygen metabolism in post 10-min CA rats. Plasma ATP levels increased significantly from 67 ± 11 nM before CA to 227 ± 103 nM 2 h after the resuscitation; however, pyruvate administration did not affect post-CA ATP levels. Notably, pyruvate improved post-CA cardiac contraction and acidemia (low pH). We also found that pyruvate increased systemic CO₂ production post-CA. These data support that pyruvate has therapeutic potential for improving CA outcomes by enhancing oxygen and energy metabolism in the brain and heart and attenuating intracellular hydrogen ion disorders, but does not exacerbate the death-signaling of eATP in the blood.

Trypanosoma cruzi is the pathogen of Chagas disease, a neglected tropical disease that affects more than 6 million people worldwide. There are no vaccines to prevent infection, and the therapeutic arsenal is very minimal and toxic. The unique E-NTPDase of T. cruzi (TcNTPDase1) plays essential roles in adhesion and infection and is a virulence factor. Quercetin is a flavonoid with antimicrobial, antiviral, and antitumor activities. Its potential as a partial inhibitor of NTPDases has also been demonstrated. In this work, we synthesized the non-natural L-glycoside derivatives of quercetin and evaluated them as inhibitors of recombinant TcNTPDase1 (rTcNTPDase1). These compounds, and quercetin and miquelianin, a natural quercetin derivative, were also tested. Compound 16 showed the most significant inhibitory effect (94%). Quercetin, miquelianin, and compound 14 showed inhibition close to 50%. We thoroughly investigated the inhibitory effect of 16. Our data suggested a competitive inhibition with a Ki of 8.39 μM (± 0.90). To better understand the interaction of compound 16 and rTcNTPDase1, we performed molecular dynamics simulations of the enzyme and docking analyses with the compounds. Our predictions show that compound 16 binds to the enzyme's catalytic site and interacts with important residues for NTPDase activity. As an inhibitor of a critical T. cruzi enzyme, (16) could be helpful as a starting point in the developing of a future treatment for Chagas disease. Furthermore, the discovery of (16) as an inhibitor of TcNTPDase1 may open new avenues in the study and development of new inhibitors of E-NTPDases.

P2X7 receptors (P2X7Rs) are membrane-bound ATP-gated ion channels that are composed of three subunits. Different subunit structures may be expressed due to alternative splicing of the P2RX7 gene, altering the receptor's function when combined with the wild-type P2X7A subunits. In this study, the application of the deep-learning method, AlphaFold2-Multimer (AF2M), for the generation of trimeric P2X7Rs was validated by comparing an AF2M-generated rat wild-type P2X7A receptor with a structure determined by cryogenic electron microscopy (cryo-EM) (Protein Data Bank Identification: 6U9V). The results suggested AF2M could firstly, accurately predict the structures of P2X7Rs and secondly, accurately identify the highest quality model through the ranking system. Subsequently, AF2M was used to generate models of heterotrimeric alternatively spliced P2X7Rs consisting of one or two wild-type P2X7A subunits in combination with one or two P2X7B, P2X7E, P2X7J, and P2X7L splice variant subunits. The top-ranking models were deemed valid based on AF2M's confidence measures, stability in molecular dynamics simulations, and consistent flexibility of the conserved regions between the models. The structure of the heterotrimeric receptors, which were missing key residues in the ATP binding sites and carboxyl terminal domains (CTDs) compared to the wild-type receptor, help to explain their observed functions. Overall, the models produced in this study (available as supplementary material) unlock the possibility of structure-based studies into the heterotrimeric P2X7Rs.