The P2Y2 Nucleotide Receptor Mediates Monocyte Tissue Factor Expression and Endotoxemia Death in Mice

Recently we reported that in human coronary artery endothelial cells, activation of the P2Y2 receptor (P2Y2R) induces up-regulation of tissue factor (TF), a vital initiator of the coagulation cascade. However, others have shown that monocyte TF is more critical than endothelial TF in provoking a pro-thrombotic state. Thus, we aimed to study whether monocytes express the P2Y2R, its role in controlling TF expression, and its relevance in vivo. RT-PCR and receptor activity assays revealed that among the eight P2Y nucleotide receptors, the P2Y2 subtype was selectively and functionally expressed in human monocytic THP-1 cells and primary monocytes. Stimulation of the cells by ATP or UTP dramatically increased TF protein expression, which was abolished by AR-C118925, a selective P2Y2R antagonist, or by siRNA silencing the P2Y2R. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA preceded with an increased TF pre-mRNA, indicating enhanced TF gene transcription. In addition, stimulation of the monocyte P2Y2R significantly activated ERK1/2, JNK, p38, and Akt, along with their downstream transcription factors including c-Jun, c-Fos, and ATF-2, whereas blocking these pathways respectively, all significantly suppressed P2Y2R-mediated TF expression. Furthermore, we found that LPS triggered ATP release and TF expression, the latter of which was suppressed by apyrase or P2Y2R blockage. Importantly, P2Y2R-null mice were more resistant than wild-type mice in response to a lethal dose of LPS, accompanied by much less TF expression in bone marrow cells. These findings demonstrate for the first time that the P2Y2R mediates TF expression in human monocytes through mechanisms involving ERK1/2, JNK, p38, and AKT, and that P2Y2R deletion protects the mice from endotoxemia-induced TF expression and death, highlighting monocyte P2Y2R may be a new drug target for the prevention and/or treatment of relevant thrombotic disease.

Extracellular Nucleotides Affect the Proangiogenic Behavior of Fibroblasts, Keratinocytes, and Endothelial Cells

Chronic wound healing is currently a severe problem due to its incidence and associated complications. Intensive research is underway on substances that retain their biological activity in the wound microenvironment and stimulate the formation of new blood vessels critical for tissue regeneration. This group includes synthetic compounds with proangiogenic activity. Previously, we identified phosphorothioate analogs of nucleoside 5′-O-monophosphates as multifunctional ligands of P2Y6 and P2Y14 receptors. The effects of a series of unmodified and phosphorothioate nucleotide analogs on the secretion of VEGF from keratinocytes and fibroblasts, as well as their influence on the viability and proliferation of keratinocytes, fibroblasts, and endothelial cells were analyzed. In addition, the expression profiles of genes encoding nucleotide receptors in tested cell models were also investigated. In this study, we defined thymidine 5′-O-monophosphorothioate (TMPS) as a positive regulator of angiogenesis. Preliminary analyses confirmed the proangiogenic potency of TMPS in vivo.

Development of Phosphodiesterase–Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–RD complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

Development of Phosphodiesterase-Protein Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (Protein Kinases, PK) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PK generates an expanded active site which enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PK, and aid in signal termination. PDEs are important drug targets and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targets PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay has been adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode, and disrupt protein-protein interactions between PDEs and cyclic nucleotide activating protein kinases in a second mode. We tested this approach with three different systems: cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG and dual-specificity RegA-RD complexes and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

Nucleotides-Induced Changes in the Mechanical Properties of Living Endothelial Cells and Astrocytes, Analyzed by Atomic Force Microscopy

Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young’s modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y2 receptor stimulation with UTP. Such exposure to UTP doubled the Young’s modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.

Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions

Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell–cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.

Performance of P2x7 and P2Y Purinergic Receptors as an Inhibiting Factor in the Progression of Pulmonary Neoplastic Cells

Different pathological processes are considered in lung cancer, such as nicotine deposition, oxidative stress, deregulation of metal ions and chronic inflammation. Purine signaling is involved in all processes, suggesting the importance of nucleotide receptors (P2X7 and P2Y) and adenosine receptors present in lung cells. The accumulation of toxic substances promotes chronic inflammation and cellular alteration, which induces the release of Adenosine Triphosphate (ATP) in the extracellular space, thus stimulating P2X7 receptors. The activation of P2X7 promotes an increase in the synthesis and release of some pro-inflammatory mediators. P2Y works by promoting the increase of intracellular calcium levels and, P2X7, changing cell permeability. Thus, drugs can be developed with a specific targeting for such recipients, thus provid additional therapeutic options to those existing.

Purinergic Signaling in Endometriosis-Associated Pain

Endometriosis is an estrogen-dependent gynecological disease, with an associated chronic inflammatory component, characterized by the presence of endometrial tissue outside the uterine cavity. Its predominant symptom is pain, a condition notably altering the quality of life of women with the disease. This review is intended to exhaustively gather current knowledge on purinergic signaling in endometriosis-associated pain. Altered extracellular ATP hydrolysis, due to changes in ectonucleotidase activity, has been reported in endometriosis; the resulting accumulation of ATP in the endometriotic microenvironment points to sustained activation of nucleotide receptors (P2 receptors) capable of generating a persistent pain message. P2X3 receptor, expressed in sensory neurons, mediates nociceptive, neuropathic, and inflammatory pain, and is enrolled in endometriosis-related pain. Pharmacological inhibition of P2X3 receptor is under evaluation as a pain relief treatment for women with endometriosis. The role of other ATP receptors is also discussed here, e.g., P2X4 and P2X7 receptors, which are involved in inflammatory cell–nerve and microglia–nerve crosstalk, and therefore in inflammatory and neuropathic pain. Adenosine receptors (P1 receptors), by contrast, mainly play antinociceptive and anti-inflammatory roles. Purinome-targeted drugs, including nucleotide receptors and metabolizing enzymes, are potential non-hormonal therapeutic tools for the pharmacological management of endometriosis-related pain.