Nucleotide Receptors Research Articles

Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the P2Y2 has been identified as an important drug target. The specificity of current P2Y2 receptor modulators is relatively poor, and currently, specific and efficient P2Y2 receptor modulators and efficient screening strategies are lacking. In this study, a cell model based on calcium-activated chloride channels (CaCCs) was established that can detect changes in intracellular calcium concentrations and can be used to high-throughput screen for P2Y2 receptor-specific regulators. This screening strategy is suitable for screening of most G-protein-coupled receptor regulators that mediate increases in intracellular calcium signals. The cell model consists of three components that include the endogenously expressed P2Y2 receptor protein, the exogenously expressed calcium-activated chloride channel Anoctamin-1 (Ano1), and a yellow fluorescent protein mutant expressed within the cell that is highly sensitive to iodine ions. This model will allow for high-throughput screening of GPCR regulators that mediate increased intracellular calcium signaling using the calcium-activated transport of iodide ions by Ano1. We verified the ability of the model to detect intracellular calcium ion concentration using fluorescence quenching kinetic experiments by applying existing P2Y2 agonists and inhibitors to validate the screening function of the model, and we also evaluated the performance of the model in the context of high-throughput screening studies. The experimental results revealed that the model could sensitively detect intracellular calcium ion concentration changes and that the model was accurate in regard to detecting P2Y2 modulators. The resultant value of the Z-factor was 0.69, thus indicating that the model possesses good sensitivity and specificity.

Nucleotide receptors mediate protection against neonatal sepsis and meningitis caused by alpha-hemolysin expressing Escherichia coli K1.

Neonatal meningitis-associated Escherichia coli (NMEC) is among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here, we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha-hemolysin (HlyA)-expressing NMEC (HlyA+ ) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA- NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA- NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activates P2XRs which in turn confers macrophage-dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.

Efficient fluorescent recognition of ATP/GTP by a water-soluble bisquinolinium pyridine-2,6-dicarboxamide compound. Crystal structures, spectroscopic studies and interaction mode with DNA.

The new dicationic pyridine-2,6-dicarboxamide-based compound 1 bearing two N-alkylquinolinium units was synthesized, structurally determined by single-crystal X-ray diffraction, and studied in-depth as a fluorescent receptor for nucleotides and inorganic phosphorylated anions in pure water. The addition of nucleotides to 1 at pH = 7.0 quenches its blue emission with a selective affinity towards adenosine 5'-triphosphate (ATP) and guanosine 5'-tripohosphate (GTP) over other nucleotides such CTP, UTP, ADP, AMP, dicarboxylates and inorganic anions. On the basis of the spectroscopic tools (1H, 31P NMR, UV-vis, fluorescence), MS measurements and DFT calculations, receptor 1 binds ATP with high affinity (log K = 5.04) through the simultaneous formation of strong hydrogen bonds and π-π interactions between the adenosine fragment and quinolinium ring with binding energy calculated in 8.7 kcal mol-1. High affinity for ATP/GTP is attributed to the high acidity of amides and preorganized rigid structure of 1. Receptor 1 is an order of magnitude more selective for ATP than GTP. An efficient photoinduced electron transfer quenching mechanism with simultaneous receptor-ATP complexation in both the excited and ground states is proposed. Additionally, multiple spectroscopic studies and molecular dynamics simulations showed that 1 can intercalate into DNA base pairs.

P2X7 receptor: the regulator of glioma tumor development and survival

P2X7 is an ionotropic nucleotide receptor, forming the cation channel upon ATP stimulation. It can also function as a large membrane pore as well as transmit ATP-dependent signal without forming a channel at all. P2X7 activity in somatic cells is well-known, but remains poorly studied in glioma tumors. The current paper presents the comprehensive study of P2X7 activity in C6 and glioma cell line showing the wide range of effects the receptor has on glioma biology. We observed that P2X7 stimulation boosts glioma cell proliferation and increases cell viability. P2X7 activation promoted cell adhesion, mitochondria depolarization, and reactive oxygen species overproduction in C6 cells. P2X7 receptor also influenced glioma tumor growth in vivo via activation of pro-survival signaling pathways and ATP release. Treatment with Brilliant Blue G, a selective P2X7 antagonist, effectively inhibited glioma tumor development; decreased the expression of negative prognostic cancer markers pro-survival and epithelial-mesenchymal transition (EMT)-related proteins; and modulated the immune response toward glioma tumor in vivo. Finally, pathway-specific enrichment analysis of the microarray data from human patients also showed an upregulation of P2X7 receptor in gliomas from grades I to III. The presented results shed more light on the role of P2X7 receptor in the biology of this 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.

Nucleotides modulate synoviocyte proliferation and osteoclast differentiation in macrophages with potential implications for rheumatoid arthritis.

The online version contains supplementary material available at 10.1007/s13205-021-03052-8.

Discovery and computational studies of 2-phenyl-benzoxazole acetamide derivatives as promising P2Y14R antagonists with anti-gout potential

The P2Y14 nucleotide receptor, a subtype of P2Y receptors, is implicated in many human inflammatory diseases. Based on the identification of favorable residues of two screening hits in the almost symmetrical P2Y14 binding domain, we describe the structural optimization of previously identified virtual screening hits 6 and 7 that result in the development of P2Y14R antagonists with a novel 2-phenyl-benzoxazole acetamide chemical scaffold. Notably, compound 52 showed potent P2Y14R antagonistic activity (IC50 = 2 nM), and a stronger inhibitory effect on MSU-induced inflammatory in vitro, better than a previously described P2Y14R antagonist PPTN. In vivo evaluation demonstrated that compound 52 also had satisfactory inhibitory activity on the inflammatory response of gout flares in mice. Moreover, P2Y14R antagonist 52 decreased paw swelling and inflammatory cell infiltration through cAMP/NLRP3/GSDMD signaling pathways in MSU-induced acute gouty arthritis mice. The discussions on the binding mechanism that employ MM/GBSA free energy calculations/decompositions also provide some useful clues for further structural designing of compound 52. Taken together, 2-phenyl-benzoxazole acetamide derivative 52 with potent P2Y14R antagonistic activity and in vivo potency could be a promising strategy for gout therapy and deserves further optimization.

Origin and Isoform Specific Functions of Exchange Proteins Directly Activated by cAMP: A Phylogenetic Analysis.

Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are one of the several families of cellular effectors of the prototypical second messenger cAMP. To understand the origin and molecular evolution of EPAC proteins, we performed a comprehensive phylogenetic analysis of EPAC1 and EPAC2. Our study demonstrates that unlike its cousin PKA, EPAC proteins are only present in multicellular Metazoa. Within the EPAC family, EPAC1 is only associated with chordates, while EPAC2 spans the entire animal kingdom. Despite a much more contemporary origin, EPAC1 proteins show much more sequence diversity among species, suggesting that EPAC1 has undergone more selection and evolved faster than EPAC2. Phylogenetic analyses of the individual cAMP binding domain (CBD) and guanine nucleotide exchange (GEF) domain of EPACs, two most conserved regions between the two isoforms, further reveal that EPAC1 and EPAC2 are closely clustered together within both the larger cyclic nucleotide receptor and RAPGEF families. These results support the notion that EPAC1 and EPAC2 share a common ancestor resulting from a fusion between the CBD of PKA and the GEF from RAPGEF1. On the other hand, the two terminal extremities and the RAS-association (RA) domains show the most sequence diversity between the two isoforms. Sequence diversities within these regions contribute significantly to the isoform-specific functions of EPACs. Importantly, unique isoform-specific sequence motifs within the RA domain have been identified.

Synthesis, characterization and biological evaluation of indomethacin derived thioureas as purinergic (P2Y1, P2Y2, P2Y4, and P2Y6) receptor antagonists

G-protein-coupled receptors for extracellular nucleotides are known as P2Y receptors and are made up of eight members that are encoded by distinct genes and can be classified into two classes based on their affinity for specific G-proteins. P2Y receptor modulators have been studied extensively, but only a few small-molecule P2Y receptor antagonists have been discovered so far and approved by drug agencies. Derivatives of indole carboxamide have been identified as P2Y12 and P2X7 antagonist, as a result, we developed and tested a series of indole derivatives4a-lhaving thiourea moiety as P2Y receptor antagonist by using a fluorescence-based assay to measure the inhibition of intracellular calcium release in 1321N1 astrocytoma cells that had been stably transfected with the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Most of the compounds exhibited moderate to excellent inhibition activity against P2Y1 receptor subtype. The series most potent compound, 4h exhibited an IC50 value of 0.36 ± 0.01 µM selectivity against other subtypes of P2Y receptor. To investigate the ligand-receptor interactions, the molecular docking studies were carried out. Compound 4h is the most potent P2Y1 receptor antagonist due to interaction with an important amino acid residue Pro105, in addition to Ile108, Phe119, and Leu102.

UTP is a regulator of in vitro and in vivo angiogenic properties of cardiac adipose-derived stem cells.

The ability of cardiac adipose-derived stem cells (cADSC) to differentiate into multiple cell types has opened new perspectives in cardiac cell-based regenerative therapies. P2Y nucleotide receptors have already been described as regulators of adipogenic differentiation of cADSC and bone marrow-derived stem cells. In this study, we defined UTP as a regulator of cADSC endothelial differentiation. A daily UTP stimulation of cADSC during endothelial predifferentiation increased their capacity to form an endothelial network in matrigel. Additionally, pro-angiogenic UTP target genes such as epiregulin and hyaluronan synthase-1 were identified in predifferentiated cADSC by RNA sequencing experiments. Their regulation by UTP was confirmed by qPCR and ELISA experiments. We then evaluated the capacity of UTP-treated predifferentiated cADSC to increase post-ischemic revascularization in mice subjected to left anterior descending artery ligation. Predifferentiated cADSC treated or not with UTP were injected in the periphery of the infarcted zone, 3days after ligation. We observed a significant increase of capillary density 14 and 30days after UTP-treated predifferentiated cADSC injection, correlated with a reduction of cardiac fibrosis. This revascularization increase was not observed after injection of UTP-treated cADSC deficient for UTP and ATP nucleotide receptor P2Y2. The present study highlights the P2Y2 receptor as a regulator of cADSC endothelial differentiation and as a potential target for the therapeutic use of cADSC in post-ischemic heart revascularization.

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.

Genesis of Antibiotic Resistance (AR) LXXI: Turbulence Modeling of Hemodynamics in Simplified Severe Sepsis Protocol‐2 (SSSP‐2) ‐ NCT01663701: Activated E‐Oligomer Complex Elicit Rapid Leucocyte‐Platelets‐Erythrocytes (LPE) Aggregation and Occlusion of Capillary by Impairing Energy Cascading and Starling Forces (Net Filtration Pressure)

Here we present a rationale for the fluid overload-induced accelerated LPE aggregates to occlude the arterial end of the capillary bed resulting in critical closing pressure (CCP). It is known that native purine/pyrimidine receptors have yet to be purified to homogeneity and characterization of receptor-ligand kinetics. Recombinant purine/pyrimidine receptors knockout genotype mice are not embryonic lethal, while its characterization in vivo is dependent of multifactorial factors of epigenetic origin namely (post-translational modification, lineage-specific developmental and hemodynamic heterogeneity; ex: meaning age, gender, morphological attribute(s) of the platelet(s), viscosity, and hematocrit). Every attempt to purify Ca2+ /Mg2+ ecto-ATPase (EC 3.6.1.15) to homogeneity, showed co-purification of CD36, PMID: 9925363); cell-CAM105 (PMID: 1637321 PMCID: PMC1132742, PMID: 8387753, PMID: 1985902); mmCGM1a (PMID: 1633107); PECAM-1,(PMID: 19558578); CBATP, (PMID: 9218468); BgP (PMID: 2527235); caldesmon (PMID: 1930128); NCAM (PMID: 8262246, PMID: 7804134); Ig gene superfamily (PMID: 2141577), Cadherin (PMID: 8489264); and chicken gizzard ecto-ATPase amino acid similarity to CD39 (PMID: 8995405). The fact remain that native CD39 has never been tested for mM [Ca2+] or [Mg2+] ecto-ATPase (EC 3.6.1.15) or E-NTPDase (NTPDase1) (EC 3.6.1.6) (PMID: 1672348, PMID: 1721639). Further, neither CAM nor CD39 knockout genotype is embryonic lethal. Experimental evidence shows that differential regulation of CD38, integrin's (CD11), and CD39 in the liver allograft, implicit of interdependency in host immune response (PMID: 31511354). We have obtained evidence that the phylogenetic tree analysis, neighbor-joining tree without distance correlation among CD36, CD38, CD39, CAM, P2Y/P2, and corona #2, receptors. The cladogram show a marked evolutionary conservancy between CD39 & P2X, CD36 & Corona virus-2, CAM & P2Y &cadherin, with a possibility of existence as an oligomer (ECpx). Taken together, it is hypothesized that nucleotide receptors (P2Y/P2X), CAM, and ectonucleotidases family of a protein complex (E-Cpx) could possibly exist as a membrane-bound oligomer. Subsequent activation of E-Cpx on LPE, aggregation, and rapid occlusion of the capillary by forming T-S waves, 3D distortion of T-S waves, in phase arrays of hairpin K-type vortices, turbulent spot formation, merging of turbulent spot formation, without energy cascading among the LPE aggregates and adherence to the damaged endothelial cells at the arterial end of the capillary, Refer Fig: 3.4, 3.5, 3.6 3.8; (ISBN: 978-0-13-127498-3), a robust anaerobic /aerobic persister (s) colonization eliciting CCP. In turn, a progressive hypotensive state induced endothelial damage and abrupt collapse of interstitial space would result in CCP ensuing hypoxia. Such a scenario may have been the cause for exacerbated mortality among the sepsis cohort in NCT01663701-SSSP-2 (PMID: 28973227).

Guanine-Based Purines as an Innovative Target to Treat Major Depressive Disorder.

Guanine-Based Purines as an Innovative Target to Treat Major Depressive Disorder.

Design, synthesis and evaluation of 3-amide-5-aryl benzoic acid derivatives as novel P2Y14R antagonists with potential high efficiency against acute gouty arthritis

P2Y14 nucleotide receptor plays important roles in series of physiological and pathologic events especially associated with immune and inflammation. Based on the 3-amide benzoic acid scaffold reported by our group previously, a series of 5-aryl-3-amide benzoic acid derivatives were designed as novel P2Y14 antagonists with improved pharmacokinetic properties. Among which compound 11m showed most potent P2Y14 antagonizing activity with an IC50 value of 2.18nM, furnishing greatly improved water solubility and bioavailability compared with PPTN. In MSU-induced acute gouty arthritis model in mice, 11m exerted promising invivo efficacy in alleviating mice paw swelling and inflammatory infiltration. Mechanistically, compound 11m notably blocked pyroptosis of macrophages through inhibiting NLRP3 inflammasome activation. This work may contribute to the identification of potential therapeutic agents to intervene in acute gouty arthritis.

Purinergic Signaling in Controlling Macrophage and T Cell Functions During Atherosclerosis Development.

Atherosclerosis is a hardening and narrowing of arteries causing a reduction of blood flow. It is a leading cause of death in industrialized countries as it causes heart attacks, strokes, and peripheral vascular disease. Pathogenesis of the atherosclerotic lesion (atheroma) relies on the accumulation of cholesterol-containing low-density lipoproteins (LDL) and on changes of artery endothelium that becomes adhesive for monocytes and lymphocytes. Immunomediated inflammatory response stimulated by lipoprotein oxidation, cytokine secretion and release of pro-inflammatory mediators, worsens the pathological context by amplifying tissue damage to the arterial lining and increasing flow-limiting stenosis. Formation of thrombi upon rupture of the endothelium and the fibrous cup may also occur, triggering thrombosis often threatening the patient’s life. Purinergic signaling, i.e., cell responses induced by stimulation of P2 and P1 membrane receptors for the extracellular nucleotides (ATP, ADP, UTP, and UDP) and nucleosides (adenosine), has been implicated in modulating the immunological response in atherosclerotic cardiovascular disease. In this review we will describe advancements in the understanding of purinergic modulation of the two main immune cells involved in atherogenesis, i.e., monocytes/macrophages and T lymphocytes, highlighting modulation of pro- and anti-atherosclerotic mediated responses of purinergic signaling in these cells and providing new insights to point out their potential clinical significance.

P2X7 receptors in the central nervous system

For the past three decades, our laboratory has conducted pioneering research to elucidate the complexity of purinergic signaling in the CNS, alone and in collaboration with other groups, inspired by the ground-breaking efforts of Geoffrey Burnstock. This review summarizes our contribution to understand the nucleotide receptor signaling in the CNS with a special focus on the P2X7 receptor.

P2Y2 receptor antagonism resolves sialadenitis and improves salivary flow in a Sjögren’s syndrome mouse model

ObjectiveSjögren’s syndrome (SS) is a chronic autoimmune exocrinopathy characterized by lymphocytic infiltration of the salivary and lacrimal glands and decreased saliva and tear production. Previous studies indicate that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is upregulated in numerous models of salivary gland inflammation (i.e., sialadenitis), where it has been implicated as a key mediator of chronic inflammation. Here, we evaluate both systemic and localized P2Y2R antagonism as a means to resolve sialadenitis in the NOD.H-2h4,IFNγ−/−,CD28−/− (NOD.H-2h4 DKO) mouse model of SS. DesignFemale 4.5 month old NOD.H-2h4 DKO mice received daily intraperitoneal injections for 10 days of the selective P2Y2R antagonist, AR-C118925, or vehicle-only control. Single-dose localized intraglandular antagonist delivery into the Wharton’s duct was also evaluated. Carbachol-induced saliva was measured and then submandibular glands (SMGs) were isolated and either fixed and paraffin-embedded for H&E staining, homogenized for RNA isolation or dissociated for flow cytometry analysis. ResultsIntraperitoneal injection, but not localized intraglandular administration, of AR-C118925 significantly enhanced carbachol-induced salivation and reduced lymphocytic foci and immune cell markers in SMGs of 5 month old NOD.H-2h4 DKO mice, compared to vehicle-injected control mice. We found that B cells represent the primary immune cell population in inflamed SMGs of NOD.H-2h4 DKO mice that express elevated levels of P2Y2R compared to C57BL/6 control mice. We further demonstrate a role for P2Y2Rs in mediating B cell migration and the release of IgM. ConclusionOur findings suggest that the P2Y2R represents a novel therapeutic target for the treatment of Sjögren’s syndrome.

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.