ShlA toxin of Serratia induces P2Y2- and α5ß1-dependent autophagy and bacterial clearance from host cells.

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.

Extracellular ATP and adenosine in tumor microenvironment: Roles in epithelial-mesenchymal transition, cell migration, and invasion.

Under nonpathological conditions, the extracellular nucleotide concentration remains constant and low (nM range) because of a close balance between ATP release and ATP consumption. This balance is completely altered in cancer disease. Adenine and uridine nucleotides are found in the extracellular space of tumors in high millimolar (mM) concentrations acting as extracellular signaling molecules. In general, although uridine nucleotides may be involved in different tumor cell responses, purinergic signaling in cancer is preferentially focused on adenine nucleotides and nucleosides. Extracellular ATP can bind to specific receptors (P receptors) triggering different responses, or it can be hydrolyzed by ectoenzymes bound to cell membranes to render the final product adenosine. The latter pathway plays an important role in the increase of adenosine in tumor microenvironment. In this study, we will focus on extracellular ATP and adenosine, their effects acting as ligands of specific receptors, activating ectoenzymes, and promoting epithelial-mesenchymal transition, migration, and invasion in cancer cells. Finding the roles that these nucleotides play in tumor microenvironment may be important to design new intervention strategies in cancer therapies.

Release of ATP by TRPV4 activation is dependent upon the expression of AQP2 in renal cells.

Increasing evidence indicates that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the transient receptor potential vanilloid 4 (TRPV4) channel. We previously found that TRPV4 physically and functionally interacts with AQP2 in cortical collecting ducts (CCD) cells, favoring cell volume regulation and cell migration. Because TRPV4 was implicated in ATP release in several tissues, we investigated the possibility that TRPV4/AQP2 interaction influences ATP release in CCD cells. Using two CCD cell lines expressing or not AQP2, we measured extracellular ATP (ATPe) under TRPV4 activation and intracellular Ca2+ under ATP addition. We found that AQP2 is critical for the release of ATP induced by TRPV4 activation. This ATP release occurs by an exocytic and a conductive route. ATPe, in turn, stimulates purinergic receptors leading to ATPe-induced ATP release by a Ca2+ -dependent mechanism. We propose that AQP2 by modulating Ca2+ and ATP differently could explain AQP2-increased cell migration.

Induction of ATP Release, PPIX Transport, and Cholesterol Uptake by Human Red Blood Cells Using a New Family of TSPO Ligands.

Two main isoforms of the Translocator Protein (TSPO) have been identified. TSPO1 is ubiquitous and is mainly present at the outer mitochondrial membrane of most eukaryotic cells, whereas, TSPO2 is specific to the erythroid lineage, located at the plasma membrane, the nucleus, and the endoplasmic reticulum. The design of specific tools is necessary to determine the molecular associations and functions of TSPO, which remain controversial nowadays. We recently demonstrated that TSPO2 is involved in a supramolecular complex of the erythrocyte membrane, where micromolar doses of the classical TSPO ligands induce ATP release and zinc protoporphyrin (ZnPPIX) transport. In this work, three newly-designed ligands (NCS1016, NCS1018, and NCS1026) were assessed for their ability to modulate the functions of various erythrocyte's and compare them to the TSPO classical ligands. The three new ligands were effective in reducing intraerythrocytic Plasmodium growth, without compromising erythrocyte survival. While NCS1016 and NCS1018 were the most effective ligands in delaying sorbitol-induced hemolysis, NCS1016 induced the highest uptake of ZnPPIX and NCS1026 was the only ligand inhibiting the cholesterol uptake. Differential effects of ligands are probably due, not only, to ligand features, but also to the dynamic interaction of TSPO with various partners at the cell membrane. Further studies are necessary to fully understand the mechanisms of the TSPO's complex activation.

On the role of ATP release, ectoATPase activity, and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells.

Hypotonicity triggered in human hepatoma cells (Huh-7) the release of ATP and cell swelling, followed by volume regulatory decrease (RVD). We analyzed how the interaction between those processes modulates cell volume. Cells exposed to hypotonic medium swelled 1.5 times their basal volume. Swelling was followed by 41% RVD(40) (extent of RVD after 40 min of maximum), whereas the concentration of extracellular ATP (ATP(e)) increased 10 times to a maximum value at 15 min. Exogenous apyrase (which removes di- and trinucleotides) did not alter RVD, whereas exogenous Na(+)-K(+)-ATPase (which converts ATP to ADP in the extracellular medium) enhanced RVD(40) by 2.6 times, suggesting that hypotonic treatment alone produced a basal RVD, whereas extracellular ADP activated RVD to achieve complete volume regulation (i.e., RVD(40) ≈100%). Under hypotonicity, addition of 2-(methylthio)adenosine 5'-diphosphate (2MetSADP; ADP analog) increased RVD to the same extent as exposure to Na(+)-K(+)-ATPase and the same analog did not stimulate RVD when coincubated with MRS2211, a blocker of ADP receptor P2Y(13). RT-PCR and Western blot analysis confirmed the presence of P2Y(13). Cells exhibited significant ectoATPase activity, which according to RT-PCR analysis can be assigned to ENTPDase2. Both carbenoxolone, a blocker of conductive ATP release, and brefeldin A, an inhibitor of exocytosis, were able to partially decrease ATP(e) accumulation, pointing to the presence of at least two mechanisms for ATP release. Thus, in Huh-7 cells, hypotonic treatment triggered the release of ATP. Conversion of ATP(e) to ADP(e) by ENTPDase 2 activity facilitates the accumulated ADP(e) to activate P2Y(13) receptors, which mediate complete RVD.

Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles.

Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs). ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space. Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases. RBCs were cultured with P. falciparum for 24-48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs. Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP. Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria. The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.

A volume regulatory response can be triggered by nucleosides in human erythrocytes, a perfect osmometer no longer.

Human erythrocytes have been regarded as perfect osmometers, which swell or shrink as dictated by their osmotic environment. In contrast, in most other cells, swelling elicits a regulatory volume decrease (RVD) modulated by the activation of purinic and pyrimidinic receptors (P receptors). For human erythrocytes this modulation has not been tested, and we thus investigated whether P receptor activation can induce RVD in these cells. Further, because ectonucleotidases may scavenge ATP or ADP or act as a source for extracellular adenosine and therefore modulate P receptor activation and RVD, we also determined their activity in intact erythrocytes. We found relatively low ectoATPase but significant ectoADPase and ectoAMPase activities. When erythrocytes were exposed to hypotonic medium alone, they swelled as expected for an osmometric response and showed no RVD. Activation of P2 receptors by exogenous ATP or ADP did not trigger RVD, whereas P1 agonists adenosine and adenosine-5'-N-ethylcarboxamide induced significant RVD. The effect of adenosine-5'-N-ethylcarboxamide was dose-dependent (maximal RVD of 27%; apparent K((1/2)) of 1.6 +/- 1.7 microM). The RVD induced by adenosine was blocked 80% with the non-selective P1 antagonist 8-(p-sulfophenyl theophylline) or the P1-A(2B) inhibitor MRS1754, but not by inhibitors of P1 subtypes A(1), A(2A), and A(3). In addition, forskolin (an inducer of intracellular cAMP formation) could mimic the effect of adenosine, supporting the idea of P1-A(2B) receptor activation. In conclusion, we report a novel P1-A(2B) receptor-mediated RVD activation in mature human erythrocytes and thus indicate that these long held perfect osmometers are not so perfect after all.

In silico Guided Drug Repurposing: Discovery of New Competitive and Non-competitive Inhibitors of Falcipain-2.

Malaria is among the leading causes of death worldwide. The emergence of Plasmodium falciparum resistant strains with reduced sensitivity to the first line combination therapy and suboptimal responses to insecticides used for Anopheles vector management have led to renewed interest in novel therapeutic options. Here, we report the development and validation of an ensemble of ligand-based computational models capable of identifying falcipain-2 inhibitors, and their subsequent application in the virtual screening of DrugBank and Sweetlead libraries. Among four hits submitted to enzymatic assays, two (odanacatib, an abandoned investigational treatment for osteoporosis and bone metastasis, and the antibiotic methacycline) confirmed inhibitory effects on falcipain-2, with Ki of 98.2 nM and 84.4 µM. Interestingly, Methacycline proved to be a non-competitive inhibitor (α = 1.42) of falcipain-2. The effects of both hits on falcipain-2 hemoglobinase activity and on the development of P. falciparum were also studied.

ATPe Dynamics in Protozoan Parasites. Adapt or Perish.

In most animals, transient increases of extracellular ATP (ATPe) are used for physiological signaling or as a danger signal in pathological conditions. ATPe dynamics are controlled by ATP release from viable cells and cell lysis, ATPe degradation and interconversion by ecto-nucleotidases, and interaction of ATPe and byproducts with cell surface purinergic receptors and purine salvage mechanisms. Infection by protozoan parasites may alter at least one of the mechanisms controlling ATPe concentration. Protozoan parasites display their own set of proteins directly altering ATPe dynamics, or control the activity of host proteins. Parasite dependent activation of ATPe conduits of the host may promote infection and systemic responses that are beneficial or detrimental to the parasite. For instance, activation of organic solute permeability at the host membrane can support the elevated metabolism of the parasite. On the other hand ecto-nucleotidases of protozoan parasites, by promoting ATPe degradation and purine/pyrimidine salvage, may be involved in parasite growth, infectivity, and virulence. In this review, we will describe the complex dynamics of ATPe regulation in the context of protozoan parasite⁻host interactions. Particular focus will be given to features of parasite membrane proteins strongly controlling ATPe dynamics. This includes evolutionary, genetic and cellular mechanisms, as well as structural-functional relationships.

Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

INTRODUCTION: The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. METHODS AND TREATMENTS: We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. RESULTS: In rbcs 10 µM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 µM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 µM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. ANALYSIS AND DISCUSSION: Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores.

The pathways for putrescine biosynthesis and the effects of polyamine biosynthesis inhibitors on the germination and hyphal development of Gigaspora rosea spores were investigated. Incubation of spores with different radioactive substrates demonstrated that both arginine and ornithine decarboxylase pathways participate in putrescine biosynthesis in G. rosea. Spermidine and spermine were the most abundant polyamines in this fungus. The putrescine biosynthesis inhibitors alpha-difluoromethylarginine and alpha-difluoromethylornithine, as well as the spermidine synthase inhibitor cyclohexylamine, slightly decreased polyamine levels. However, only the latter interfered with spore germination. The consequences of the use of putrescine biosynthesis inhibitors for the control of plant pathogenic fungi on the viability of G. rosea spores in soil are discussed.