Alpha hemolysin of Escherichia coli induces a necrotic-like procoagulant state in platelets.

Uropathogenic strains of E. coli (UPEC) is a leading cause of sepsis, deploying multiple virulence factors to evade host immune responses. Notably, alpha-hemolysin (HlyA) produced by UPEC is implicated in septic symptoms associated with bacteremia, correlating with thrombocytopenia, a critical indicator of organ dysfunction and a predictor of poorer patient prognosis. This study meticulously explores the impact of sublytic concentrations of HlyA on platelets. Findings reveal that HlyA triggers an increase in intracellular calcium, activating calpain and exposing phosphatidylserine to the cell surface, as validated by flow cytometric experiments. Electron microscopy reveals a distinctive balloon-like shape in HlyA-treated platelets, indicative of a procoagulant state. The toxin induces the release of procoagulant extracellular vesicles and the secretion of alpha and dense granules. Overall, the results point to HlyA inducing a necrotic-like procoagulant state in platelets. The effects of sublytic concentrations of HlyA on both erythrocytes and platelets could have a potential impact on capillary microcirculation. Targeting HlyA emerges as a viable therapeutic strategy to mitigate the adverse effects of UPEC infections, especially in South American countries where these infections are endemic, underscoring its significance as a potential therapeutic target.

Alpha hemolysin of E. coli induces hemolysis of human erythrocytes independently of toxin interaction with membrane proteins.

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has been debated. Experiments using anti-GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GP binding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyA remained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonance experiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA (inactive protoxin), HlyAΔ914-936 (mutant of HlyA lacking the binding domain to GPA) or human serum albumin, indicating that the binding between the proteins and GPA is not specific. Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts with Band 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicated these proteins do not mediate the hemolytic process. Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediate the cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This finding holds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of the growing antibiotic resistance exhibited by bacteria.

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.

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 erythrocyte microvesicles by Escherichia Coli Alpha hemolysin.

Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains - which proteolyse cytoskeleton proteins - and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter ∼200 nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might act as a protective mechanism on the part of erythrocytes that removes the toxin from the membrane to prevent the spread of infection. Although erythrocytes have been found to eliminate denatured hemoglobin and several membrane proteins by shedding MVs, the present work has revealed for the first time that an exogenous protein, such as a toxin, is eliminated by this process. This finding sheds light on the mechanism of action of the toxin and serves to further elucidate the consequences of UPEC infection in patients exhibiting HlyA-related diseases.

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.

A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell.

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde-3-phosphate-dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low-cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi-well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):502-515, 2018.

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.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.

BACKGROUND: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10µM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

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 human erythrocytes.

We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ß-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ((10)Panx1) decreased [ATP]e by 75-84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ∼1 pmol × (10(6) cells)(-1). A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (10(6) cells)(-1), followed by a slower exponential decay (t(½) = 3.7 min) to a constant value of 1.3 pmol × (10(6) cells)(-1). Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (∼28 fmol × (10(6) cells min)(-1)), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.

Regulatory volume decrease and P receptor signaling in fish cells: mechanisms, physiology, and modeling approaches.

For animal cell plasma membranes, the permeability of water is much higher than that of ions and other solutes, and exposure to hyposmotic conditions almost invariably causes rapid water influx and cell swelling. In this situation, cells deploy regulatory mechanisms to preserve membrane integrity and avoid lysis. The phenomenon of regulatory volume decrease, the partial or full restoration of cell volume following cell swelling, is well-studied in mammals, with uncountable investigations yielding details on the signaling network and the effector mechanisms involved in the process. In comparison, cells from other vertebrates and from invertebrates received little attention, despite of the fact that e.g. fish cells could present rewarding model systems given the diversity in ecology and lifestyle of this animal group that may be reflected by an equal diversity of physiological adaptive mechanisms, including those related to cell volume regulation. In this review, we therefore present an overview on the most relevant aspects known on hypotonic volume regulation presently known in fish, summarizing transporters and signaling pathways described so far, and then focus on an aspect we have particularly studied over the past years using fish cell models, i.e. the role of extracellular nucleotides in mediating cell volume recovery of swollen cells. We, furthermore, present diverse modeling approaches developed on the basis of data derived from studies with fish and other models and discuss their potential use for gaining insight into the theoretical framework of volume regulation.

Perspective--from describing to understanding environment-physiology relations: 50th birthday of a branch in ecophysiology.

Animals generally show various adaptation features that render them fit for survival in their specific environment or, turned the other way round, specific environments can only be inhabited by animals that have developed corresponding adaptations. While this seems obvious nowadays to every biologist, 50years ago this concept still needed to be validated for each specific case. In a brief historical perspective we highlight an outstanding example of an article where such environment-physiology relations have been examined in detail and where in fact the foundations of a new branch in ecophysiology have been established, the Ecophysiology of the Marine Meiofauna.

Negative feedback of extracellular ADP on ATP release in goldfish hepatocytes: a theoretical study.

A mathematical model was built to account for the kinetic of extracellular ATP (ATPe) and extracellular ADP (ADPe) concentrations from goldfish hepatocytes exposed to hypotonicity. The model was based on previous experimental results on the time course of ATPe accumulation, ectoATPase activity, and cell viability [Pafundo et al., 2008]. The kinetic of ATPe is controlled by a lytic ATP flux, a non-lytic ATP flux, and ecto-ATPase activity, whereas ADPe kinetic is governed by a lytic ADP flux and both ecto-ATPase and ecto-ADPase activities. Non-lytic ATPe efflux was included as a diffusion equation modulated by ATPe activation (positive feedback) and ADPe inhibition (negative feedback). The model yielded physically meaningful and stable steady-state solutions, was able to fit the experimental time evolution of ATPe and simulated the concomitant kinetic of ADPe. According to the model during the first minute of hypotonicity the concentration of ATPe is mainly governed by both lytic and non-lytic ATP efflux, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of ATPe levels. ADPe inhibition of the non-lytic ATP efflux was strong, whereas ATPe activation was minimal. Finally, the model was able to predict the consequences of partial inhibition of ecto-ATPase activity on the ATPe kinetic, thus emulating the exposure of goldfish cells to hypotonic medium in the presence of the ATP analog AMP-PCP. The model predicts this analog to both inhibit ectoATPase activity and increase non-lytic ATP release.

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.

[Purinergic signals]. / Señales purinérgicas.

In the last decade evidence accumulated that nucleosides and nucleotides of both uridine and adenine can act as extracellular signaling factors. Their action is mediated by two main types of surface receptors commonly known as purinergic. P1 receptors are metabotropic and activated by adenosine, whereas receptors for nucleotides (ATP, ADP, UTP and UDP) and nucleotide-sugars (UDP-glucose and UDP-galactose) can be either metabotropic (P2Y) or ionotropic (P2X). The importance and complexity of this signaling system is evidenced by various mechanisms of nucleotide release, as well as by the ibiquitous distribution of various types of ectonucleotidases which catalyze and convert extracellular nucleotides. Up to now about twenty receptors have been cloned and found to modulate the nerve impulse, inflammatory response, insuline secretion, the regulation of the vascular tone and nociception, among other processes. In the present review we describe the main structural and pharmacological features of purinergic receptors, and analyze how the dynamic interaction between these receptors, nucleotides and nucleosides, and ectonucleotidases modulate several biological responses. Particular focus is given to platelet aggregation and thrombus formation, the immune response and the hydration of the mucosal linings of the respiratory tract.

Señales purinérgicas / Purinergic signals

En la última década se ha aportado clara evidencia de que tanto nucleósidos como nucleótidos de adenina y uridina pueden funcionar como factores de señalización extracelular. Su acción es mediada por dos tipos principales de receptores de superficie denominados purinérgicos. Los receptores P1 se activan por adenosina, y son todos metabotrópicos, mientras que los receptores de nucleótidos (ATP, ADP, UTP y UDP) y nucleótidos-azúcares (UDP-glucosa y UDP-galactosa) pueden ser metabotrópicos (P2Y) o ionotrópicos (P2X). La importancia y complejidad de este sistema de señalización se evidencia por la diversidad de mecanismos de liberación de nucleótidos al medio extracelular y por la distribución ubicua de varios grupos de ectonucleotidasas capaces de catalizar la degradación y conversión de nucleótidos. Hasta el momento se han descrito y clonado una veintena de estos receptores que modulan una variedad de respuestas, como el impulso nervioso, la respuesta inflamatoria, la secreción de insulina, la regulación del tono vascular y la percepción del dolor. En la presente revisión se describen las características estructurales y farmacológicas de los receptores purinérgicos y se analiza la interacción dinámica entre estos receptores, los nucleósidos y nucleótidos, y las ectonucleotidasas, con especial atención a la dinámica de la agregación plaquetaria, la respuesta inmune y la hidratación de las mucosas respiratorias.

In the last decade evidence accumulated that nucleosides and nucleotides of both uridine and adenine can act as extracellular signaling factors. Their action is mediated by two main types of surface receptors commonly known as purinergic. P1 receptors are metabotropic and activated by adenosine, whereas receptors for nucleotides (ATP, ADP, UTP and UDP) and nucleotide-sugars (UDP-glucose and UDP-galactose) can be either metabotropic (P2Y) or ionotropic (P2X). The importance and complexity of this signaling system is evidenced by various mechanisms of nucleotide release, as well as by the ibiquitous distribution of various types of ectonucleotidases which catalyze and convert extracellular nucleotides. Up to now about twenty receptors have been cloned and found to modulate the nerve impulse, inflammatory response, insuline secretion, the regulation of the vascular tone and nociception, among other processes. In the present review we describe the main structural and pharmacological features of purinergic receptors, and analyze how the dynamic interaction between these receptors, nucleotides and nucleosides, and ectonucleotidases modulate several biological responses. Particular focus is given to platelet aggregation and thrombus formation, the immune response and the hydration of the mucosal linings of the respiratory tract.

Kinetics of extracellular ATP from goldfish hepatocytes: a lesson from mathematical modeling.

In goldfish hepatocytes, hypotonic exposure leads to cell swelling, followed by a compensatory shrinkage termed RVD. It has been previously shown that ATP is accumulated in the extracellular medium of swollen cells in a non-linear fashion, and that extracellular ATP (ATPe) is an essential intermediate to trigger RVD. Thus, to understand how RVD proceeds in goldfish hepatocytes, we developed two mathematical models accounting for the experimental ATPe kinetics reported recently by Pafundo et al. in Am. J. Physiol. 294, R220-R233, 2008. Four different equations for ATPe fluxes were built to account for the release of ATP by lytic (J(L)) and nonlytic mechanisms (J(NL)), ATPe diffusion (J(D)), and ATPe consumption by ectonucleotidases (J(V)). Particular focus was given to J(NL), defined as the product of a time function (J(R)) and a positive feedback mechanism whereby ATPe amplifies J(NL). Several J (R) functions (Constant, Step, Impulse, Gaussian, and Lognormal) were studied. Models were tested without (model 1) or with (model 2) diffusion of ATPe. Mathematical analysis allowed us to get a general expression for each of the models. Subsequently, by using model dependent fit (simulations) as well as model analysis at infinite time, we observed that: - use of J(D) does not lead to improvements of the models. - Constant and Step time functions are only applicable when J(R)=0 (and thus, J(NL)=0), so that the only source of ATPe would be J(L), a result incompatible with experimental data. - use of impulse, Gaussian, and lognormal J(R)s in the models led to reasonable good fits to experimental data, with the lognormal function in model 1 providing the best option. Finally, the predictive nature of model 1 loaded with a lognormal J(R) was tested by simulating different putative in vivo scenarios where J(V) and J(NL) were varied over ample ranges.

Immunocytochemical localization of NTPDases1 and 2 in the neural retina of mouse and zebrafish.

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are a family of membrane-bound enzymes that hydrolyze extracellular di- and triphosphate nucleosides. E-NTPDases have been proposed to control extracellular nucleotide levels that mediate intercellular communication by binding to specific membrane receptors. Here we show a detailed immunocytochemical localization of two enzymes of the E-NTPDase family in the retinal layers of two vertebrate species, namely, the mouse and the zebrafish. In the mouse retina, NTPDase2 was chiefly localized in Müller glia and ganglion cell processes. NTPDase1 was located on neurons as well, since it was expressed by horizontal and ganglion cell processes, suggesting that nucleotides such as ATP and ADP can be hydrolyzed at the surface of these cells. NTPDase1 was also detected in intraretinal blood vessels of the mouse. Regarding zebrafish, NTPDases1 and 2 seem to be differentially localized in horizontal cell processes, photoreceptor segments, and ganglion cell dendrites and axons, but absent from Müller glia. Moreover, NTPDases1 and 2 appear to be expressed within the germinal margin of the zebrafish retina that contains proliferative and differentiating cells. Retinal homogenates from both species exhibited ecto-ATPase activity which might be attributed at least to NTPDases1 and 2, whose expression is described in this report. Our results suggest a compartmentalized regulation of extracellular nucleotide/nucleoside concentration in the retinal layers, supporting a relevant role for extracellular nucleotide mediated-signaling in vertebrate retinas.

Kinetics of ATP release and cell volume regulation of hyposmotically challenged goldfish hepatocytes.

In most animal cells, hypotonic swelling is followed by a regulatory volume decrease (RVD) thought to prevent cell death. In contrast, goldfish hepatocytes challenged with hypotonic medium (180 mosM, HYPO) increase their volume 1.7 times but remain swollen and viable for at least 5 h. Incubation with ATPgammaS (an ATP analog) in HYPO triggers a 42% volume decrease. This effect is concentration dependent (K(1/2) = 760 nM) and partially abolished by P2 receptor antagonists (64% inhibition). A similar induction of RVD is observed with ATP, UTP, and UDP, whereas adenosine inhibits RVD. Goldfish hepatocytes release more than 500 nM ATP during the first minutes of HYPO with no induction of RVD. The fact that similar concentrations of ATPgammaS did trigger RVD could be explained by showing that ATPgammaS induced ATP release. Finally, we observed that in a very small extracellular volume, hepatocytes do show a 56% RVD. This response was diminished by P2 receptor antagonists (73%) and increased (73%) when the extracellular ATP hydrolysis was inhibited 72%. Using a mathematical model, we predict that during the first 2 min of HYPO exposure the extracellular [ATP] is mainly governed by ATP diffusion and by both nonlytic and lytic ATP release, with almost no contribution from ecto-ATPase activity. We show that goldfish hepatocytes under standard HYPO (large volume) do not display RVD unless this is triggered by the addition of micromolar concentrations of nucleotides. However, under very low assay volumes, sufficient endogenous extracellular [ATP] can build up to induce RVD.