High Concentrations Of ATP Research Articles

Mass-Specific Metabolic Rate Influences Sperm Performance through Energy Production in Mammals.

Mass-specific metabolic rate, the rate at which organisms consume energy per gram of body weight, is negatively associated with body size in metazoans. As a consequence, small species have higher cellular metabolic rates and are able to process resources at a faster rate than large species. Since mass-specific metabolic rate has been shown to constrain evolution of sperm traits, and most of the metabolic activity of sperm cells relates to ATP production for sperm motility, we hypothesized that mass-specific metabolic rate could influence sperm energetic metabolism at the cellular level if sperm cells maintain the metabolic rate of organisms that generate them. We compared data on sperm straight-line velocity, mass-specific metabolic rate, and sperm ATP content from 40 mammalian species and found that the mass-specific metabolic rate positively influences sperm swimming velocity by (a) an indirect effect of sperm as the result of an increased sperm length, and (b) a direct effect independent of sperm length. In addition, our analyses show that species with higher mass-specific metabolic rate have higher ATP content per sperm and higher concentration of ATP per μm of sperm length, which are positively associated with sperm velocity. In conclusion, our results suggest that species with high mass-specific metabolic rate have been able to evolve both long and fast sperm. Moreover, independently of its effect on the production of larger sperm, the mass-specific metabolic rate is able to influence sperm velocity by increasing sperm ATP content in mammals.

Abstract 3648: A high-throughput fluorescence anisotropy screening for discovery of inhibitors that target the D-recruitment site of ERK in vitro and in cells

Abstract Background: Docking sites on mitogen-activated protein kinases (MAPKs) direct protein binding interactions and generate signal specificity. Therapeutically targeting these sites in cancer offers a way to potentially overcome many issues associated with ATP-competitive MAPK inhibitor design, such as high cellular ATP concentrations and highly conserved active sites among MAPKs. Extracellular signal-regulated kinases 1/2 (ERK1/2) are MAPKs that have a pro-tumorigenic role in numerous cancers and possess two protein docking sites that are distinct from the active site: the D-recruitment site (DRS) and F-recruitment site (FRS). In this study, we have developed a high-throughput fluorescence anisotropy screening to identify small molecule inhibitors that target the DRS of ERK. Methods: DRS inhibitors were detected in the high-throughput screening by using fluorescence anisotropy to measure the ability of over 22,000 molecules to competitively displace a fluorescent peptide from the DRS of inactive ERK2. The top 100 compounds from this screening were validated with a secondary radioactive kinase assay. In this assay, the ability of each compound to prevent the phosphorylation of a DRS-specific peptide by active ERK2 was assessed using [γ-32P]-labeled ATP substrate to measure enzyme activity. The top nine compounds were characterized by in vitro dose-response assays against wild-type and C159S ERK2, a DRS mutant. Inhibition of ERK2 phosphorylation of an FRS substrate and JNK2 phosphorylation of c-Jun was evaluated in vitro to determine compound specificity. The potency and specificity of the compounds were also assessed in EGF-stimulated HEK293T cells for their ability to prevent ERK1/2, JNK1/2, and p38α phosphorylation using Western blot. The compounds were tested for irreversible inhibition in vitro through ERK activity tests after exposure to, and subsequent removal of the compounds. Results: Of the nine hit compounds found from the screening, four reversibly inhibited the DRS of ERK2 with in vitro IC50 and Ki values under 5 μM, and did not show significant inhibition of the FRS or C159S ERK2. Two of these compounds also reduced ERK activation to basal levels in MAPK pathway-stimulated HEK293T cells at concentrations of 5 μM. However, in specificity tests these inhibitors demonstrate ability to also target JNK2 and p38α. Conclusions: The fluorescence anisotropy screening developed here can be used to identify potent inhibitors that target the DRS of ERK. These inhibitors can be used to probe ERK signaling events that are mediated by the DRS and can be further optimized to yield highly selective ERK DRS inhibitors with potential therapeutic value. Citation Format: Rachel M. Sammons, Tamer S. Kaoud, Ashwini K. Devkota, Eun J. Cho, Kevin N. Dalby. A high-throughput fluorescence anisotropy screening for discovery of inhibitors that target the D-recruitment site of ERK in vitro and in cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3648. doi:10.1158/1538-7445.AM2015-3648

Photosynthesis-dependent/independent control of stomatal responses to CO2 in mutant barley with surplus electron transport capacity and reduced SLAH3 anion channel transcript

The mechanisms of stomatal sensitivity to CO2 are yet to be fully understood. The role of photosynthetic and non-photosynthetic factors in stomatal responses to CO2 was investigated in wild-type barley (Hordeum vulgare var. Graphic) and in a mutant (G132) with decreased photochemical and Rubisco capacities. The CO2 and DCMU responses of stomatal conductance (gs), gas exchange, chlorophyll fluorescence and levels of ATP, with a putative transcript for stomatal opening were analysed. G132 had greater gs than the wild-type, despite lower photosynthesis rates and higher intercellular CO2 concentrations (Ci). The mutant had Rubisco-limited photosynthesis at very high CO2 levels, and higher ATP contents than the wild-type. Stomatal sensitivity to CO2 under red light was lower in G132 than in the wild-type, both in photosynthesizing and DCMU-inhibited leaves. Under constant Ci and red light, stomatal sensitivity to DCMU inhibition was higher in G132. The levels of a SLAH3-like slow anion channel transcript, involved in stomatal closure, decreased sharply in G132. The results suggest that stomatal responses to CO2 depend partly on the balance of photosynthetic electron transport to carbon assimilation capacities, but are partially regulated by the CO2 signalling network. High gs can improve the adaptation to climate change in well-watered conditions.

P2X7 receptors and Fyn kinase mediate ATP-induced oligodendrocyte progenitor cell migration.

Recruitment of oligodendrocyte precursor cells (OPCs) to the lesions is the most important event for remyelination after central nervous system (CNS) injury or in demyelinating diseases. However, the underlying molecular mechanism is not fully understood. In the present study, we found high concentrations of ATP could increase the number of migrating OPCs in vitro, while after pretreatment with oxidized ATP (a P2X7 receptor antagonist), the promotive effect was attenuated. The promotive effect of 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) (a P2X7 receptor agonist) was more potent than ATP. After incubation with BzATP, the activity of Fyn, one member of the Src family of kinases, was enhanced. Moreover, the interaction between P2X7 and Fyn was identified by co-immunoprecipitation. After blocking the activity of Fyn or down-regulating the expression of Fyn, the migration of OPCs induced by BzATP was inhibited. These data indicate that P2X7 receptors/Fyn may mediate ATP-induced OPC migration under pathological conditions.

An Improved Method for P2X7R Antagonist Screening.

ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimer's disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC50 value of the known agonist ATP and the ICs50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 mM and 173-285 μM for ATP, BBG and OATP, respectively). The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist

P2X7 receptor stimulates breast cancer cell invasion and migration via the AKT pathway.

Purinergic signaling has been implicated in the regulation of many cellular processes. A high concentration of ATP has been observed in the tumor microenvironment, suggesting a possible role of extracellular ATP in tumor progression. The P2X7 receptor, which belongs to the ligand-gated ion channel receptor family, is involved in tumor development and metastasis. In the present study, we found that extracellular ATP stimulated the invasion and migration of human T47D breast cancer cells, in a dose-dependent manner. BzATP (ATP analogue), but not ADP, also promoted invasion and migration. We further found that the P2X7 receptor was highly expressed in the T47D cells. After knockdown of the P2X7 receptor, ATP-stimulated invasion and migration were markedly inhibited. Moreover, activation of the P2X7 receptor by ATP downregulated the protein level of E-cadherin and upregulated the production of MMP-13. In addition, ATP time-dependently induced the activation of AKT via the P2X7 receptor, and the AKT pathway was required for the ATP-mediated invasion and migration. Taken together, our results revealed that activation of the P2X7 receptor by ATP promotes breast cancer cell invasion and migration, possibly via activation of the AKT pathway and regulation of E-cadherin and MMP-13 expression. Therefore, the P2X7 receptor may be a useful therapeutic target for the treatment of breast cancer.

(340) Target-derived IL-1β mediates ischemia and reperfusion injury-induced sensitization of group III and IV muscle afferents

We have previously shown that ischemia and reperfusion injury (I/R) in the forepaw muscles of adult Swiss Webster mice decreased mechanical thresholds and increased firing to chemical stimuli in group III and IV muscle afferents as assessed by an ex vivo forepaw muscle-median and ulnar nerve-DRG-spinal cord recording preparation. Additionally, I/R induced a significant increase in the number of cells that responded to both non-noxious (low lactic acid and ATP concentrations; pH 7.0) and noxious (high lactic acid and ATP concentrations, pH 6.6) metabolite solutions.

Physiological and biochemical responses to saline-alkaline stress in two halophytic grass species with different photosynthetic pathways

We examined the physiological and biochemical responses of two halophytic grasses with different photosynthetic pathways, Puccinellia tenuiflora (C3) and Chloris virgata (C4), to saline-alkaline stresses. Plants were grown at different Na2CO3 concentrations (from 0 to 200 mM). Low Na2CO3 (< 12.5 mM) enhanced seed germination and plant growth, whereas high Na2CO3 concentrations (> 100 mM) reduced seed germination by 45% in P. tenuiflora and by 30% in C. virgata. Compared to C. virgata, P. tenuiflora showed lower net photosynthesis, stomatal conductance, intercellular CO2 concentration, and water-use efficiency under the same treatment. C. virgata exhibited also relatively higher ATP content, K+ concentration, and the K+/Na+ ratio under the stress treatments implying that salt tolerance may be the main mechanism for salt resistance in this species. Our results demonstrated that the C. virgata was relatively more resistant to saline-alkaline stress than the co-occurring P. tenuiflora; both two species adapt to their native saline-alkaline habitat by different physiological mechanisms.

Is Ecto-nucleoside Triphosphate Diphosphohydrolase (NTPDase)-based Therapy of Central Nervous System Disorders Possible?

Extracellular nucleotides and nucleosides are signalling molecules acting in all tissues and organs, including the central nervous system (CNS). A wide variety of effects, exerted by ecto-purines, requires that their levels, and ATP in particular, must be precisely controlled. Under physiological conditions, concentration of ecto-purines is regulated by a complex cascade of ecto-enzymes, including ecto-NTPDases (nucleoside triphosphate diphosphohydrolases), ecto-NPPs (nucleotide pyrophosphohydrolases/phosphodiesterases), ectoalkaline phosphatases, and ecto-5'nucleotidase. Adenylate kinase, transferring the phosphate moiety between nucleotides, also plays a role in controlling ecto-purines concentration. Disturbances in the elements of purinergic pathway within the CNS underlie the induction and amplification of many neurological pathologies. ATP released in bulk from the cells, and not degraded by less efficient or dysfunctional ecto-nucleotidases, triggers excitotoxic damage and neuro-inflammation in the brain tissue. High ATP concentration activating specific receptors has been shown to be involved in various disorders throughout CNS, including brain injury and ischemia, neuro-inflammation, epilepsy as well as neuropathic pain and migraine. Taking the above mentioned influence of ATP into consideration, the modulation of ecto-NTPDase activity or its site-targeted delivery seems a good therapeutic method. The availability of effective brain-targeted drug-delivery system is one of the most significant challenges facing potential NTPDase-based treatment of CNS disorders. The application of genetically engineered stem cells as carrier vehicles offers a promising strategy for the efficient delivery of the enzyme to CNS tissues.

Rapid Activation of Distinct Conducting States in P2X Receptor Channels

P2X receptors are trimeric cation channels that are activated by extracellular ATP. These purinergic receptors have been reported to undergo pore dilation following activation by high concentrations of ATP, a phenomenon thought to mediate apoptosis in the immune system. Here we demonstrate that the widely reported slow time-dependent increase in the relative permeability of NMDG to Na, thought to reflect pore dilation, results from a gradual inhibition of small-cation-selective channels following depletion of intracellular alkali ions. Moreover, we find that P2X receptors enter both small-cation-selective open states and large-cation-permeable open states within milliseconds of ATP application. Taken together, our results demonstrate that P2X receptors can rapidly enter a large-cation-permeable open state without requiring either high ATP concentrations or high channel density, indicating that the ability of ATP to permeabilize cells can occur under physiologically realistic conditions.

Abstract A53: The specific FMS kinase inhibitor, DCC-3014, durably inhibits FMS kinase in vivo and blocks cancer bone invasiveness

Abstract Within the tumor microenvironment, tumor-associated macrophages (TAMs) rely on signaling through FMS kinase to promote tumor growth, angiogenesis, vasculogenesis, and metastasis. In addition, FMS-expressing macrophages communicate with the adaptive immune system, which can lead to cancer immunotolerance. Osteoclasts also depend on FMS kinase for differentiation and growth, and play a direct role in the ability of a tumor to metastasize to the bone and progress. Taken together, FMS kinase is an excellent target for small molecule therapy of tumor growth and bone metastasis. Using Deciphera Pharmaceuticals' approach to kinase inhibition, potent and specific FMS kinase inhibitors have been identified that achieve single digit nanomolar inhibition in biochemical and cellular assays of FMS activity. Due to their binding mode, these inhibitors retain potency in the presence of high ATP concentrations and also exhibit long off-rates from FMS kinase. Development candidate DCC-3014 is highly selective, inhibiting no other kinases within 100-fold of FMS potency, including related kinases KIT, PDGFRs, FLT3, and VEGFR2. DCC-3014 exhibits potent and durable inhibition of FMS in vivo in a pharmacokinetic/pharmacodynamic model. In a variety of cancer models, DCC-3014 or related analogs slow tumor growth, decrease tumor-promoting macrophages, and protect against osteolytic bone invasion. DCC-3014 is currently undergoing formal preclinical development. Citation Format: Bryan D. Smith, Michael D. Kaufman, Cynthia B. Leary, Molly M. Hood, Wei-Ping Lu, Benjamin A. Turner, Subha Vogeti, Scott C. Wise, Daniel L. Flynn. The specific FMS kinase inhibitor, DCC-3014, durably inhibits FMS kinase in vivo and blocks cancer bone invasiveness. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A53. doi:10.1158/1538-7445.CHTME14-A53

Motility, mitochondrial membrane potential and ATP content of rabbit spermatozoa stored in extender supplemented with GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide.

The present study was aimed to determine the effect of GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide on the quality of rabbit spermatozoa stored at 17°C for 3 days. Semen from 5 bucks (13 ejaculates) was used in the experiment. Ejaculates were divided and diluted at a 1:10 ratio with rabbit semen extender Galap (IMV, France) (Control) or with Galap extender supplemented with GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide (50 μg/ml) and stored for 3 days. Sperm motility parameters, mitochondrial membrane potential (MMP) and ATP content were as- sessed on each day of the experiment. Motility analysis was performed using a computer-assisted sperm analysis (CASA) system. The following sperm motility parameters were recorded: total motile spermatozoa, progressively motile spermatozoa, curvilinear velocity, straight-line velocity, average path velocity, linearity, straightness and amplitude of lateral head displacement. MMP was evaluated using JC-1 fluorescent dye. ATP content was assessed using a bioluminescence method. The addition of GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide to Galap extender did not affect any of the quality parameters studied. However, in both groups (Control and GnRH), significant changes in motility parameters (except straight-line velocity) and proportion of spermatozoa showing high MMP and ATP content were observed throughout 3 days of storage.

Torque Generation of Enterococcus hirae V-ATPase

V-ATPase (V(o)V1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V(o)V1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V(o)V1 (EhV(o)V1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV(o)V1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV(o)V1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhV(o)V1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhV(o)V1 showed faster rotation than EhV1, and the torque of EhV(o)V1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhV(o)V1. These results indicate that rotor-stator interactions of the V(o) moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhV(o)V1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV(o)V1.

Nucleotide receptors control IL-8/CXCL8 and MCP-1/CCL2 secretions as well as proliferation in human glioma cells

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.

Thermodynamic analysis of F1-ATPase rotary catalysis using high-speed imaging.

F1-ATPase (F1) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F1 rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F1 catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F1 rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (Pi). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, Pi release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG(‡) = 53-56 kJ mol(-1), the contributions of the enthalpy (ΔH(‡)), and entropy (ΔS(‡)) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH(‡) and ΔS(‡). The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.

Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities

SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG–SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg2+, mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes.

Mass-specific metabolic rate influences sperm performance through energy production in mammals

Mass-specific metabolic rate, the rate at which organisms consume energy per gram of body weight, is negatively associated with body size in metazoans. As a consequence, small species have higher cellular metabolic rates and are able to process resources at a faster rate than large species. Since mass-specific metabolic rate has been shown to constrain evolution of sperm traits, and most of the metabolic activity of sperm cells relates to ATP production for sperm motility, we hypothesized that mass-specific metabolic rate could influence sperm energetic metabolism at the cellular level if sperm cells maintain the metabolic rate of organisms that generate them. We compared data on sperm straight-line velocity, mass-specific metabolic rate, and sperm ATP content from 40 mammalian species and found that the mass-specific metabolic rate positively influences sperm swimming velocity by (a) an indirect effect of sperm as the result of an increased sperm length, and (b) a direct effect independent of sperm length. In addition, our analyses show that species with higher mass-specific metabolic rate have higher ATP content per sperm and higher concentration of ATP per μm of sperm length, which are positively associated with sperm velocity. In conclusion, our results suggest that species with high mass-specific metabolic rate have been able to evolve both long and fast sperm. Moreover, independently of its effect on the production of larger sperm, the mass-specific metabolic rate is able to influence sperm velocity by increasing sperm ATP content in mammals.

The C Ring of the F1Fo ATP Synthase Forms the Mitochondrial Permeability Transition Pore: A Critical Appraisal.

The C Ring of the F1Fo ATP Synthase Forms the Mitochondrial Permeability Transition Pore: A Critical Appraisal.

CD39 is a negative regulator of P2X7-mediated inflammatory cell death in mast cells.

BackgroundMast cells (MCs) are major contributors to an inflammatory milieu. One of the most potent drivers of inflammation is the cytokine IL-1β, which is produced in the cytoplasm in response to danger signals like LPS. Several controlling mechanisms have been reported which limit the release of IL-1β. Central to this regulation is the NLRP3 inflammasome, activation of which requires a second danger signal with the capacity to subvert the homeostasis of lysosomes and mitochondria. High concentrations of extracellular ATP have the capability to perturb the plasma membrane by activation of P2X7 channels and serve as such a danger signal. In this study we investigate the role of P2X7 channels and the ecto-5´-nucleotidase CD39 in ATP-triggered release of IL-1β from LPS-treated mast cells.ResultsWe report that in MCs CD39 sets an activation threshold for the P2X7-dependent inflammatory cell death and concomitant IL-1β release. Knock-out of CD39 or stimulation with non-hydrolysable ATP led to a lower activation threshold for P2X7-dependent responses. We found that stimulation of LPS-primed MCs with high doses of ATP readily induced inflammatory cell death. Yet, cell death-dependent release of IL-1β yielded only minute amounts of IL-1β. Intriguingly, stimulation with low ATP concentrations augmented the production of IL-1β in LPS-primed MCs in a P2X7-independent but caspase-1-dependent manner.ConclusionOur study demonstrates that the fine-tuned interplay between ATP and different surface molecules recognizing or modifying ATP can control inflammatory and cell death decisions.

Interception of the endotoxin-induced arterial hyporeactivity to vasoconstrictors

Septic shock is a severe pathophysiologic condition characterized by vasodilation, hypotension, hypoperfusion, tissue hypoxia, multiple organ failure and death. It is unclear what causes the septic vasodilation that may result from general dysfunction of vascular smooth muscles (VSMs) or selective disruption of vasomotor balances in VSMs. The latter could be due to enhanced vasorelaxation and/or depressed vasoconstriction. Understanding these may lead to pharmacological interventions to septic vasodilation. Therefore, we performed studies in isolated and perfused mesenteric arterial rings. A 20-h exposure of the rings to lipopolysaccharide (LPS, 1μg/ml) led to hyporeactivity to phenylephrine (PE). However, the responses of the LPS-treated rings to high concentrations of KCl (60mM) and ATP remained comparable to control rings, suggesting that contractility of VSMs is retained. The hyporeactivity was marginally affected by atropine, indomethacin and L-NAME, suggesting that endothelium-dependent vasorelaxation does not play a major role. In addition to PE, the LPS-treated rings were hyporeactive to dopamine, histamine and angiotensin II. They showed intermediate hyporeactivity to the thromboxane-A2 receptor agonist U46619. Little hyporeactivity to endothelin-1 (ET-1), serotonin (5-HT) and vasopressin was found. ET-1-induced vasoconstriction occurred without endothelium, whereas the effect of 5-HT was endothelium dependent. Although rings were hyporeactive to some of the vasopressors, their vasoconstriction effects were significantly potentiated by PE co-application. Taken together, these data suggest that the endotoxin-induced vasodilation may not result from general dysfunction of VSMs, neither from the endothelium-dependent vasorelaxation. The promising vascular response to various vasoconstrictors found in this study warrants further investigations of therapeutic potentials of these agents.