Adenosine Triphosphate Stimulation Research Articles

Disruption of P2Y2 signaling promotes breast tumor cell dissemination by reducing ATP-dependent calcium elevation and actin localization to cell junctions.

The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca 2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca 2+ responses. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca 2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca 2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response.

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation.

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results showthat substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

Neuronal Panx1 drives peripheral sensitization in experimental plantar inflammatory pain

BackgroundThe channel-forming protein Pannexin1 (Panx1) has been implicated in both human studies and animal models of chronic pain, but the underlying mechanisms remain incompletely understood.MethodsWild-type (WT, n = 24), global Panx1 KO (n = 24), neuron-specific Panx1 KO (n = 20), and glia-specific Panx1 KO (n = 20) mice were used in this study at Albert Einstein College of Medicine. The von Frey test was used to quantify pain sensitivity in these mice following complete Freund’s adjuvant (CFA) injection (7, 14, and 21 d). The qRT-PCR was employed to measure mRNA levels of Panx1, Panx2, Panx3, Cx43, Calhm1, and β-catenin. Laser scanning confocal microscopy imaging, Sholl analysis, and electrophysiology were utilized to evaluate the impact of Panx1 on neuronal excitability and morphology in Neuro2a and dorsal root ganglion neurons (DRGNs) in which Panx1 expression or function was manipulated. Ethidium bromide (EtBr) dye uptake assay and calcium imaging were employed to investigate the role of Panx1 in adenosine triphosphate (ATP) sensitivity. β-galactosidase (β-gal) staining was applied to determine the relative cellular expression levels of Panx1 in trigeminal ganglia (TG) and DRG of transgenic mice.ResultsGlobal or neuron-specific Panx1 deletion markedly decreased pain thresholds after CFA stimuli (7, 14, and 21 d; P < 0.01 vs. WT group), indicating that Panx1 was positively correlated with pain sensitivity. In Neuro2a, global Panx1 deletion dramatically reduced neurite extension and inward currents compared to the WT group (P < 0.05), revealing that Panx1 enhanced neurogenesis and excitability. Similarly, global Panx1 deletion significantly suppressed Wnt/β-catenin dependent DRG neurogenesis following 5 d of nerve growth factor (NGF) treatment (P < 0.01 vs. WT group). Moreover, Panx1 channels enhanced DRG neuron response to ATP after CFA injection (P < 0.01 vs. Panx1 KO group). Furthermore, ATP release increased Ca2+ responses in DRGNs and satellite glial cells surrounding them following 7 d of CFA treatment (P < 0.01 vs. Panx1 KO group), suggesting that Panx1 in glia also impacts exaggerated neuronal excitability. Interestingly, neuron-specific Panx1 deletion was found to markedly reduce differentiation in cultured DRGNs, as evidenced by stunted neurite outgrowth (P < 0.05 vs. Panx1 KO group; P < 0.01 vs. WT group or GFAP-Cre group), blunted activation of Wnt/β-catenin signaling (P < 0.01 vs. WT, Panx1 KO and GFAP-Cre groups), and diminished cell excitability (P < 0.01 vs. GFAP-Cre group) and response to ATP stimulation (P < 0.01 vs. WT group). Analysis of β-gal staining showed that cellular expression levels of Panx1 in neurons are significantly higher (2.5-fold increase) in the DRG than in the TG.ConclusionsThe present study revealed that neuronal Panx1 is a prominent driver of peripheral sensitivity in the setting of inflammatory pain through cell-autonomous effects on neuronal excitability. This hyperexcitability dependence on neuronal Panx1 contrasts with inflammatory orofacial pain, where similar studies revealed a prominent role for glial Panx1. The apparent differences in Panx1 expression in neuronal and non-neuronal TG and DRG cells are likely responsible for the distinct impact of these cell types in the two pain models.

Abstract A044: P2Y2 signaling disruption reduces ATP-dependent calcium elevation and actin localization to promote breast tumor cell dissemination

Abstract The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neural cell signaling. Here we report the importance of ATP-dependent P2Y2 signaling in breast epithelial cells and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular calcium (Ca2+) in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca2+ responses. The non-tumorigenic cells respond to increased Ca2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. Citation Format: Makenzy Mull, Stephen Pratt, Keyata Thompson, David Annis, Julia Ju, Megan Stemberger, Liron Boyman, Michele Vitolo, W. Jonathan Lederer, Stuart Martin. P2Y2 signaling disruption reduces ATP-dependent calcium elevation and actin localization to promote breast tumor cell dissemination [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A044.

Styrax (Liquidambar orientalis Mill.) promotes mitochondrial function and reduces cardiac damage following myocardial ischemic injury: the role of the AMPK-PGC1α signaling pathway.

To explore the effect of extract of Styrax (ES) on myocardial ischemic injury and its molecular mechanism, indirectly providing a theoretical basis for the development of ES. In order to assess the impact of ES treatment on ischemic heart disease, both a left anterior descending ligation-induced myocardial infarction (MI) model and an ischemia/hypoxia (I/H)-induced H9c2 cell injury model have been constructed. Specifically, Sprague-Dawley rats were randomly assigned to the following groups (n = 8) and administered intragastrically once a day for seven consecutive days: Sham group, MI group, ES-L (0.2 g/kg) group, ES-M (0.4 g/kg) group, ES-H (0.8 g/kg) group, and trimetazidine (TMZ, 0.02 g/kg) group. The cardiac functions and biochemical assessment of rats were detected. Then, we validated experimentally the targets and mechanism of ES on these pathological processes in I/H-induced H9c2 cell injury model. These results showed that different doses of ES (0.2 g/kg, 0.4 g/kg, 0.8 g/kg, intragastric) significantly improved myocardial structure and function when compared to the MI group. The results of 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin-eosin, and masson staining indicated that ES could significantly reduce infarct size, inhibit myocardium apoptosis, and decrease myocardial fibrosis. Moreover, ES distinctly suppressed the serum levels of lactate dehydrogenase (LDH), cardiac troponin T (cTnT), and creatine kinase-MB (CK-MB), alleviated myocardial mitochondrial morphology, and stimulated adenosine triphosphate (ATP) production, increased the level of succinate dehydrogenase (SDH), complex I and complex V activity. Different doses of ES (5 μg/ml, 10 μg/ml, 20 μg/ml) also improved cardiomyocyte morphology and decreased the apoptosis rate in H9c2 cells that had been exposed to I/H. Furthermore, the results of western blotting and qRT-PCR indicated that ES promoted the expression of proteins and mRNA related to energy metabolism, including phosphorylated adenosine monophosphate activated protein kinase (p-AMPK), peroxisome proliferator activated receptor gamma coactivator 1 alpha (PCG-1α), nuclear respiratory factor 1, and mitochondrial transcription factor A (TFAM). Mechanically, after the administration of Compound C (dorsomorphin), an AMPK inhibitor, these effects of myocardial protection produced by ES were reversed. Collectively, these results demonstrated that ES could improve myocardial mitochondrial function and reduce ischemic injury by activating AMPK/PCG-1α signaling pathway, while indicating its potential advantages as a dietary supplement.

Intracellular calcium ion transients evoked by cell poking independently of released autocrine ATP in Madin-Darby canine kidney cells.

The mechanical stimulation induced by poking cells with a glass needle activates Piezo1 receptors and the adenosine triphosphate (ATP) autocrine pathway, thus increasing intracellular Ca2+ concentration. The differences between the increase in intracellular Ca2+ concentration induced by cell poking and by ATP-only stimulation have not been investigated. In this study, we investigated the Ca2+ signaling mechanism induced by autocrine ATP release during Madin-Darby Canine Kidney cell membrane deformation by cell poking. The results suggest that the pathways for supplying Ca2+ into the cytoplasm were not identical between cell poking and conventional ATP stimulation. The functions of the G protein-coupled receptor (GPCR) subunits (G q, G ), ATP-activated receptor and the upstream Ca2+ release signal from the intracellular endoplasmic reticulum Ca2+ store, were investigated. The results show that G q plays a major role in the Ca2+ response evoked by ATP-only stimulation, while cell poking induces a Ca2+ response requiring the involvement of both G q and G units simultaneously. These results suggest that GPCR are not only activated by ATP-only stimulation or autocrine ATP release during Ca2+ signaling, but also activated by the mechanical effects of cell poking.

Prevention of M2 polarization and temporal limitation of differentiation in monocytes by extracellular ATP

BackgroundElevated levels of extracellular adenosine triphosphate (ATP) modulate immunologic pathways and are considered to be a danger signal in inflammation, lung fibrosis and cancer. Macrophages can be classified into two main types: M1 macrophages are classically activated, pro-inflammatory macrophages, whereas M2 macrophages are alternatively activated, pro-fibrotic macrophages. In this study, we examined the effect of ATP on differentiation of native human monocytes into these macrophage subtypes. We characterized M1 and M2 like macrophages by their release of Interleukin-1beta (IL-1β) and Chemokine (C–C motif) ligand 18 (CCL18), respectively.ResultsMonocytes were stimulated with ATP or the P2X7 receptor agonist Benzoylbenzoyl-ATP (Bz-ATP), and the production of various cytokines was analyzed, with a particular focus on CCL18 and IL-1β, along with the expression of different purinergic receptors. Over a 72 h period of cell culture, monocytes spontaneously differentiated to M2 like macrophages, as indicated by an increased release of CCL18. Immediate stimulation of monocytes with ATP resulted in a dose-dependent reduction in CCL18 release, but had no effect on the concentration of IL-1β. In contrast, delayed stimulation with ATP had no effect on either CCL18 or IL-1β release. Similar results were observed in a model of inflammation using lipopolysaccharide-stimulated human monocytes. Stimulation with the P2X7 receptor agonist Bz-ATP mimicked the effect of ATP on M2-macrophage differentiation, indicating that P2X7 is involved in ATP-induced inhibition of CCL18 release. Indeed, P2X7 was downregulated during spontaneous M2 differentiation, which may partially explain the ineffectiveness of late ATP stimulation of monocytes. However, pre-incubation of monocytes with PPADS, Suramin (unselective P2X- and P2Y-receptor blockers) and KN62 (P2X7-antagonist) failed to reverse the reduction of CCL18 by ATP.ConclusionsATP prevents spontaneous differentiation of monocytes into M2-like macrophages in a dose- and time-dependent manner. These effects were not mediated by P2X and P2Y receptors.

Salicornia herbacea Aqueous Extracts Regulate NLRP3 Inflammasome Activation in Macrophages and Trophoblasts.

Salicornia herbacea L. (Chenopodiaceae), an edible salt marsh plant with anti-inflammatory effects, was examined in macrophages and trophoblasts whether it modulates NLRP3 inflammasome activity. Pretreatment and delayed treatment of S. herbacea extract (SHE) in bone marrow-derived macrophages (BMDMs) reduced the activity of NLRP3 inflammasome induced by lipopolysaccharide (LPS) and adenosine triphosphate stimulation and downregulated interleukin (IL)-1β production. SHE also inhibited pyroptotic cell death, the adaptor molecule apoptosis-associated speck-like protein containing a CARD (ASC), oligomerization, and speck by NLRP3 inflammasome activity in BMDM. Similarly, SHE decreased the mRNA expression of NLRP3, ASC, IL-1β, and IL-6 in the LPS-stimulated human trophoblast cell line, Swan 71 cells. In addition, SHE inhibited the production of IL-6 and IL-1β and decreased the expression of cyclooxygenase-2 and prostaglandin E2 in stimulated Swan 71 cells. Finally, 3,5-dicaffeoylquinic acid (3,5-DCQA), one of the components of S. herbacea, inhibited IL-1β produced by NLRP3 inflammasome activity. In conclusion, SHE downregulated the activity of the NLRP3 inflammasome in macrophages and trophoblasts.

Sex Difference in Renal Artery Contractility in a Novel CRISPR/Cas9‐Generated P2X7 Knockout Rat

Purinergic signaling regulates numerous physiological processes, including renal hemodynamics. The P2X7 receptor (P2X7) is a non‐selective cation channel activated by extra‐cellular adenosine triphosphate (ATP) which plays an important role in immune cell response and inflammation. P2X7 is also expressed in endothelial cells. In the kidney, acute pharmacological blockade of P2X7 increases renal perfusion in healthy rats (Kidney Int. 88(5): 1079–1087, 2015). Here, we generated a novel P2X7 knockout (KO) rat and examined ex vivo contractility of the renal artery in male and female rats.A P2rx7 global KO rat was generated on a F344 IcoCrl background using CRISPR/Cas9 technology. KO was achieved via a two‐adenine insertion predicted to introduce a premature stop codon in exon 2. RNA and protein were extracted from KO and wild‐type (WT) kidneys to detect P2X7 by RTqPCR (n=4‐5/group) and western blotting (n=2/group). Bone marrow‐derived macrophages (BMDM) from KO and WT rats were used to assess P2X7 function by measuring interleukin (IL)‐1β release (ELISA) after stimulation with lipopolysaccharide (LPS) (1μg/mL, 4h) and ATP (3mM, 1h) (n=5‐6/group). RNA from BMDM was extracted after LPS to detect P2rx7, Il1b, Tnf and Il6 mRNA by RTqPCR (n=3/group), and Griess reaction was used to assess BMDM nitric oxide (NO) production (n=5‐6/group). One main renal artery from 2.5‐to‐4.5‐month‐old male and female KO and WT rats (n=8‐11/group) was mounted on a wire myograph to assess the response to 125 mM potassium physiological solution (KPSS) and to cumulative doses (1 nM‐30 μM) of phenylephrine (PE), acetylcholine (ACh) and sodium nitroprusside (SNP).The P2rx7 mutation in exon 2 was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was decreased by ~60% in kidney and ~53% in BMDM from KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. P2X7 KO did not alter BMDM polarization after LPS stimulation. The induction of NO production was not different between genotypes (2.14±0.26 vs 2.31±0.43 μM, NS) and mRNA expression of M1 markers Il1b, Tnf and Il6 was similarly increased. In BMDM from male WT rats, ATP stimulation induced IL‐1β release; this response was markedly suppressed in KO BMDM (2577±70 vs 413±19 pg/mL, p&lt;0.0001). In the renal artery, the constriction induced by KPSS depolarization or by PE was not different between genotypes for either sex. In male rats, the maximum vasodilation induced by ACh was significantly decreased in KO compared to WT (IC50=0.25 vs 0.20 μM, NS; %Imax= 24±3 vs 10±2%, p=0.0015); this was not observed in females (IC50=0.11 vs 8.28 μM, NS; %Imax= 19±4 vs 12±4%, NS). The dilation induced by the direct NO donor, SNP was not affected by either genotype or sex.Global genetic deletion of P2X7 impaired endothelial‐dependent vasodilation in the renal artery of male rats that may reflect diminished NO production in response to ACh. Thus, the increased renal perfusion induced by P2X7 inhibitor may arise from off‐target effects. The potential sexual dimorphism in vascular expression and/or function of P2X7 requires further investigation.

Role of ATP in Extracellular Vesicle Biogenesis and Dynamics.

Adenosine triphosphate (ATP) is among the molecules involved in the immune response. It acts as danger signal that promotes inflammation by activating both P2X and P2Y purinergic receptors expressed in immune cells, including microglia, and tumor cells. One of the most important receptors implicated in ATP-induced inflammation is P2X7 receptor (P2X7R). The stimulation of P2X7R by high concentration of ATP results in cell proliferation, inflammasome activation and shedding of extracellular vesicles (EVs). EVs are membrane structures released by all cells, which contain a selection of donor cell components, including proteins, lipids, RNA and ATP itself, and are able to transfer these molecules to target cells. ATP stimulation not only promotes EV production from microglia but also influences EV composition and signaling to the environment. In the present review, we will discuss the current knowledge on the role of ATP in the biogenesis and dynamics of EVs, which exert important functions in physiology and pathophysiology.

Epoxyeicosatrienoic acids inhibit the activation of NLRP3 inflammasome in murine macrophages.

Epoxyeicosatrienoic acids (EETs) derived from arachidonic acid exert anti-inflammation effects. We have reported that blocking the degradation of EETs with a soluble epoxide hydrolase (sEH) inhibitor protects mice from lipopolysaccharide (LPS)-induced acute lung injury (ALI). The underlying mechanisms remain essential questions. In this study, we investigated the effects of EETs on the activation of nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome in murine macrophages. In an LPS-induced ALI murine model, we found that sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl), TPPU, profoundly attenuated the pathological injury and inhibited the activation of the NLRP3 inflammasome, characterized by the reduction of the protein expression of NLRP3, ASC, pro-caspase-1, interleukin precursor (pro-IL-1β), and IL-1β p17 in the lungs of LPS-treated mice. In vitro, primary peritoneal macrophages from C57BL/6 were primed with LPS and activated with exogenous adenosine triphosphate (ATP). TPPU treatment remarkably reduced the expression of NLRP3 inflammasome-related molecules and blocked the activation of NLRP3 inflammasome. Importantly, four EETs (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) inhibited the activation of NLRP3 inflammasome induced by LPS + ATP or LPS + nigericin in macrophages in various degree. While the inhibitory effect of 5,6-EET was the weakest. Mechanismly, EETs profoundly decreased the content of reactive oxygen species (ROS) and restored the calcium overload in macrophages receiving LPS + ATP stimulation. In conclusion, this study suggests that EETs inhibit the activation of the NLRP3 inflammasome by suppressing calcium overload and ROS production in macrophages, contributing to the therapeutic potency to ALI.

A role for neurokinin 1 receptor expressing neurons in the paratrigeminal nucleus in bradykinin-evoked cough in guinea-pigs.

Airway projecting sensory neurons arising from the jugular vagal ganglia terminate centrally in the brainstem paratrigeminal nucleus, synapsing upon neurons expressing the neurokinin 1 receptor. This study aimed to assess the involvement of paratrigeminal neurokinin 1 receptor neurons in the regulation of cough, breathing and airway defensive responses. Lesioning neurokinin 1 receptor expressing paratrigeminal neurons significantly reduced cough evoked by inhaled bradykinin but not inhaled ATP or tracheal mechanical stimulation. The reduction in bradykinin-evoked cough was not accompanied by changes in baseline or evoked respiratory variables (e.g. frequency, volume or timing), animal avoidance behaviours or the laryngeal apnoea reflex. These findings warrant further investigations into targeting the jugular ganglia and paratrigeminal nucleus as a therapy for treating cough in disease. Jugular vagal ganglia sensory neurons innervate the large airways and are thought to mediate cough and associated perceptions of airway irritations to a range of chemical irritants. The central terminals of jugular sensory neurons lie within the brainstem paratrigeminal nucleus, where postsynaptic neurons can be differentiated based on the absence or presence of the neurokinin 1 (NK1) receptor. Therefore, in the present study, we set out to test the hypothesis that NK1 receptor expressing paratrigeminal neurons play a role in cough evoked by inhaled chemical irritants. To test this, we performed selective neurotoxin lesions of NK1 receptor expressing neurons in the paratrigeminal nucleus in guinea-pigs using substance P conjugated to saporin (SSP-SAP). Sham lesion control or SSP-SAP lesion guinea-pigs received nebulised challenges, with the pan-nociceptor stimulant bradykinin or the nodose ganglia specific stimulant adenosine 5'-triphosphate (ATP), in conscious whole-body plethysmography to study cough and associated behaviours. Laryngeal apnoea reflexes and cough evoked by mechanical stimulation of the trachea were additionally investigated in anaesthetised guinea-pigs. SSP-SAP significantly and selectively reduced the number of NK1 receptor expressing neurons in the paratrigeminal nucleus. This was associated with a significant reduction in bradykinin-evoked cough, but not ATP-evoked cough, mechanical cough or laryngeal apnoeic responses. These data provide further evidence for a role of jugular vagal pathways in cough, and additionally suggest an involvement of NK1 receptor expressing neurons in the paratrigeminal nucleus. Therefore, this neural pathway may provide novel therapeutic opportunities to treat conditions of chronic cough.

ATP and repetitive electric stimulation increases leukemia inhibitory factor expression in astrocytes: A potential role for astrocytes in the action mechanism of electroconvulsive therapy.

Electroconvulsive therapy (ECT) is effective for psychiatric disorders. However, its action mechanism remains unclear. We previously reported that transcription factor 7 (TCF7) was increased in patients successfully treated with ECT. TCF7 regulates Wnt pathway, which regulates adult hippocampal neurogenesis. Adult hippocampal neurogenesis is involved in the pathophysiology of psychiatric disorders. Astrocytes play a role in adult hippocampal neurogenesis via neurogenic factors. Of astrocyte-derived neurogenic factors, leukemia inhibitory factor (LIF) and fibroblast growth factor 2 (FGF2) activate Wnt pathway. In addition, adenosine triphosphate (ATP), released from excited neurons, activates astrocytes. Therefore, we hypothesized that ECT might increase LIF and/or FGF2 in astrocytes. To test this, we investigated the effects of ATP and electric stimulation (ES) on LIF and FGF2 expressions in astrocytes. Astrocytes were derived from neonatal mouse forebrain and administered ATP and ES. The mRNA expression was estimated with quantitative reverse-transcription polymerase chain reaction. Protein concentration was measured with ELISA. ATP increased LIF, but not FGF2, expression. Multiple ES, but not single, increased LIF expression. Knockdown of P2X2 receptor (P2X2R) attenuated ATP-induced increase of LIF mRNA expression. In contrast, P2X3 and P2X4 receptors intensified it. P2X2R may mediate ATP-induced LIF expression in astrocytes and multiple ES directly increases LIF expression in astrocytes. Therefore, both ATP/P2X2R and multiple ES-induced increases of LIF expression in astrocytes might mediate the efficacy of ECT on psychiatric disorders. Elucidating detailed mechanisms of ATP/P2X2R and multiple ES-induced LIF expression is expected to result in the identification of new therapeutic targets for psychiatric disorders.

TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy.

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca2+-dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5′-triphosphate (ATP), is also known as most potent Ca2+-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2+ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2+/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

Effect of interleukin-31 on septic shock through regulating inflammasomes and interleukin-1β.

Sepsis with severe systemic inflammation remains a great challenge for the intensive care unit in clinics. Although biomarkers have been identified to diagnose, monitor and predict these syndromes, novel therapeutic approaches are required for the amelioration of symptoms of sepsis and septic shock. The present study demonstrated that interleukin (IL)-31 was able reduce the mortality rate of lipopolysaccharide (LPS)-induced sepsis with the reduction of inflammatory cytokines in the sera. IL-31 also inhibited IL-1β production in the peritoneal lavage fluid in LPS-induced or cecal ligation and puncture-induced sepsis. The in vitro mechanism responsible for IL-31 regulation on peritoneal IL-1β activation following LPS challenge was explored. It was demonstrated that IL-1β secretion was suppressed by IL-31 treatment from LPS-challenged peritoneal macrophages following adenosine triphosphate stimulation, which is an activator of NLR family, pyrin domain-containing 3 (NLRP3). Furthermore, IL-31 inhibited the expression of NLRP3 at the transcriptional level. In human THP-1 cells, anti-IL-31/anti-IL-31 receptor (R) neutralizing antibody enhanced NLRP3 expression as well as IL-1β activation, suggesting a role of the IL-31-IL-31R-NLRP3-IL-1β signaling axis in the physiological status of sepsis. On the other hand, IL-31 displayed a negative effect on the NLRP1 inflammasome, but not on NLRP3 on the LPS-primed human peripheral blood monocytes, resulting in reduction of the inflammatory cytokine, tumor necrosis factor (TNF)-α, in the supernatant. Taken together, the present data implied that T helper 2-type cytokine, IL-31, may be a promising therapeutic option for treatment of sepsis and septic shock in clinics.

Variations in Cellular Responses of Mouse T Cells to Adenosine-5'-Triphosphate Stimulation Do Not Depend on P2X7 Receptor Expression Levels but on Their Activation and Differentiation Stage.

A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5′-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RBlow Tconvs, but not CD45RBhigh Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlow Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44low to effector/memory CD45RBlowCD44high stage. Maximum levels of upregulation are reached on recently activated CD69+ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+ CD45RBhighCD44low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RBhighCD44low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlow Tconvs, CD45RBlowFoxp3+ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.

P2Y12 Receptors in Tumorigenesis and Metastasis

Platelets, beyond their role in hemostasis and thrombosis, may sustain tumorigenesis and metastasis. These effects may occur via direct interaction of platelets with cancer and stromal cells and by the release of several platelet products. Platelets and tumor cells release several bioactive molecules among which a great amount of adenosine triphosphate (ATP) and adenosine diphosphate (ADP). ADP is also formed extracellularly from ATP breakdown by the ecto-nucleoside-triphosphate-diphosphohydrolases. Under ATP and ADP stimulation the purinergic P2Y1 receptor (R) initiates platelet activation followed by the ADP-P2Y12R-mediated amplification. P2Y12R stimulation amplifies also platelet response to several platelet agonists and to flow conditions, acting as a key positive feed-forward signal in intensifying platelet responses. P2Y12R represents a potential target for an anticancer therapy due to its involvement in platelet-cancer cell crosstalk. Thus, P2Y12R antagonists, including clopidogrel, ticagrelor, and prasugrel, might represent potential anti-cancer agents, in addition to their role as effective antithrombotic drugs. However, further studies, in experimental animals and patients, are required before the recommendation of the use of P2Y12R antagonists in cancer prevention and progression can be made.

The Antioxidant Procyanidin Reduces Reactive Oxygen Species Signaling in Macrophages and Ameliorates Experimental Colitis in Mice.

Management of inflammatory bowel disease (IBD) is a real clinical challenge. Despite intense investigation, the mechanisms of IBD remain substantially unidentified. Some inflammatory conditions, such as matrix metalloproteinases (MMPs) and the nuclear factor-κB (NF-κB) and NOD-like receptor protein 3 (NLRP3) inflammasome signaling pathways, are reported to contribute to the development and maintenance of IBD. Regulation of their common upstream signaling, that is, reactive oxygen species (ROS), may be important to control the progression of IBD. In the present study, we found that procyanidin, a powerful antioxidation flavonoid, has a significant effect on ROS clearance on THP-1 macrophages after lipopolysaccharide (LPS) or LPS-combined adenosine triphosphate stimulation, thus downregulating MMP9 expression, suppressing NF-κB signaling, and interrupting the formation of the NLRP3 inflammasome. Moreover, our in vivo data showed that procyanidin attenuated Dextran sulfate sodium-induced experimental colitis in a dose-dependent fashion by suppressing the expression of MMP9, NF-κB, and NLRP3 inflammasome signaling in colonic tissues in mice. Overall, our results suggested that targeting ROS could be a potential therapeutic choice for colonic inflammation.

Periodically Self-Pulsating Microcapsule as Programmed Microseparator via ATP-Regulated Energy Dissipation.

Living systems can experience time‐dependent dynamic self‐assembly for periodic, adaptive behavior via energy dissipation pathway. Creating in vitro mimics is a daunting mission. Here a “living” giant vesicle system that can perform a periodic pulsating motion using adenosine‐5'‐triphosphate (ATP)‐fuelled dissipative self‐assembly is described. This dynamic system is built on transient supramolecular interactions between the polymer and cellular energy currency ATP. The vesicles capturing ATPs will deviate away from equilibrium, leading to an energy ascent that drives a continuous vesicular expansion, until a competitive ATP hydrolysis predominates to break the ATP–polymer interactions and deplete the energy stored in the vesicles, leading to an opposing vesicular contraction. The input of ATP energy can sustain that these vesicles run periodically along this reciprocating expansile–contractile process, resembling a “pulsating” behavior. ATP level can orchestrate the rhythm, amplitude, and lifetime of this biomimetic pulsation. By pre‐programming the ATP stimulation protocol, this kind of adaptive microcapsules can function as high‐performance microseparators to perform size‐selective sieving of different nanoparticles through ATP‐mediated transmembrane traffic. This man‐made system offers a primitive model of time‐dependent dynamic self‐assembly and may offer new ways to build life‐like materials with biomimetic functions.

Analysis of differential expression of tight junction proteins in cultured oral epithelial cells altered by Porphyromonas gingivalis, Porphyromonas gingivalis lipopolysaccharide, and extracellular adenosine triphosphate

Tight junctions (TJs) are the most apical intercellular junctions of epithelial cells formed by occludin, claudins, junctional adhesion molecules (JAMs), and zonula occludens (ZO). Tight junction proteins can sense the presence of bacteria and regulate the transcription of target genes that encode effectors and regulators of the immune response. The aim of this study was to determine the impact of TJ proteins in response to Porphyromonas gingivalis (P. gingivalis), P. gingivalis lipopolysaccharide (P. gingivalis LPS), and extracellular adenosine triphosphate (ATP) in the oral epithelial cell culture model. Quantified real time-polymerase chain reaction (RT-PCR), immunoblots, and immunostaining were performed to assess the gene and protein expression in TJs. It was found that P. gingivalis infection led to transient upregulation of the genes encoding occludin, claudin-1, and claudin-4 but not JAM-A, claudin-15, or ZO-1, while P. gingivalis LPS increased claudin-1, claudin-15, and ZO-1 and decreased occludin, JAM-A, and claudin-4. Tight junction proteins showed significant upregulation in the above two groups when cells were pretreated with ATP for 3 h. The findings indicated that P. gingivalis induced the host defence responses at an early stage. P. gingivalis LPS exerted a more powerful stimulatory effect on the disruption of the epithelial barrier than P. gingivalis. ATP stimulation enhanced the reaction of TJ proteins to P. gingivalis invasion and LPS destruction of the epithelium.