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Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

ABSTRACT: Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (Fc?RI) and trigger type I allergic reactions. Fc?RI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced Fc?RI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.

SUBMITTER: Yoshida K

PROVIDER: S-EPMC6829402 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Yoshida Kazuki K Tajima Makoto M Nagano Tomoki T Obayashi Kosuke K Ito Masaaki M Yamamoto Kimiko K Matsuoka Isao I

International journal of molecular sciences 20191017 20

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degran ...[more]

PMID: 31627451

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Project description:Extracellular nucleotides have been recognized as important mediators of activation, triggering multiple responses via plasma membrane receptors known as P2 receptors. P2 receptors comprise P2X ionotropic receptors and G protein-coupled P2Y receptors. P2X receptors are expressed in many tissues, where they are involved in a number of functions including synaptic transmission, muscle contraction, platelet aggregation, inflammation, macrophage activation, differentiation and proliferation, neuropathic and inflammatory pain. P2X4 is one of the most sensitive purinergic receptors (at nanomolar ATP concentrations), about one thousand times more than the archetypal P2X7. P2X4 is widely expressed in central and peripheral neurons, in microglia, and also found in various epithelial tissues and endothelial cells. It localizes on the plasma membrane, but also in intracellular compartments. P2X4 is preferentially localized in lysosomes, where it is protected from proteolysis by its glycosylation. High ATP concentration in the lysosomes does not activate P2X4 at low pH; P2X4 gets activated by intra-lysosomal ATP only in its fully dissociated tetra-anionic form, when the pH increases to 7.4. Thus, P2X4 is functioning as a Ca2+-channel after the fusion of late endosomes and lysosomes. P2X4 modulates major neurotransmitter systems and regulates alcohol-induced responses in microglia. P2X4 is one of the key receptors mediating neuropathic pain. However, injury-induced upregulation of P2X4 expression is gender dependent and plays a key role in pain difference between males and females. P2X4 is also involved in inflammation. Extracellular ATP being a pro-inflammatory molecule, P2X4 can trigger inflammation in response to high ATP release. It is therefore involved in multiple pathologies, like post-ischemic inflammation, rheumatoid arthritis, airways inflammation in asthma, neurodegenerative diseases and even metabolic syndrome. Although P2X4 remains poorly characterized, more studies are needed as it is likely to be a potential therapeutic target in these multiple pathologies.

Project description:P2X4 and P2X7 are members of the P2X receptor family, comprising seven isoforms (P2X1-P2X7) that form homo- and heterotrimeric non-specific cation channels gated by extracellular ATP. P2X4 and P2X7 are widely coexpressed, particularly in secretory epithelial cells and immune and inflammatory cells, and regulate inflammation and nociception. Although functional heteromerization has been established for P2X2 and P2X3 subunits expressed in sensory neurons, there are contradictory reports regarding a functional interaction between P2X4 and P2X7 subunits. To resolve this issue, we coexpressed P2X4 and P2X7 receptor subunits labeled with green (EGFP) and red (TagRFP) fluorescent proteins in Xenopus laevis oocytes and investigated a putative physical interaction between the fusion proteins by Förster resonance energy transfer (FRET). Coexpression of P2X4 and P2X7 subunits with EGFP and TagRFP located in the extracellular receptor domains led to significant FRET signals. Significant FRET signals were also measured between C-terminally fluorophore-labeled full-length P2X41-384 and C-terminally truncated fluorescent P2X71-408 subunits. We furthermore used the two-electrode voltage clamp technique to investigate whether human P2X4 and P2X7 receptors (hP2X4, hP2X7) functionally interact at the level of ATP-induced whole-cell currents. Concentration-response curves and effects of ivermectin (P2X4-potentiating drug) or BzATP (P2X7-specific agonist) were consistent with a model in which coexpressed hP2X4 and hP2X7 do not interact. Similarly, the effect of adding specific inhibitors of P2X4 (PSB-15417) or P2X7 (oATP, A438079) could be explained by a model in which only homomers exist, and that these are blocked by the respective antagonist. In conclusion, we show that P2X4 and P2X7 subunits can form heterotrimeric P2X4/P2X7 receptors. However, unlike observations for P2X2 and P2X3, coexpression of P2X4 and P2X7 subunits does not result in a novel electrophysiologically discriminable P2X receptor phenotype.

Dataset Information

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Publications

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

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