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Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells.

ABSTRACT: BACKGROUND AND PURPOSE: P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs). EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools. KEY RESULTS: RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1-1000 microM) to cells clamped at -60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by alphabetamethylene ATP (10 microM, 94% cells, n= 31) and was antagonized in LAD2 cells by NF 449 (1 microM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1-10 microM). A P2X7-like non-desensitizing current in response to high concentrations of ATP (1-5 mM) was also seen in both cell types (96% LAD2, n= 24; 54% HLMCs, n= 24) which was antagonized by AZ11645373 (1 microM). P2X7-like responses were also evoked in LAD2 cells by 2'(3')-0-(4-benzoylbenzoyl)ATP (300 microM). A P2X4-like current was evoked by 100 microM ATP (80% LAD2, n= 10; 21% HLMCs, n= 29), the amplitude and duration of which was potentiated by ivermectin (3 microM). CONCLUSION AND IMPLICATIONS: Our data confirmed the presence of functional P2X1, P2X4 and P2X7 receptors in LAD2 cells and HLMCs.

SUBMITTER: Wareham K

PROVIDER: S-EPMC2743840 | biostudies-other | 2009 Aug

REPOSITORIES: biostudies-other

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Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells.

Wareham K K Vial C C Wykes R C E RC Bradding P P Seward E P EP

British journal of pharmacology 20090622 7

<h4>Background and purpose</h4>P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs).<h4>Experimental approach</h4>Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools.<h4>Key results</h4>RT-PCR revealed P2X1, P2X4 and ...[more]

PMID: 19552691

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Project description:Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen activated protein kinase. These findings support a novel role for P2X7 and P2X4 coupled to induction of inflammatory molecules in modulating high glucose and palmitate-induced endothelial cell activation and dysfunction.

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.

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Project description:Endometriosis is an inflammatory disease characterized by the presence of ectopic endometrial tissue outside the uterus. A diffuse infiltration of mast cells (MCs) is observed throughout endometriotic lesions, but little is known about how these cells contribute to the network of molecules that modulate the growth of ectopic endometrial implants and promote endometriosis-associated inflammation. The aryl hydrocarbon receptor (AhR), a transcription factor known to respond to environmental toxins and endogenous compounds, is present in MCs. In response to AhR activation, MCs produce IL-17 and reactive oxygen species, highlighting the potential impact of AhR ligands on inflammation via MCs. Here, we investigated the possibility that endometrial MCs promote an inflammatory microenvironment by sensing AhR ligands, thus sustaining endometriosis development. Using human endometriotic tissue (ET) samples, we performed the following experiments: (i) examined the cytokine expression profile; (ii) counted AhR-expressing MCs; (iii) verified the phenotype of AhR-expressing MCs to establish whether MCs have a tolerogenic (IL-10-positive) or inflammatory (IL-17-positive) phenotype; (iv) measured the presence of AhR ligands (tryptophan-derived kynurenine) and tryptophan-metabolizing enzymes (indoleamine 2,3-dioxygenase 1 (IDO1)); (v) treated ET organ cultures with an AhR antagonist in vitro to measure changes in the cytokine milieu; and (vi) measured the growth of endometrial stromal cells cultured with AhR-activated MC-conditioned medium. We found that ET tissue was conducive to cytokine production, orchestrating chronic inflammation and a population of AhR-expressing MCs that are both IL-17 and IL-10-positive. ET was rich in IDO1 and the AhR-ligand kynurenine compared with control tissue, possibly promoting MC activation through AhR. ET was susceptible to treatment with an AhR antagonist, and endometrial stromal cell growth was improved in the presence of soluble factors released by MCs on AhR activation. These results suggest a new mechanistic role of MCs in the pathogenesis of endometriosis.

Dataset Information

Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells.

Publications

Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells.

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