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P2X4 Receptor-Dependent Ca(2+) Influx in Model Human Monocytes and Macrophages
Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5′-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X re...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713231/ https://www.ncbi.nlm.nih.gov/pubmed/29077063 http://dx.doi.org/10.3390/ijms18112261 |
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author | Layhadi, Janice A. Fountain, Samuel J. |
author_facet | Layhadi, Janice A. Fountain, Samuel J. |
author_sort | Layhadi, Janice A. |
collection | PubMed |
description | Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5′-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X receptors) or by mobilizing intracellular calcium stores (metabotropic P2Y receptors). Here, a side-by-side comparison to investigate the contribution of P2X4 receptor activation in ATP-evoked calcium responses in model human monocytes and macrophages was performed. The expression of P2X1, P2X4, P2X5 and P2X7 was confirmed by qRT-PCR and immunocytochemistry in both model monocyte and macrophage. ATP evoked a concentration-dependent increase in intracellular calcium in both THP-1 monocyte and macrophages. The sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thasigargin (Tg) responses to the maximal ATP concentration (100 μM) in THP-1 monocytes, and responses in macrophage were significantly attenuated. Tg-resistant ATP-evoked calcium responses in the model macrophage were dependent on extracellular calcium, suggesting a requirement for calcium influx. Ivermectin (IVM) potentiated the magnitude of Tg-resistant component and slowed the decay of response in the model macrophage. The Tg-resistant component was attenuated by P2X4 antagonists 5-BDBD and PSB-12062 but not by the P2X1 antagonist Ro0437626 or the P2X7 antagonist A438079. shRNA-mediated P2X4 knockdown resulted in a significant reduction in Tg-resistant ATP-evoked calcium response as well as reduced sensitivities towards P2X4-specific pharmacological tools, IVM and PSB-12062. Inhibition of endocytosis with dynasore significantly reduced the magnitude of Tg-resistant component but substantially slowed decay response. Inhibition of calcium-dependent exocytosis with vacuolin-1 had no effect on the Tg-resistant component. These pharmacological data suggest that P2X4 receptor activation contributed significantly towards the ionotropic calcium response evoked by ATP of the model human macrophage. |
format | Online Article Text |
id | pubmed-5713231 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-57132312017-12-07 P2X4 Receptor-Dependent Ca(2+) Influx in Model Human Monocytes and Macrophages Layhadi, Janice A. Fountain, Samuel J. Int J Mol Sci Article Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5′-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X receptors) or by mobilizing intracellular calcium stores (metabotropic P2Y receptors). Here, a side-by-side comparison to investigate the contribution of P2X4 receptor activation in ATP-evoked calcium responses in model human monocytes and macrophages was performed. The expression of P2X1, P2X4, P2X5 and P2X7 was confirmed by qRT-PCR and immunocytochemistry in both model monocyte and macrophage. ATP evoked a concentration-dependent increase in intracellular calcium in both THP-1 monocyte and macrophages. The sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thasigargin (Tg) responses to the maximal ATP concentration (100 μM) in THP-1 monocytes, and responses in macrophage were significantly attenuated. Tg-resistant ATP-evoked calcium responses in the model macrophage were dependent on extracellular calcium, suggesting a requirement for calcium influx. Ivermectin (IVM) potentiated the magnitude of Tg-resistant component and slowed the decay of response in the model macrophage. The Tg-resistant component was attenuated by P2X4 antagonists 5-BDBD and PSB-12062 but not by the P2X1 antagonist Ro0437626 or the P2X7 antagonist A438079. shRNA-mediated P2X4 knockdown resulted in a significant reduction in Tg-resistant ATP-evoked calcium response as well as reduced sensitivities towards P2X4-specific pharmacological tools, IVM and PSB-12062. Inhibition of endocytosis with dynasore significantly reduced the magnitude of Tg-resistant component but substantially slowed decay response. Inhibition of calcium-dependent exocytosis with vacuolin-1 had no effect on the Tg-resistant component. These pharmacological data suggest that P2X4 receptor activation contributed significantly towards the ionotropic calcium response evoked by ATP of the model human macrophage. MDPI 2017-10-27 /pmc/articles/PMC5713231/ /pubmed/29077063 http://dx.doi.org/10.3390/ijms18112261 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Layhadi, Janice A. Fountain, Samuel J. P2X4 Receptor-Dependent Ca(2+) Influx in Model Human Monocytes and Macrophages |
title | P2X4 Receptor-Dependent Ca(2+) Influx in Model Human Monocytes and Macrophages |
title_full | P2X4 Receptor-Dependent Ca(2+) Influx in Model Human Monocytes and Macrophages |
title_fullStr | P2X4 Receptor-Dependent Ca(2+) Influx in Model Human Monocytes and Macrophages |
title_full_unstemmed | P2X4 Receptor-Dependent Ca(2+) Influx in Model Human Monocytes and Macrophages |
title_short | P2X4 Receptor-Dependent Ca(2+) Influx in Model Human Monocytes and Macrophages |
title_sort | p2x4 receptor-dependent ca(2+) influx in model human monocytes and macrophages |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713231/ https://www.ncbi.nlm.nih.gov/pubmed/29077063 http://dx.doi.org/10.3390/ijms18112261 |
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