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Large-conductance channel formation mediated by P2X7 receptor activation is regulated through distinct intracellular signaling pathways in peritoneal macrophages and 2BH4 cells

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Abstract

The P2X7 receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X7 receptor is induced by increasing the intracellular Ca2+ concentration (Faria et al., Am J Physiol Cell Physiol 297:C28–C42, 2005). Here, we investigated the intracellular signaling pathways associated with P2X7 large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca2+-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X7 receptor large-channel formation in 2BH4 cells and peritoneal macrophages.

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Acknowledgements

We would like to thank Dr. Andrea Henrique Pons (IOC/FIOCRUZ) for helping us in the initial phase of this work. This work was supported by grants from FAPERJ, PAPES V/CNPq, and the Oswaldo Cruz Institute.

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Authors and Affiliations

  1. Laboratory of Cellular Communication, Department of Immunology, Oswaldo Cruz Institute, FIOCRUZ (Oswaldo Cruz Foundation), Av. Brazil, 4365, Manguinhos, Rio de Janeiro, 21045-900, Brazil

    R. X. Faria & Luiz Anastácio Alves

  2. Laboratory of Cell Biology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil

    C. M. Cascabulho

  3. Program in Neurobiology, Institute of Biophysics, UFRJ, Rio de Janeiro, Brazil

    R. A. M. Reis

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  1. R. X. Faria
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  2. C. M. Cascabulho
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  3. R. A. M. Reis
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  4. Luiz Anastácio Alves
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Correspondence to R. X. Faria.

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Faria, R.X., Cascabulho, C.M., Reis, R.A.M. et al. Large-conductance channel formation mediated by P2X7 receptor activation is regulated through distinct intracellular signaling pathways in peritoneal macrophages and 2BH4 cells. Naunyn-Schmied Arch Pharmacol 382, 73–87 (2010). https://doi.org/10.1007/s00210-010-0523-8

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