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Stimulation of Xenopus P2Y1 receptor activates CFTR in A6 cells

  • Ion Channels, Transporters
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Abstract

Nucleotide binding to purinergic P2Y receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Here, we investigate the regulatory mechanism of the P2Y1 receptor agonist 2-methylthioadenosine diphosphate (2-MeSADP) on Cl transport in A6 cells, a commonly used model of the distal section of the Xenopus laevis nephron. Protein and mRNA expression analysis together with functional measurements demonstrated the basolateral location of the Xenopus P2Y1 receptor. 2-MeSADP increased intracellular [Ca2+] and cAMP and Cl efflux, responses that were all inhibited by the specific P2Y1 receptor antagonist MRS 2179. Cl efflux was also inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Inhibition of either protein kinase A (PKA) or the binding between A-kinase-anchoring proteins (AKAPs) and the regulatory PKA RII subunit blocked the 2-MeSADP-induced activation of CFTR, suggesting that PKA mediates P2Y1 receptor regulation of CFTR through one or more AKAPs. Further, the truncation of the PDZ1 domain of the scaffolding protein Na+/H+ exchanger regulatory factor-2 (NHERF-2) inhibited 2-MeSADP-dependent stimulation of Cl efflux, suggesting the involvement of this scaffolding protein. Activation or inhibition of PKC had no effect per se on basal Cl efflux but potentiated or reduced the 2-MeSADP-dependent stimulation of Cl efflux, respectively. These data suggest that the X. laevis P2Y1 receptor in A6 cells can increase both cAMP/PKA and Ca2+/PKC intracellular levels and that the PKC pathway is involved in CFTR activation via potentiation of the PKA pathway.

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Acknowledgements

This work was supported by grants from: Telethon, Italy, grant E.1125; the Italian Cystic Fibrosis Research Foundation and CEGBA (Centro di Eccellenza di Genomica in Campo Biomedico ed Agrario). We thank Dr. Pann-Ghill Suh of Life Science and School of Environmental Engineering, Pohang University of Science and Tecnology, Pohang 790-784, South Korea, for the kind gift of NHERF-2-ΔPDZ1.

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Correspondence to V. Casavola.

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Guerra, L., Favia, M., Fanelli, T. et al. Stimulation of Xenopus P2Y1 receptor activates CFTR in A6 cells. Pflugers Arch - Eur J Physiol 449, 66–75 (2004). https://doi.org/10.1007/s00424-004-1293-2

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