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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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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DOI: https://doi.org/10.1007/s00424-004-1293-2