Stimulation of Xenopus P2Y1 receptor activates CFTR in A6 cells

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 comm...

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Bibliographic Details
Published in:	Pflügers Archiv Vol. 449; no. 1; pp. 66 - 75
Main Authors:	Guerra, L, Favia, M, Fanelli, T, Calamita, G, Svetlo, M, Bagorda, A, Jacobson, K A, Reshkin, S J, Casavola, V
Format:	Journal Article
Language:	English
Published:	Germany Springer Nature B.V 01-10-2004
Subjects:	Adenosine Diphosphate - analogs & derivatives Adenosine Diphosphate - metabolism Animals Calcium - metabolism Cell Line Chlorides - metabolism Cyclic AMP - metabolism Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors Cyclic AMP-Dependent Protein Kinases - metabolism Cystic fibrosis Cystic Fibrosis Transmembrane Conductance Regulator - genetics Cystic Fibrosis Transmembrane Conductance Regulator - metabolism Epithelial Cells - cytology Epithelial Cells - metabolism Glyburide - metabolism Indomethacin - metabolism Isoquinolines - metabolism Kidney Tubules - cytology Kinases Phosphoproteins - metabolism Proteins Purinergic P2 Receptor Antagonists Receptors, Purinergic P2 - genetics Receptors, Purinergic P2 - metabolism Receptors, Purinergic P2Y1 Regulation Sodium-Hydrogen Exchangers Sulfonamides - metabolism Thionucleotides - metabolism Xenopus laevis
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0031-6768 1432-2013
DOI:	10.1007/s00424-004-1293-2