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 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.
Guerra
L
L
Department of General and Environmental Physiology, University of Bari, Via Amendola 165/A, 70126 Bari, Italy.
Favia
M
M
Fanelli
T
T
Calamita
G
G
Svetlo
M
M
Bagorda
A
A
Jacobson
K A
KA
Reshkin
S J
SJ
Casavola
V
V
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005905:Glyburide / Q000378:metabolism; D007213:Indomethacin / Q000378:metabolism; D007546:Isoquinolines / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology; D010750:Phosphoproteins / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism; D014982:Xenopus laevis
D002712:Chlorides; D007546:Isoquinolines; C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; D010750:Phosphoproteins; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides; D013873:Thionucleotides; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C002480:methylthio-ADP; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D005905:Glyburide; D002118:Calcium; D007213:Indomethacin