Vasopressin regulates the phosphorylation state of AMP-activated protein kinase (AMPK) in MDCK-C7 cells.
AMP-activated protein kinase (AMPK) is a regulatory kinase coupling cellular metabolism with ion transport. Madin-Darby Canine Kidney-Clone 7 (MDCK-C7) cells possess characteristics of the renal principal cell type, express the cystic fibrosis transmembrane regulator and the epithelial Na(+) channel, and display NPPB and amiloride-sensitive transepithelial transport when stimulated with [Arg(8)]-vasopressin. [Arg(8)]-vasopressin binding to its receptor on the basolateral membrane of MDCK-C7 results in cAMP production, activation of cAMP-dependent protein kinase A (PKA), and increases in Cl(-) and Na(+) transport. Ussing-style electrophysiology showed that the PKA inhibitor, H89, blocked Cl(-) and Na(+) transport. Unexpectedly, [Arg(8)]-vasopressin stimulation resulted in the dephosphorylation of pAMPK(thr172). H89 did not prevent this, suggesting that the dephosphorylation is independent of PKA. 24 hour, but not 15 minute, incubation with the AMPK activator, AICAR, also blocked [Arg(8)]-vasopressin-stimulated currents. Contrary to previous studies, immunoblotting revealed that AICAR did not increase abundance of the active, phosphorylated form of AMPK (pAMPK(thr172)); although, AICAR treatment significantly blocked [Arg(8)]-vasopressin -stimulated cAMP production. [Arg(8)]-vasopressin still caused pAMPK(thr172) dephosphorylation in the presence of AICAR, suggesting that this effect is also independent of cAMP. In summary, these data suggest [Arg(8)]-vasopressin regulates AMPK phosphorylation and that AICAR inhibits ion transport independently of AMPK in MDCK-C7 cells.
Nofziger
Charity
C
Department of Biology, Indiana University, Indianapolis, USA.
Kalsi
Kameljit
K
West
T Aaron
TA
Baines
Deborah
D
Blazer-Yost
Bonnie L
BL
D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004285:Dogs; D053503:Epithelial Sodium Channels / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D009711:Nucleotides / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010539:Permeability / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D012265:Ribonucleotides / Q000494:pharmacology; D013997:Time Factors
D002712:Chlorides; D053503:Epithelial Sodium Channels; D009711:Nucleotides; D012265:Ribonucleotides; D001127:Arginine Vasopressin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D019319:Okadaic Acid; D000620:Aminoimidazole Carboxamide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide