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RECEPTORS AND SIGNAL TRANSDUCTION

Regulation of ERK1/2 by ouabain and Na-K-ATPase-dependent energy utilization and AMPK activation in parotid acinar cells

Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts

Submitted 31 March 2008 ; accepted in final form 21 July 2008

We previously found that the phosphorylation of ERK1/2 by submaximal concentrations of the muscarinic receptor ligand carbachol was potentiated in rat parotid acinar cells exposed to ouabain, a cardiac glycoside that inhibits the Na-K-ATPase. We now report that this signaling phenomenon involves the prevention of negative regulation of extracellular signal-regulated kinase-1/2 (ERK1/2) that is normally mediated by AMP-activated protein kinase (AMPK). Carbachol increases the turnover of the ATP-consuming Na-K-ATPase, reducing intracellular ATP and promoting the phosphorylation/activation of the energy sensor AMPK. Ouabain blocks the reduction in ATP and subsequent AMPK phosphorylation, which is regulated by the AMP-to-ATP ratio. The ouabain-promoted enhancement of ERK1/2 phosphorylation was not reproduced in Par-C10 cells, an immortalized rat parotid cell line that did not respond to carbachol with an ATP reduction and that employs an upstream AMPK kinase (Ca²⁺/calmodulin-dependent protein kinase kinase, CaMKK) different from that (LKB1) in native cells. In native parotid cells, inhibitory effects of AMPK on ERK1/2 signaling were examined by activating AMPK with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which is converted to an AMP mimetic but does not alter parotid ATP levels. AICAR-treated cells display increases in AMPK phosphorylation and a reduced phosphorylation of ERK1/2 subsequent to activation of muscarinic and P2X₇ receptors, which promote increases in Na-K-ATPase turnover, but not upon epidermal growth factor receptor activation. These results suggest that carbachol-initiated AMPK activation can produce a negative feedback on ERK1/2 signaling in response to submaximal muscarinic receptor activation and that increases in fluid secretion can modulate receptor-initiated signaling events indirectly by producing ion transport-dependent decreases in ATP.

adenosine 5'-triphosphatase; calmodulin-dependent protein kinase kinase; 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; carbachol; 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate

This article has been cited by other articles:

				K. J. Sweadner A third mode of ouabain signaling. Focus on "Regulation of ERK1/2 by ouabain and Na-K-ATPase-dependent energy utilization and AMPK activation in parotid acinar cells" Am J Physiol Cell Physiol, September 1, 2008; 295(3): C588 - C589. [Full Text] [PDF]