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EDITORIAL FOCUS
Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
IN RECENT YEARS evidence has emerged that the Na-K-ATPase, a canonical active ion pump, has parallel roles as a platform for cell signaling. Na-K-ATPase has been shown to associate directly with other proteins in signaling complexes in cardiac myocytes, renal cells, and several other cell types (4). Signals transmitted through the Na-K-ATPase and associated Src have been implicated in hypertrophy, proliferation or stasis, and resistance to apoptotic events. The signaling variously activates phopholipase C, Akt, epidermal growth factor receptor, and reactive oxygen species, and signaling by interaction with inositol-1,4,5-trisphosphate receptor has been implicated in Ca2+ oscillations and nuclear factor-
B activation (1) All of these signaling events occur with the specific inhibitor ouabain, a cardiac glycoside. Cardiac glycosides and bufadienolides, the only well-characterized extracellular ligands of the Na-K-ATPase, were originally discovered as plant and amphibian toxins, but a body of evidence shows that some function as endogenous regulators in mammals (8). In many investigations of ouabain-mediated signaling, a principal experimental readout has been detection of activated (phosphorylated) mitogen-activated protein kinase ERK1/2.
Ouabain-elicited signaling has been viewed by some investigators as occurring at concentrations too low to inhibit the Na-K-ATPase, but this needs to be understood in a physiological context. From biochemical studies, ouabain is not known to interact with purified Na-K-ATPase without inhibiting it, but effective signaling may require the occupancy of only a fraction of the Na-K-ATPase present in a cell (6). In intact cells, ouabain at very low concentrations has been shown to activate trafficking of other transporters, and in some cases, it actually increases measured Na-K-ATPase activity (2, 8). There is evidence for separate (and slowly interconvertible) pools of Na-K-ATPase with pumping or signaling functions and different locations or different associated proteins (5). Thus, in some experimental paradigms, the signaling effect of ouabain is amplified relative to its effect on ion transport. In many other studies, however, signaling events have been investigated at high enough concentrations of ouabain to inhibit a substantial fraction of the Na-K-ATPase. The higher the ouabain concentration used, the more intracellular signaling pathways may be called into play.
In a previous paper, Plourde and Soltoff (7) observed in parotid acinar cells that the phosphorylation of ERK1/2 in response to muscarinic cholinergic agonists was potentiated by treatment of the cells with ouabain at concentrations that do inhibit the enzyme. Ouabain treatment by itself, however, without concomitant muscarinic cholinergic receptor activation, caused a much more limited phosphorylation of ERK1/2. In this way parotid acinar cells were different from the tissues and cells where ouabain alone robustly activated ERK1/2. A salient observation was that blocking Na-K-ATPase by eliminating extracellular K+ resulted in ERK1/2 phosphorylation similar to that with 10–5 M carbachol. This suggested that it was inhibition of Na-K-ATPase activity, not necessarily signaling, that affected ERK1/2 activation in parotid cells. Activated ERK1/2 also played a positive role in modulating Na-K-ATPase activity, suggesting bidirectional interactions.
In a new paper published by Soltoff and Hedden (9), carbachol was shown to cause phosphorylation and activation of AMPK, the AMP-activated, energy state-sensing kinase (9) (Fig. 1) . Concomitant treatment with ouabain blocked the phosphorylation of AMPK. This is consistent with the idea that the muscarinic cholinergic activation of the Na-K-ATPase results in enough hydrolysis of ATP to elevate the AMP-to-ATP ratio (presumably assisted by adenylate kinase to redistribute the ATPase product ADP to ATP and AMP). To test whether activated AMPK regulates ERK1/2 phosphorylation, the investigators used 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), a cell-permeable AMP mimetic, and found that it reduced the ERK1/2 stimulation seen not only in response to carbachol but also in response to another agent that stimulates the Na-K-ATPase, a purinergic agonist. AICAR did not affect ERK1/2 phosphorylation in response to the Src-signaling growth factor EGF, which is independent of Na-K-ATPase activity. AICAR treatment also blocked, or rather bypassed, the effect of ouabain, as predicted since it was used to mimic the elevated hydrolysis of ATP that ouabain inhibits. Consistent with this model, measured ATP levels were markedly reduced by carbachol and protected by ouabain. All of this was seen in freshly dissociated parotid acinar cells but not in a cell line derived from parotid cells. This cell line proved to have lower Na-K-ATPase activity, lower LKB1, the kinase that normally phosphorylates AMPK in energy signaling, and no detectable reduction in ATP concentration in response to carbachol. Instead, the cell line appeared to lack these metabolic features of the highly secretory native parotid acinar cell.
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2. Khundmiri SJ, Amin V, Henson J, Lewis J, Ameen M, Rane MJ, Delamere NA. Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway. Am J Physiol Cell Physiol 293: C1171–C1180, 2007.
3. Lee MY, Song H, Nakai H, Ohkura M, Kotlikoff MI, Kinsey SP, Golovina VA, Blaustein MP. Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor. Proc Natl Acad Sci USA 102: 13232–13237, 2006.
4. Li Z, Xie Z. The Na/K-ATPase/Src complex and cardiotonic steroid-activated protein kinase cascades. Pfluegers Arch. In press.
5. Liang M, Tian J, Liu L, Pierre S, Liu J, Shapiro J, Xie ZJ. Identification of a pool of non-pumping Na/K-ATPase. J Biol Chem 282: 10585–10593, 2007.
6. Liu L, Askari A. On the importance and mechanism of amplification of digitalis signal through Na+/K+-ATPase. Cell Mol Biol (Noisy-le-grand) 52: 28–30, 2006.[Medline]
7. Plourde D, Soltoff SP. Ouabain potentiates the activation of ERK1/2 by carbachol in parotid gland epithelial cells; inhibition of ERK1/2 reduces Na+-K+-ATPase activity. Am J Physiol Cell Physiol 290: C702–C710, 2006.
8. Schoner W, Scheiner-Bobis G. Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth. Am J Physiol Cell Physiol 293: C509–C536, 2007.
9. Soltoff SP, Hedden L. 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 (July 16, 2008). doi:10.1152/ajpcell.00140.2008.
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