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".

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 Ca oscillations and nuclear factorB 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-KATPase, 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) mitogenactivated 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 NaK-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 Address for reprint requests and other correspondence: K. J. Sweadner, Thier 415, 55 Fruit St., Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 (e-mail: [email protected]). Fig. 1. Three basic ways the Na-K-ATPase participates in signaling. 1) Na gradient established by the Na-K-ATPase is used to drive Na /Ca2 exchange by NCX, the Na /Ca2 exchanger. By inhibiting the Na-K-ATPase, ouabain reduces the Na gradient and less Ca2 is driven out of the cell. This is the simplest form of Ca2 modulation by Na-K-ATPase activity, and recent research has revealed much more detail and regulatory complexity than suggested in the diagram. 2) Ouabain binds to the Na-K-ATPase and activates Src interacting with the pump in a signaling complex. The Na-K-ATPase can also interact with inositol 1,4,5 trisphosphate receptor (not shown). Again, much more detail is known than shown in the diagram. The activation of ERK1/2 by phosphorylation is an easily measured response to ouabain signalplex formation. 3) It is the hydrolysis of ATP by Na-K-ATPase that activates AMPK in stimulated salivary acinar cells. Activated AMPK then brings about a reduction in the phosphorylation of ERK1/2. By inhibiting the Na-KATPase, ouabain blocks this process and results in a higher level of activation of ERK1/2. Although the final result resembles the events in 2, the mechanism is different. Am J Physiol Cell Physiol 295: C588–C589, 2008; doi:10.1152/ajpcell.00388.2008.