Na,K-ATPase as a signal transducer.

Recent studies have indicated that Na,K-ATPase may, in addition to being the key regulator of intracellular Na(+) and K(+) concentration, act as a signal transducer. Despite extensive research, the biological role for ouabain, a natural ligand of Na,K-ATPase, is not well understood. We have reported that exposure of rat proximal tubular cells (RPTC) to doses of ouabain that inhibit the Na,K-ATPase activity by less than 50% (10 nM - 500 micro M), will induce intracellular [Ca(2+)](i) oscillations and that this calcium signal leads to activation of the transcription factor NF-kappaB. The ouabain-induced calcium oscillations were blocked by an inhibitor of the IP(3) receptors but not by phospholipase C inhibitors nor by cellular depletion of IP(3), suggesting that the calcium signal is not due to phospholipase C-mediated IP(3) release. Fluorescence resonance energy transfer (FRET) studies suggested a close proximity between the Na,K-ATPase and IP(3) receptor. Our findings demonstrate a novel principle for calcium signaling via Na,K-ATPase.

Low doses of ouabain protect from serum deprivation-triggered apoptosis and stimulate kidney cell proliferation via activation of NF-kappaB.

It now generally is agreed that Na,K-ATPase, in addition to its role in the maintenance of Na+ and K+ gradients across the cell membrane, plays a role in communicating information from the extracellular environment to intracellular signaling pathways. It was reported recently that interaction between ouabain-bound Na,K-ATPase and the 1,4,5-trisphosphate receptor (IP3R) triggers slow calcium oscillations and activation of NF-kappaB. Here it is demonstrated that this signaling pathway can serve to prevent cell death and promote cell growth. Rat renal proximal tubular cells in primary culture first were grown in the presence of 10% serum and then exposed to 0.2% serum for 24 h to induce apoptosis. Serum starvation increased the apoptotic index from 1.21 +/- 0.26 to 14.01 +/- 1.17%. Ouabain in concentrations that did not inhibit Na,K-ATPase activity (1 to 10 nM) completely abolished the apoptotic effect of serum starvation. Ouabain protection from apoptosis was not observed when release of calcium from intracellular stores via the IP3R was prevented. It was shown that the NH2 terminal tail of the Na,K-ATPase alpha subunit plays a key role in ouabain-triggered calcium oscillations. It was found that ouabain did not protect from apoptosis in serum-deprived cells that expressed a mutant Na,K-ATPase alpha subunit with deletion of the NH2 terminal tail. Ouabain exposure (10 nM for 24 h) significantly increased translocation of NF-kappaB from cytoplasm to nucleus. Helenalin, an inhibitor of NF-kappaB, abolished the antiapoptotic effect of ouabain. Ouabain (0.1 to 10 nM) also was found to stimulate proliferation and increase the viability of kidney cells. These effects were abolished when release of calcium via the IP3R was prevented.

Distinct role of the N-terminal tail of the Na,K-ATPase catalytic subunit as a signal transducer.

Mounting evidence suggests that the ion pump, Na,K-ATPase, can, in the presence of ouabain, act as a signal transducer. A prominent binding motif linking the Na,K-ATPase to intracellular signaling effectors has, however, not yet been identified. Here we report that the N-terminal tail of the Na,K-ATPase catalytic alpha-subunit (alphaNT-t) binds directly to the N terminus of the inositol 1,4,5-trisphosphate receptor. Three amino acid residues, LKK, conserved in most species and most alpha-isoforms, are essential for the binding to occur. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-kappaB and protect from apoptosis. All of these effects are suppressed in cells overexpressing a peptide corresponding to alphaNT-t but not in cells overexpressing a peptide corresponding to alphaNT-t deltaLKK. Thus we have identified a well conserved Na,K-ATPase motif that binds to the inositol 1,4,5-trisphosphate receptor and can trigger an anti-apoptotic calcium signal.

Changes in Na(+)-K(+)-ATPase activity influence cell attachment to fibronectin.

Most vital cellular functions are dependent on a fine-tuned regulation of intracellular ion homeostasis. Here we have demonstrated, using COS cells that were untransfected or transfected with wild-type rat ouabain-resistant Na(+)-K(+)-ATPase, that partial inhibition of Na(+)-K(+)-ATPase has a dramatic influence on cell attachment to fibronectin. Ouabain dose-dependently decreased attachment in untransfected cells and in cells expressing wild-type Na(+)-K(+)-ATPase, but not in cells expressing ouabain-insensitive Na(+)-K(+)-ATPase, whereas inhibition of Na(+)-K(+)-ATPase by lowering extracellular K(+) concentration decreased attachment in all three cell types. Thirty percent inhibition of Na(+)-K(+)-ATPase significantly attenuated attachment. Na(+)-K(+)-ATPase inhibition caused a sustained increase in the intracellular Ca(2+) concentration that obscured Ca(2+) transients observed in untreated cells during attachment. Inhibitors of Ca(2+) transporters significantly decreased attachment, but inhibition of Na(+)/H(+) exchanger did not. Ouabain reduced focal adhesion kinase autophosphorylation but had no effect on cell surface integrin expression. These results suggest that the level of Na(+)-K(+)-ATPase activity strongly influences cell attachment, possibly by an effect on intracellular Ca(2+).

Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations.

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.