The plant-derived steroid, digoxin, a specific inhibitor of Na,KATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium (

Despite the importance of dopamine signaling, it remains unknown if the two major subclasses of dopamine receptors exist on the same or distinct populations of neurons. Here we used confocal microscopy to demonstrate that virtually all striatal neurons, both in vitro and in vivo, contained dopamine receptors of both classes. We also provide functional evidence for such colocalization: in essentially all neurons examined, fenoldopam, an agonist of the D1 subclass of receptors, inhibited both the Na+/K+ pump and tetrodotoxin (TTX)-sensitive sodium channels, and quinpirole, an agonist of the D2 subclass of receptors, activated TTX-sensitive sodium channels. Thus D1 and D2 classes of ligands may functionally interact in virtually all dopamine-responsive neurons within the basal ganglia.

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 Ca

Mounting evidence suggests that the ion pump, Na,KATPase, 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,KATPase catalytic ␣-subunit (␣NT-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 ␣-isoforms, are essential for the binding to occur. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-B and protect from apoptosis. All of these effects are suppressed in cells overexpressing a peptide corresponding to ␣NT-t but not in cells overexpressing a peptide corresponding to ␣NT-t⌬LKK. 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.The Na,K-ATPase is an integral plasma membrane protein that establishes the electrochemical gradient across the plasma membrane in all mammalian cells. Ouabain is a steroid derivative that binds specifically to Na,K-ATPase. Several recent studies suggest that the ouabain⅐Na,K-ATPase complex may act as a signal transducer and transcription activator (1-4) modulating cell growth (5, 6), apoptosis (7), and cell motility (8). These effects have been ascribed to the activation of a number of intracellular signaling pathways (for review see Refs. 9 and 10). Most, if not all, of these pathways involve the release of calcium (Ca 2ϩ ) from intracellular stores via the inositol 1,4,5-trisphosphate receptor (InsP 3 R), 4 and results from recent studies indicate that Na,K-ATPase tethers the InsP 3 R into a Ca 2ϩ regulatory complex (3, 4). The exact mechanisms by which Na,K-ATPase activates the InsP 3 R remains to be elucidated.Here we report that the N-terminal tail of the Na,K-ATPase catalytic ␣-subunit binds to the N terminus of the InsP 3 R. Interaction between Na,K-ATPase and InsP 3 R modulates the Ca 2ϩ oscillatory signal, which serves to protect the cell from apoptosis (11). The identification of a distinct motif in the Na,KATPase that via protein-protein interaction transmits this signal to the InsP 3 R has important implications for the many vital cell functions that are regulated by ouabain⅐Na,K-ATPase signaling. EXPERIMENTAL PROCEDURESCells and Tissue-Two types of cells were used. COS-7 cells, a cell line derived from fetal monkey kidney, were used in most protocols. Because transformed cells are not suitable for serum deprivation-induced apoptosis, rat proximal tubule (RPT) cells in primary culture were used in these protocols. COS-7 cells were purchased from the European Collection of Cell Cultures and were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum and 2 mM L-glutamine. RPT cells were prepared as described previously (1). Briefly, the kidneys of 20-day-old male Sprague-Dawley rats...

It is well documented that the activity of Na