Through their ion-pumping and non-ion-pumping functions Na+-K+-ATPase protein complexes in the plasma membrane are critical to intracellular homeostasis and to the physiological and pharmacological actions of cardiotonic steroids. followed by reperfusion (I-R). Biotinylation studies suggested that I-R itself acted as an inducer of Na+-K+-ATPase internalization and that surface expression of the mutant was higher than the native Na+-K+-ATPase before and after ischemia. Annexin V/propidium iodide staining and lactate dehydrogenase release suggested that I-R injury was reduced in α1-L499V-expressing cells compared with α1-expressing cells. Hence modulation of Na+-K+-ATPase cell surface abundance through structural determinants on the α-subunit is an important mechanism of regulation of cellular Na+-K+-ATPase in various physiological and pathophysiological conditions with a significant impact on cell survival in face of an ischemic stress. is a negatively charged residue and KW-2478 is a polar residue (26). The sequence is well conserved among all the known mammalian α1 sequences (Table 2) and our studies revealed that mutations targeting this motif such as L499V or E495S resulted in an increased abundance of Na+-K+-ATPase α1-units at the cell surface (44). Table 1. Summary of domains and sites of posttranslational modifications involved in the regulation of rat Na+-K+-ATPase α1 surface expression Table 2. Conserved dileucine motif of the form [D/E]XXXL[L/I] motif in Na+-K+-ATPase α1 sequences in various species Using a Na+-K+-ATPase α1 Structural Determinant of Surface Abundance as a Target for Protection Against Ischemia-Reperfusion Injury We reckoned that an increased abundance of Na+-K+-ATPase pump units at the cell surface could be salutary to cells with critically high levels of intracellular Na+ such as those reported during ischemia-reperfusion (I-R) injury and may result in protection against I-R-induced cell death (34 35 This hypothesis was tested in opossum kidney (OK) cells stably expressing native or L499V-mutated forms of Na+-K+-ATPase α1 polypeptide exposed to substrate/coverslip-induced I-R. METHODS Cell Lines OK cells stably expressing native and L499V-mutated forms of Na+-K+-ATPase α1 were used. Details on the experimental procedures related to expression vectors and site-directed mutagenesis heterologous expression and preliminary characterization of Na+-K+-ATPase enzyme properties in these cells are available in Sottejeau et al. (44). Substrate and Coverslip-Induced Ischemia-Reperfusion Ischemia was induced by removal of the substrate and keeping a cup coverslip over some from the Alright cell monolayers as referred to previously (38). Quickly 70 confluent Alright cells expanded in 100-mm meals had been rinsed once with PBS and incubated in Krebs-Henseleit (KH) buffer including (in mmol/l) 118.0 NaCl 4 KCl 1.8 CaCl2 1.3 KH2PO4 1.2 MgSO4 0.3 EGTA 25 NaHCO3 and 37 d-glucose for 20 min at 37°C. Ischemia was after that simulated by changing the Emcn KH buffer by PBS and putting two 22 × 44 mm LifterSlips and one 22 × 63 mm LifterSlip (Thermo medical) on the cell monolayers for 30 min at 37°C. Reperfusion was initiated by mild removal of the LifterSlips and came KW-2478 back to KH buffer at 37°C. For confocal imaging research Alright cells had been expanded on square coverslip KW-2478 22 × 22 mm (Fisher) in six-well plates and I-R was induced as referred to KW-2478 above using 18-mm size round cup coverslips (Fisher). Annexin V/Propidium Iodide Staining By the end from the experimental process Alright cells had been set in 2% paraformaldehyde for 10 min at space temperatures after a clean in PBS 1×. Cells had been after that incubated with Alexa Fluor 488 annexin V and reddish colored fluorescent propidium iodide (PI) (Vybrant Apoptosis Assay Package no. 2 Invitrogen) based on the manufacturer’s suggestions. The coverslips had been installed with ProLong Yellow metal antifade reagent with 4 6 (DAPI Invitrogen). Confocal images were captured by sequential scanning with no overlap using a Leica TCS SP5 broadband confocal microscope system coupled to a DMI 6000CS inverted microscope equipped with multiple continuous wave lasers and a ×63/1.3 oil objective. Measurement of Lactate Dehydrogenase Activity At the end of a 60-min long reperfusion period the cell incubation buffer was collected and lactate dehydrogenase (LDH) activity was determined colorimetrically.