Angiotensin II stimulates elution of Na-K-ATPase from a digoxin-affinity column by increasing the kinetic response to ligands that trigger the decay of E₂-P

Abstract

We earlier observed that treating rat proximal tubules with concentrations of angiotensin II (ANG II) that directly stimulate Na-K-ATPase activity changed how Na-K-ATPase subsequently eluted from an ouabain-affinity column. In this study we tested whether ANG II increases the rate of elution in response to ligands that trigger the decay of E(2)-P, which implies a change in functional properties of Na-K-ATPase, or by decreasing the amount subsequently eluted with SDS, which suggests a change in how Na-K-ATPase interacts with other proteins. We utilized a new digoxin-affinity column and novel lines of opossum kidney (OK) cells that coexpress the rat AT(1a) receptor and either the wild-type rat alpha(1)-isoform of Na-K-ATPase or a truncation mutant missing the first 32 amino acids of its NH(2) terminus. We characterized how rat kidney microsomes bind to and elute from the digoxin-affinity column and demonstrated that they are heterogeneous in the rate at which they release digoxin in response to ligands that trigger the decay of E(2)-P. Incubating OK cells with ANG II stimulated the ensuing elution of wild-type rat alpha(1)-subunit by increasing the kinetic response to ligands that cause a decay of E(2)-P without affecting the amount later eluted with SDS. In contrast, ANG II had no effect on the kinetic response of the truncation mutant but decreased the amount eluted with SDS. These data suggest that ANG II regulates both the kinetic properties of Na-K-ATPase and its interaction with other proteins by a mechanism(s) involving its NH(2) terminus.

Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

“…It has, however, never been demonstrated that Ang II-dependent phosphorylation of the Na +/ K + -ATPase can change any of the basic biochemical properties of the Na +/ K + -ATPase. We were, therefore, intrigued by the evidence that exposing rat proximal tubule cells to Ang II changes the rate at which the Na +/ K + -ATPase is released from a digoxin-affinity column in response to Na + and ATP [7], because this response means that Ang II must have changed the conformation of Na +/ K + -ATPase before it bound to digoxin. Furthermore, the Na +/ K + -ATPase in the plasma membranes from rat kidney elutes in two distinct peaks in response to different concentrations of Na + and each peak is differentially regulated by Ang II [7].…”

Decreased renal perfusion rapidly increases plasma membrane Na-K-ATPase in rat cortex by an angiotensin II-dependent mechanism

“…The samples were centrifuged at 16,000 RCF for 10 min at 4°C, and the supernatant was discarded. A volume of 0.5 ml of cold RIPA buffer containing 2% SDS and protease inhibitors (22) was added to the pellet, and the tube was vortexed for 10 min at 4°C. Another 0.25 ml of the same solution was added, and the mixing continued.…”

“…The final protein concentration was ϳ4 mg/ml. An aliquot containing 0.3 mg of protein was removed, and the volume was increased to 0.5 ml by adding RIPA buffer supplemented with protease inhibitors (22). This sample was combined with 0.5 ml of immobilized streptavidin that had been previously washed twice with ice-cold RIPA buffer.…”

“…The relative amount of biotinylated Na-K-ATPase in the right and left kidneys from each experiment was measured by immunoblotting (15) for the ␣-subunit of the Na-K-ATPase by using chemiluminescence as previously described (22). On each blot we ran multiple samples from the left and right kidneys from a given experiment along with known amounts of rat kidney microsomes (22).…”

“…On each blot we ran multiple samples from the left and right kidneys from a given experiment along with known amounts of rat kidney microsomes (22). The total amount of protein added per lane was ϳ5 g or less.…”