Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

Abstract

Cellular signaling can inhibit the membrane Na؉ -K ؉ pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the ␤ 1 subunit of the pump ␣/␤ heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47 phox and p22 phox , required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na ؉ -K ؉ pump ␤ 1 subunit and decreased its co-immunoprecipitation with the ␣ 1 subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the ␣ 1 /␤ 1 subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na ؉ -K ؉ pump current (arising from the 3:2 Na ؉ :K ؉ exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the ⑀-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMPand PKC-dependent inhibition of the cardiac Na ؉ -K ؉ pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump ␤ 1 subunit.

FXYD Proteins Reverse Inhibition of the Na+-K+ Pump Mediated by Glutathionylation of Its β1 Subunit

“…Identifying location and orientation of Cys 46 in the crystal structure of the E 1 conformation of the Na ϩ -K ϩ pump may explain its reactivity. Physiological and Pathophysiological Implications-Physiological, receptor-coupled pathways can induce glutathionylation of the ␤ 1 subunit and Na ϩ -K ϩ pump inhibition by activating NADPH oxidase (11)(12)(13). Reversal of glutathionylation and pump inhibition is equally important, and we show that the reversal depends on FXYD proteins.…”

“…There may be pathophysiological implications of FXYD-dependent redox regulation of membrane transport. Raised levels of neurohormones that activate redox signaling (12,13,25), increased myocardial oxidative stress, and dysregulation of cytosolic Na ϩ and Ca 2ϩ handling contribute to the pathophysiology of heart failure (38). Decreased FXYD1 expression (39) may accentuate such abnormalities.…”

“…Exposure of myocytes to angiotensin II for 15 min to activate cardiac NADPH oxidase (12) increased glutathionylation. The adenyl cyclase activator forskolin induces NADPH oxidase-dependent glutathionylation of the ␤ 1 subunit (13), and forskolin also increased the glutathionylation of FXYD1 (Fig. 1D).…”

“…Patch clamped myocytes were exposed to angiotensin II or CL316,243 in the superfusate after the whole cell configuration was established. I p was measured at 37°C identified as the ouabain-induced shift in holding currents (12,13).…”

β 3 Adrenergic Stimulation of the Cardiac Na + -K + Pump by Reversal of an Inhibitory Oxidative Modification

“…The concentration of H-89 we used abolishes the effect of forskolin-induced PKA activation to induce a decrease in I p . 20 The combined blockade of ␤ 1 /␤ 2 ARs and the downstream pathway coupled to them had no effect on the 〉RL-induced increase in I p (Figure 2A). The increase also persisted in the presence of the ␣ AR antagonist prazosin ( Figure 2A).…”