Reversible Oxidative Modification

Abstract

Abstract-Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na ϩ -K ϩ pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of ␤ 1 , but not ␣ 1 , subunits in rabbit ventricular myocytes at baseline. ␤ 1 Subunit glutathionylation was increased by peroxynitrite (ONOO Ϫ ), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased ␣ 1 /␤ 1 subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with ␤ 1 subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na ϩ -K ϩ pump current (I p ). Cysteine (Cys46) of the ␤ 1 subunit was the likely candidate for glutathionylation. We expressed Na ϩ -K ϩ pump ␣ 1 subunits with wild-type or Cys46-mutated ␤ 1 subunits in Xenopus oocytes. ONOO Ϫ induced glutathionylation of ␤ 1 subunit and a decrease in Na ϩ -K ϩ pump turnover number. This was eliminated by mutation of Cys46. ONOO Ϫ also induced glutathionylation of the Na ϩ -K ϩ ATPase ␤ 1 subunit from pig kidney. This was associated with a Ϸ2-fold decrease in the rate-limiting E 2 3 E 1 conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na ϩ -K ϩ pump occurs via glutathionylation of its ␤ 1 subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na Key Words: glutathionylation Ⅲ glutaredoxin Ⅲ Na ϩ -K ϩ pump Ⅲ angiotensin Ⅲ NADPH oxidase T he ATP-dependent Na ϩ -K ϩ pump transports Na ϩ out of cells in exchange for extracellular K ϩ , against their respective electrochemical gradients. The Na ϩ and K ϩ gradients that it generates maintain the membrane potential essential for cellular electric excitability. However, the gradients also have an important broader role because they serve in coupled co-and countertransport processes for other ions and organic compounds. Regulation of the Na ϩ -K ϩ pump, usually attributed to phosphorylation, is poorly understood. We have found that the isoform of protein kinase C (PKC) mediates angiotensin II (Ang II)-induced inhibition of the pump in rabbit ventricular myocytes. 1 However, inhibition could not be attributed to phosphorylation of the pump molecule or of the closely associated "FXYD protein" phospholemman that has been implicated in PKC-dependent pump regulation in cardiac myocytes. 2 Instead, the dependence of Ang II-induced pump inhibition on NADPH oxidase and superoxide (O 2 ⅐Ϫ ) 1 strongly suggests a role for oxidative signaling.Oxidative signaling can occur via modifications of susceptible protein sulfhydryl groups (protein-SH). Their reactivities are too low to make them realistic targets for oxidative modification induced by O 2 ⅐Ϫ , 3 b...

β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia

“…This increase in pump current was abolished by L‐ N G ‐nitroarginine methyl ester, showing that stimulation was mediated by eNOS‐derived NO production enhanced by ex vivo supplementation of BH 4 . In previous reports we have shown that NO generation from eNOS stimulates the Na + ‐K + pump,10, 38 and conditions that cause eNOS uncoupling inhibit the Na + ‐K + pump 24, 3940 where changes in redox milieu are sensed by eNOS and transduced to regulation of the Na + ‐K + pump function via redox‐dependent mechanisms.…”

“…We tested this by examining glutathionylation of the highly abundant, ubiquitous membrane protein Na + ‐K + pump, which mediates inhibition of the pump function in cardiac myocytes24 and vascular smooth muscle cells 23. Alloxan, at high concentrations in vitro (1 mmol/L, 37°C, 30 minutes), reflecting the pancreatotoxic diabetogenic doses used in vivo, increased glutathionylation of the β 1 Na + ‐K + pump subunit (Figure 1A).…”

“…CL decreased hyperglycemia‐induced glutathionylation of β 1 Na + ‐K + pump subunit, a redox modification that inhibits the pump,23, 24 and enhanced pump activity as implied by increased K + ‐induced vasorelaxation. This should largely reflect the signal from vascular smooth muscle cells as the media layer constitutes the major part of the vascular wall.…”

Role of phospholemman and the 70 kDa inhibitor protein in regulating Na+/K+ ATPase activity in pulmonary artery smooth muscle cells under U46619 stimulation

“…Glutathionylation inhibits the pump by stabilizing its E1 conformation. Figtree et al [27] showed that glutathionylation of NKA is reversible in nature where glutaredoxin1 (GRX1) facilitated deglutathionylation of the pump. In BPASMCs, we determined the presence of GRX1 (Fig.…”

S-Glutathionylation of Ion Channels: Insights into the Regulation of Channel Functions, Thiol Modification Crosstalk, and Mechanosensing

“…In addition, the application of peroxynitrite (ONOO -) or Snitrosoglutathione (GSNO, 10-500 lM) to induce S-glutathionylation is also found in the literature (12,38,125,129). The application of peroxynitrite is of high physiological relevance but as peroxynitrite can also induce other modification including S-nitrosylation, additional experiments should be performed to further dissect the exact modification mechanism.…”