N. Fry

Glutathionylation mediates angiotensin II-induced eNOS uncoupling, amplifying NADPH oxidase-dependent endothelial dysfunction.

Background Glutathionylation of endothelial nitric oxide synthase (eNOS) “uncouples” the enzyme, switching its function from nitric oxide (NO) to O2•− generation. We examined whether this reversible redox modification plays a role in angiotensin II (Ang II)‐induced endothelial dysfunction. Methods and Results Ang II increased eNOS glutathionylation in cultured human umbilical vein endothelial cells (HUVECs), rabbit aorta, and human arteries in vitro. This was associated with decreased NO bioavailability and eNOS activity as well as increased O2•− generation. Ang II‐induced decrease in eNOS activity was mediated by glutathionylation, as shown by restoration of function by glutaredoxin‐1. Moreover, Ang II‐induced increase in O2•− and decrease in NO were abolished in HUVECs transiently transfected, with mutant eNOS rendered resistant to glutathionylation. Ang II effects were nicotinamide adenine dinucleotide phosphate (NADPH) oxidase dependent because preincubation with gp 91ds‐tat, an inhibitor of NADPH oxidase, abolished the increase in eNOS glutathionylation and loss of eNOS activity. Functional significance of glutathionylation in intact vessels was supported by Ang II‐induced impairment of endothelium‐dependent vasorelaxation that was abolished by the disulfide reducing agent, dithiothreitol. Furthermore, attenuation of Ang II signaling in vivo by administration of an angiotensin converting enzyme (ACE) inhibitor reduced eNOS glutathionylation, increased NO, diminished O2•−, improved endothelium‐dependent vasorelaxation and reduced blood pressure. Conclusions Uncoupling of eNOS by glutathionylation is a key mediator of Ang II‐induced endothelial dysfunction, and its reversal is a mechanism for cardiovascular protection by ACE inhibition. We suggest that Ang II‐induced O2•− generation in endothelial cells, although dependent on NADPH oxidase, is amplified by glutathionylation‐dependent eNOS uncoupling.

Susceptibility of β1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump

Background: Glutathionylation of a cysteine in the membrane Na+-K+ pump β subunit occurs despite its lipid bulk phase location in the currently known structure of the pump molecule. Results: Glutathionylation was dependent on the conformational changes that occur in the catalytic cycle of the Na+-K+ pump. Conclusion: Na+-K+ pump cycle phase determines glutathionylation. Significance: Cysteine glutathionylation can depend on protein conformational state. Glutathionylation of cysteine 46 of the β1 subunit of the Na+-K+ pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K+·Pi configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na+-K+ pump cycle as described by the Albers-Post scheme. We measured β1 subunit glutathionylation and function of Na+-K+-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na+-K+-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na3 conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO−). Inhibition of Na+-K+-ATPase activity after exposure to ONOO− was greater when the enzyme had been in the E1Na3 than the E2 conformation. We exposed myocytes to different extracellular K+ concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K+ concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO−-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na+-K+ pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na+-K+ pump.

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: Implications for angiotensin II-induced vascular dysfunction

Glutathionylation of the Na(+)-K(+) pumps β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pumps β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an important protective role in the vasculature under conditions of oxidative stress.

Protein kinase-dependent oxidative regulation of the cardiac Na+–K+ pump: evidence from in vivo and in vitro modulation of cell signalling

•  The widely believed effects of β1 adrenergic receptors and protein kinase A (PKA) to stimulate the membrane Na+–K+ pump in cardiac myocytes are not easily reconciled with the effects of activation of the receptor and PKA on contractility in normal heart or with the benefit of β1 adrenergic blockade in heart failure. •  We show that reduction in PKA activity by β1 adrenergic blockade in vivo stimulates the Na+–K+ pump by reducing glutathionylation of one of its subunits, a reversible oxidative modification that inhibits pump activity. •  Na+–K+ pump stimulation induced by β1 adrenergic blockade in vivo is reversed by activation of PKA‐dependent signalling in isolated cardiac myocytes studied ex vivo. •  Inhibition of the myocyte Na+–K+ pump mediated by PKA‐ and redox‐dependent signalling pathways and downstream glutathionylation of a subunit of the Na+–K+ pump is readily reconciled with effects of PKA on contractility in normal heart and efficacy of β1 adrenergic blockade in heart failure.

The first-in-man randomized trial of a beta3 adrenoceptor agonist in chronic heart failure: the BEAT-HF trial: β3 adrenoceptor agonist in human heart failure

The third isotype of beta adrenergic receptors (β3 ARs) has distinctly different effects on cardiomyocytes compared with β1 and β2 ARs. Stimulation of β3 ARs may reduce cardiomyocyte Na+ overload and reduce oxidative stress in heart failure (HF). We examined if treatment with the β3 AR agonist mirabegron increases LVEF in patients with HF.

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

Background Perturbed balance between NO and O2 •−. (ie, NO/redox imbalance) is central in the pathobiology of diabetes‐induced vascular dysfunction. We examined whether stimulation of β3 adrenergic receptors (β3 ARs), coupled to endothelial nitric oxide synthase (eNOS) activation, would re‐establish NO/redox balance, relieve oxidative inhibition of the membrane proteins eNOS and Na+‐K+ (NK) pump, and improve vascular function in a new animal model of hyperglycemia. Methods and Results We established hyperglycemia in male White New Zealand rabbits by infusion of S961, a competitive high‐affinity peptide inhibitor of the insulin receptor. Hyperglycemia impaired endothelium‐dependent vasorelaxation by “uncoupling” of eNOS via glutathionylation (eNOS‐GSS) that was dependent on NADPH oxidase activity. Accordingly, NO levels were lower while O2 •− levels were higher in hyperglycemic rabbits. Infusion of the β3 AR agonist CL316243 (CL) decreased eNOS‐GSS, reduced O2 •−, restored NO levels, and improved endothelium‐dependent relaxation. CL decreased hyperglycemia‐induced NADPH oxidase activation as suggested by co‐immunoprecipitation experiments, and it increased eNOS co‐immunoprecipitation with glutaredoxin‐1, which may reflect promotion of eNOS de‐glutathionylation by CL. Moreover, CL reversed hyperglycemia‐induced glutathionylation of the β1 NK pump subunit that causes NK pump inhibition, and improved K+‐induced vasorelaxation that reflects enhancement in NK pump activity. Lastly, eNOS‐GSS was higher in vessels of diabetic patients and was reduced by CL, suggesting potential significance of the experimental findings in human diabetes. Conclusions β3 AR activation restored NO/redox balance and improved endothelial function in hyperglycemia. β3 AR agonists may confer protection against diabetes‐induced vascular dysfunction.

β3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade

Dysregulated nitric oxide (NO)- and superoxide (O2 (·-))-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of β3-adrenergic receptors (β3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na(+)-K(+) pump β1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47(phox) to p22(phox) with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na(+)-K(+) pump β1-subunit and decrease in pump current. In vivo treatment with the β3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22(phox) with p47(phox), abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na(+)-K(+) pump β1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na(+)-K(+) pump β1-subunit, which may reflect facilitation of deglutathionylation. In vivo β3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes.