Elisha Hamilton | The University of Sydney (original) (raw)

Papers by Elisha Hamilton

Research paper thumbnail of cGMP Compartmentalization Accounts for the Bi-phasic Effect of ANP on Na +–K + Pump in Cardiac Myocytes

Heart Lung Circ, 2009

Atrial natriuretic peptide (ANP) at a physiological tissue concentration stimulates Na + -K + pum... more Atrial natriuretic peptide (ANP) at a physiological tissue concentration stimulates Na + -K + pump current (I p ) in rabbit myocytes via cGMP dependent protein kinase but stimulation is lost at higher (200 nM) ANP concentrations. Since cyclic nucleotide signaling in myocytes is restricted to microdomians defined by phosphodiesterases (PDEs) we examined the effect of increasing endogenously available cGMP. We measured I p in myocytes voltage-clamped using pipette solutions that included T-1032, an inhibitor of the cGMP-selective PDE V. T-1032 reproduced the stimulation of I p seen at the low (10 nM) ANP concentration but stimulation was lost when myocytes were exposed to both T-1032 and 10 nM ANP. We induced cGMP synthesis with YC-1, which induced an increase in I p that was abolished by PDE V inhibition with T-1032. Since cGMP inhibits PDE III, the main PDE that breaks down cAMP, we examined the effect of inhibiting cAMP-activated protein kinase (PKA) with H-89. H-89 restored stimulation of I p at high ANP concentrations or with combined YC-1/T-1032 exposure. Since subunits of the Na + -K + pump and NADPH oxidase co-localize in cardiac myocytes and PKA activation inhibits I p via NADPH oxidase in myocytes, we examined the effect of inhibiting NADPH oxidase. We included gp91ds, an inhibitory peptide for NADPH oxidase in pipette solutions and exposed myocytes 200 nM ANP. NADPH oxidase inhibition restored stimulation of I p at high ANP concentration. Our study suggests that cGMP-dependent stimulation of the Na + -K + pump is compartmentalized, and that the loss of pump stimulation at high ANP concentrations is due to NADPH oxidase.

Research paper thumbnail of PKA/PKC cross-talk mediates NADPH oxidase-induced Na +�K + pump inhibition in cardiac myocytes

Heart Lung Circ, 2007

lung weight/body weight ratio [0.33 vs. 0.40, p < 0.05]). Cell culture experiments demonstrated a... more lung weight/body weight ratio [0.33 vs. 0.40, p < 0.05]). Cell culture experiments demonstrated a direct dose dependent reduction of collagen synthesis with FT-011 in response to AII and TGF-␤ stimulation (p < 0.01). Conclusion: Treatment with FT-011 post MI prevents adverse LV remodelling and heart failure at least in part by reducing pathological remodelling of collagen in the LV.

Research paper thumbnail of β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia

Journal of the American Heart Association, Jan 19, 2016

Perturbed balance between NO and O2 (•-). (ie, NO/redox imbalance) is central in the pathobiology... more 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. 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 improv...

Research paper thumbnail of PKA inhibits the Na +�K +pump in cadiac myocytes via superoxide

J Mol Cell Cardiol, 2006

morphine-treated (CMP) hearts (75 mg pellet, 5 days) displayed a significant recovery of RPP (83 ... more morphine-treated (CMP) hearts (75 mg pellet, 5 days) displayed a significant recovery of RPP (83 T 3%) and an almost complete return of EDP. As expected, co-administration of opioid antagonist, naloxone, via osmotic minipump for 5 days prevented the initiation (''trigger'' phase), and thus, protection of CMP (RPP, 46 T 2%). Moreover, 5 day infusion of the primary morphine metabolites, morphine-3-glucuronide and morphine-6-glucuronide, failed to induced any form of cardioprotection, suggesting that neither the mu-opioid receptor nor metabolites are involved. Concurrent infusion of the PKA inhibitor, PKI, during the activation phase failed to abolish the CMP-mediated protection (69 T 5% RPP). Interestingly, naloxone, administered pre-and post-ischemia (the ''mediation'' phase), following 5 days of morphine exposure, failed to modify the CMP-induced phenotype, while PKI completely abolished CMP protection, with no effect on placebo-treated hearts.

Research paper thumbnail of ANP activates the sarcolemmal sodium pump via nitric oxide synthase

Journal of Molecular and Cellular Cardiology

Research paper thumbnail of NPR-C, the natriuretic peptide "clearance receptor," mediates cardiac myocyte Na+-K+ pump activation

Research paper thumbnail of Phospholemman enhances the sensitivity of the rabbit ventricular myocyte Na+-K+ pump to intracellular Na+

Research paper thumbnail of Nitric oxide stimulates the cardiac sarcolemmal Na+-K+ pump

Research paper thumbnail of Bi-phasic effects of sodium nitroprusside on the sodium pump - Role of superoxide and NO mediated pathways

Journal of Molecular and Cellular Cardiology

Research paper thumbnail of Stimulation of the Cardiac Myocyte Na+-K+ Pump Due to Reversal of Its Constitutive Oxidative Inhibition

American journal of physiology. Cell physiology, Jan 17, 2015

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1 Na(+)-K(+) pum... more Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1 Na(+)-K(+) pump subunit, inhibiting activity of the catalytic α subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na(+)-K(+) pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition cardiac myocytes were exposed to Ang II to activate NADPH oxidase and inhibit Na(+)-K(+) pump current (Ip). Co-exposure to 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases 2A (PP2A) but not by inhibition of PP1 and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1 Na(+)-K(+) pump subunit glutathionylation and pump activity, YC-1 decreased Ang II-induced β1 subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decrea...

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

American journal of physiology. Cell physiology, Jan 10, 2015

Dysregulated nitric oxide (NO) and superoxide (O2 (.-))-dependent signaling contributes to the pa... more 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 nitric oxide synthase (eNOS) activation, relieves oxidative inhibition of eNOS and Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male White New Zealand rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indices of oxidative stress. It induced glutathionylation of the β1 Na(+)-K(+) pump 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 that causes its uncoupling, and it increased co-immunoprecipitation of cytosolic p47(phox) -and membranous p22(phox) NAPDH oxidase subunits consistent with NADPH oxidas...

Research paper thumbnail of Protein kinase-dependent oxidative regulation of the cardiac Na + -K + pump: Evidence from in vivo and in vitro modulation of cell signalling

The Journal of Physiology, 2013

• The widely believed effects of β 1 adrenergic receptors and protein kinase A (PKA) to stimulate... more • The widely believed effects of β 1 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 β 1 adrenergic blockade in heart failure. • We show that reduction in PKA activity by β 1 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 β 1 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 β 1 adrenergic blockade in heart failure.

Research paper thumbnail of Redox-dependent regulation of the Na+–K+ pump: New twists to an old target for treatment of heart failure

Journal of Molecular and Cellular Cardiology, 2013

By the time it was appreciated that the positive inotropic effect of cardiac glycosides is due to... more By the time it was appreciated that the positive inotropic effect of cardiac glycosides is due to inhibition of the membrane Na(+)-K(+) pump, glycosides had been used for treatment of heart failure on an empiric basis for ~200 years. The subsequent documentation of their lack of clinical efficacy and possible harmful effect largely coincided with the discovery that a raised Na(+) concentration in cardiac myocytes plays an important role in the electromechanical phenotype of heart failure syndromes. Consistent with this, efficacious pharmacological treatments for heart failure have been found to stimulate the Na(+)-K(+) pump, effectively the only export route for intracellular Na(+) in the heart failure. A paradigm has emerged that implicates pump inhibition in the raised Na(+) levels in heart failure. It invokes protein kinase-dependent activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and glutathionylation, a reversible oxidative modification, of the Na(+)-K(+) pump molecular complex that inhibits its activity. Since treatments of proven efficacy reverse the oxidative Na(+)-K(+) pump inhibition, the pump retains its status as a key pharmacological target in heart failure. Its role as a target is well integrated with the paradigms of neurohormonal abnormalities, raised myocardial oxidative stress and energy deficiency implicated in the pathophysiology of the failing heart. We propose that targeting oxidative inhibition of the pump is useful for the exploration of future treatment strategies. This article is part of a Special Issue entitled &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Na(+)Regulation in Cardiac Myocytes&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;.

Research paper thumbnail of FXYD Proteins Reverse Inhibition of the Na+-K+ Pump Mediated by Glutathionylation of Its  1 Subunit

Journal of Biological Chemistry, 2011

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its ... more The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

Research paper thumbnail of Susceptibility of  1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump

Journal of Biological Chemistry, 2012

Background: Glutathionylation of a cysteine in the membrane Na + -K + pump subunit occurs despite... more Background: Glutathionylation of a cysteine in the membrane Na + -K + pump 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 Na + -K + pump's catalytic cycle. Conclusion: Na + -K + pump cycle phase determines glutathionylation. Significance: Cysteine glutathionylation can depend on protein conformational state.

Research paper thumbnail of Stimulation of β3 Adrenergic Signalling Reverses Diabetes-Induced Oxidative Inhibition of Sarcolemmal Na+–K+ pump

Heart, Lung and Circulation, 2013

Research paper thumbnail of Antioxidant Effect of Beta-1 Adrenergic Receptor Blockade Through Macromolecular Remodelling of An Oxidative Pathway: New Tricks of An Old Dog

Heart, Lung and Circulation, 2012

Research paper thumbnail of β3 Adrenergic Receptor Stimulates the Cardiac Na/K Pump via an NO-dependent Pathway and Reduces Oxidative Modification of the β1 Pump Subunit

Heart, Lung and Circulation, 2009

Research paper thumbnail of cGMP Compartmentalization Accounts for the Bi-phasic Effect of ANP on Na+–K+ Pump in Cardiac Myocytes

Heart, Lung and Circulation, 2009

Atrial natriuretic peptide (ANP) at a physiological tissue concentration stimulates Na + -K + pum... more Atrial natriuretic peptide (ANP) at a physiological tissue concentration stimulates Na + -K + pump current (I p ) in rabbit myocytes via cGMP dependent protein kinase but stimulation is lost at higher (200 nM) ANP concentrations. Since cyclic nucleotide signaling in myocytes is restricted to microdomians defined by phosphodiesterases (PDEs) we examined the effect of increasing endogenously available cGMP. We measured I p in myocytes voltage-clamped using pipette solutions that included T-1032, an inhibitor of the cGMP-selective PDE V. T-1032 reproduced the stimulation of I p seen at the low (10 nM) ANP concentration but stimulation was lost when myocytes were exposed to both T-1032 and 10 nM ANP. We induced cGMP synthesis with YC-1, which induced an increase in I p that was abolished by PDE V inhibition with T-1032. Since cGMP inhibits PDE III, the main PDE that breaks down cAMP, we examined the effect of inhibiting cAMP-activated protein kinase (PKA) with H-89. H-89 restored stimulation of I p at high ANP concentrations or with combined YC-1/T-1032 exposure. Since subunits of the Na + -K + pump and NADPH oxidase co-localize in cardiac myocytes and PKA activation inhibits I p via NADPH oxidase in myocytes, we examined the effect of inhibiting NADPH oxidase. We included gp91ds, an inhibitory peptide for NADPH oxidase in pipette solutions and exposed myocytes 200 nM ANP. NADPH oxidase inhibition restored stimulation of I p at high ANP concentration. Our study suggests that cGMP-dependent stimulation of the Na + -K + pump is compartmentalized, and that the loss of pump stimulation at high ANP concentrations is due to NADPH oxidase.

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

Free Radical Biology and Medicine, 2013

Glutathionylation of the Na(+)-K(+) pump&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;... more Glutathionylation of the Na(+)-K(+) pump&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s 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…

Research paper thumbnail of cGMP Compartmentalization Accounts for the Bi-phasic Effect of ANP on Na +–K + Pump in Cardiac Myocytes

Heart Lung Circ, 2009

Atrial natriuretic peptide (ANP) at a physiological tissue concentration stimulates Na + -K + pum... more Atrial natriuretic peptide (ANP) at a physiological tissue concentration stimulates Na + -K + pump current (I p ) in rabbit myocytes via cGMP dependent protein kinase but stimulation is lost at higher (200 nM) ANP concentrations. Since cyclic nucleotide signaling in myocytes is restricted to microdomians defined by phosphodiesterases (PDEs) we examined the effect of increasing endogenously available cGMP. We measured I p in myocytes voltage-clamped using pipette solutions that included T-1032, an inhibitor of the cGMP-selective PDE V. T-1032 reproduced the stimulation of I p seen at the low (10 nM) ANP concentration but stimulation was lost when myocytes were exposed to both T-1032 and 10 nM ANP. We induced cGMP synthesis with YC-1, which induced an increase in I p that was abolished by PDE V inhibition with T-1032. Since cGMP inhibits PDE III, the main PDE that breaks down cAMP, we examined the effect of inhibiting cAMP-activated protein kinase (PKA) with H-89. H-89 restored stimulation of I p at high ANP concentrations or with combined YC-1/T-1032 exposure. Since subunits of the Na + -K + pump and NADPH oxidase co-localize in cardiac myocytes and PKA activation inhibits I p via NADPH oxidase in myocytes, we examined the effect of inhibiting NADPH oxidase. We included gp91ds, an inhibitory peptide for NADPH oxidase in pipette solutions and exposed myocytes 200 nM ANP. NADPH oxidase inhibition restored stimulation of I p at high ANP concentration. Our study suggests that cGMP-dependent stimulation of the Na + -K + pump is compartmentalized, and that the loss of pump stimulation at high ANP concentrations is due to NADPH oxidase.

Research paper thumbnail of PKA/PKC cross-talk mediates NADPH oxidase-induced Na +�K + pump inhibition in cardiac myocytes

Heart Lung Circ, 2007

lung weight/body weight ratio [0.33 vs. 0.40, p < 0.05]). Cell culture experiments demonstrated a... more lung weight/body weight ratio [0.33 vs. 0.40, p < 0.05]). Cell culture experiments demonstrated a direct dose dependent reduction of collagen synthesis with FT-011 in response to AII and TGF-␤ stimulation (p < 0.01). Conclusion: Treatment with FT-011 post MI prevents adverse LV remodelling and heart failure at least in part by reducing pathological remodelling of collagen in the LV.

Research paper thumbnail of β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia

Journal of the American Heart Association, Jan 19, 2016

Perturbed balance between NO and O2 (•-). (ie, NO/redox imbalance) is central in the pathobiology... more 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. 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 improv...

Research paper thumbnail of PKA inhibits the Na +�K +pump in cadiac myocytes via superoxide

J Mol Cell Cardiol, 2006

morphine-treated (CMP) hearts (75 mg pellet, 5 days) displayed a significant recovery of RPP (83 ... more morphine-treated (CMP) hearts (75 mg pellet, 5 days) displayed a significant recovery of RPP (83 T 3%) and an almost complete return of EDP. As expected, co-administration of opioid antagonist, naloxone, via osmotic minipump for 5 days prevented the initiation (''trigger'' phase), and thus, protection of CMP (RPP, 46 T 2%). Moreover, 5 day infusion of the primary morphine metabolites, morphine-3-glucuronide and morphine-6-glucuronide, failed to induced any form of cardioprotection, suggesting that neither the mu-opioid receptor nor metabolites are involved. Concurrent infusion of the PKA inhibitor, PKI, during the activation phase failed to abolish the CMP-mediated protection (69 T 5% RPP). Interestingly, naloxone, administered pre-and post-ischemia (the ''mediation'' phase), following 5 days of morphine exposure, failed to modify the CMP-induced phenotype, while PKI completely abolished CMP protection, with no effect on placebo-treated hearts.

Research paper thumbnail of ANP activates the sarcolemmal sodium pump via nitric oxide synthase

Journal of Molecular and Cellular Cardiology

Research paper thumbnail of NPR-C, the natriuretic peptide "clearance receptor," mediates cardiac myocyte Na+-K+ pump activation

Research paper thumbnail of Phospholemman enhances the sensitivity of the rabbit ventricular myocyte Na+-K+ pump to intracellular Na+

Research paper thumbnail of Nitric oxide stimulates the cardiac sarcolemmal Na+-K+ pump

Research paper thumbnail of Bi-phasic effects of sodium nitroprusside on the sodium pump - Role of superoxide and NO mediated pathways

Journal of Molecular and Cellular Cardiology

Research paper thumbnail of Stimulation of the Cardiac Myocyte Na+-K+ Pump Due to Reversal of Its Constitutive Oxidative Inhibition

American journal of physiology. Cell physiology, Jan 17, 2015

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1 Na(+)-K(+) pum... more Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1 Na(+)-K(+) pump subunit, inhibiting activity of the catalytic α subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na(+)-K(+) pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition cardiac myocytes were exposed to Ang II to activate NADPH oxidase and inhibit Na(+)-K(+) pump current (Ip). Co-exposure to 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases 2A (PP2A) but not by inhibition of PP1 and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1 Na(+)-K(+) pump subunit glutathionylation and pump activity, YC-1 decreased Ang II-induced β1 subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decrea...

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

American journal of physiology. Cell physiology, Jan 10, 2015

Dysregulated nitric oxide (NO) and superoxide (O2 (.-))-dependent signaling contributes to the pa... more 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 nitric oxide synthase (eNOS) activation, relieves oxidative inhibition of eNOS and Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male White New Zealand rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indices of oxidative stress. It induced glutathionylation of the β1 Na(+)-K(+) pump 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 that causes its uncoupling, and it increased co-immunoprecipitation of cytosolic p47(phox) -and membranous p22(phox) NAPDH oxidase subunits consistent with NADPH oxidas...

Research paper thumbnail of Protein kinase-dependent oxidative regulation of the cardiac Na + -K + pump: Evidence from in vivo and in vitro modulation of cell signalling

The Journal of Physiology, 2013

• The widely believed effects of β 1 adrenergic receptors and protein kinase A (PKA) to stimulate... more • The widely believed effects of β 1 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 β 1 adrenergic blockade in heart failure. • We show that reduction in PKA activity by β 1 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 β 1 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 β 1 adrenergic blockade in heart failure.

Research paper thumbnail of Redox-dependent regulation of the Na+–K+ pump: New twists to an old target for treatment of heart failure

Journal of Molecular and Cellular Cardiology, 2013

By the time it was appreciated that the positive inotropic effect of cardiac glycosides is due to... more By the time it was appreciated that the positive inotropic effect of cardiac glycosides is due to inhibition of the membrane Na(+)-K(+) pump, glycosides had been used for treatment of heart failure on an empiric basis for ~200 years. The subsequent documentation of their lack of clinical efficacy and possible harmful effect largely coincided with the discovery that a raised Na(+) concentration in cardiac myocytes plays an important role in the electromechanical phenotype of heart failure syndromes. Consistent with this, efficacious pharmacological treatments for heart failure have been found to stimulate the Na(+)-K(+) pump, effectively the only export route for intracellular Na(+) in the heart failure. A paradigm has emerged that implicates pump inhibition in the raised Na(+) levels in heart failure. It invokes protein kinase-dependent activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and glutathionylation, a reversible oxidative modification, of the Na(+)-K(+) pump molecular complex that inhibits its activity. Since treatments of proven efficacy reverse the oxidative Na(+)-K(+) pump inhibition, the pump retains its status as a key pharmacological target in heart failure. Its role as a target is well integrated with the paradigms of neurohormonal abnormalities, raised myocardial oxidative stress and energy deficiency implicated in the pathophysiology of the failing heart. We propose that targeting oxidative inhibition of the pump is useful for the exploration of future treatment strategies. This article is part of a Special Issue entitled &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Na(+)Regulation in Cardiac Myocytes&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;.

Research paper thumbnail of FXYD Proteins Reverse Inhibition of the Na+-K+ Pump Mediated by Glutathionylation of Its  1 Subunit

Journal of Biological Chemistry, 2011

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its ... more The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

Research paper thumbnail of Susceptibility of  1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump

Journal of Biological Chemistry, 2012

Background: Glutathionylation of a cysteine in the membrane Na + -K + pump subunit occurs despite... more Background: Glutathionylation of a cysteine in the membrane Na + -K + pump 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 Na + -K + pump's catalytic cycle. Conclusion: Na + -K + pump cycle phase determines glutathionylation. Significance: Cysteine glutathionylation can depend on protein conformational state.

Research paper thumbnail of Stimulation of β3 Adrenergic Signalling Reverses Diabetes-Induced Oxidative Inhibition of Sarcolemmal Na+–K+ pump

Heart, Lung and Circulation, 2013

Research paper thumbnail of Antioxidant Effect of Beta-1 Adrenergic Receptor Blockade Through Macromolecular Remodelling of An Oxidative Pathway: New Tricks of An Old Dog

Heart, Lung and Circulation, 2012

Research paper thumbnail of β3 Adrenergic Receptor Stimulates the Cardiac Na/K Pump via an NO-dependent Pathway and Reduces Oxidative Modification of the β1 Pump Subunit

Heart, Lung and Circulation, 2009

Research paper thumbnail of cGMP Compartmentalization Accounts for the Bi-phasic Effect of ANP on Na+–K+ Pump in Cardiac Myocytes

Heart, Lung and Circulation, 2009

Atrial natriuretic peptide (ANP) at a physiological tissue concentration stimulates Na + -K + pum... more Atrial natriuretic peptide (ANP) at a physiological tissue concentration stimulates Na + -K + pump current (I p ) in rabbit myocytes via cGMP dependent protein kinase but stimulation is lost at higher (200 nM) ANP concentrations. Since cyclic nucleotide signaling in myocytes is restricted to microdomians defined by phosphodiesterases (PDEs) we examined the effect of increasing endogenously available cGMP. We measured I p in myocytes voltage-clamped using pipette solutions that included T-1032, an inhibitor of the cGMP-selective PDE V. T-1032 reproduced the stimulation of I p seen at the low (10 nM) ANP concentration but stimulation was lost when myocytes were exposed to both T-1032 and 10 nM ANP. We induced cGMP synthesis with YC-1, which induced an increase in I p that was abolished by PDE V inhibition with T-1032. Since cGMP inhibits PDE III, the main PDE that breaks down cAMP, we examined the effect of inhibiting cAMP-activated protein kinase (PKA) with H-89. H-89 restored stimulation of I p at high ANP concentrations or with combined YC-1/T-1032 exposure. Since subunits of the Na + -K + pump and NADPH oxidase co-localize in cardiac myocytes and PKA activation inhibits I p via NADPH oxidase in myocytes, we examined the effect of inhibiting NADPH oxidase. We included gp91ds, an inhibitory peptide for NADPH oxidase in pipette solutions and exposed myocytes 200 nM ANP. NADPH oxidase inhibition restored stimulation of I p at high ANP concentration. Our study suggests that cGMP-dependent stimulation of the Na + -K + pump is compartmentalized, and that the loss of pump stimulation at high ANP concentrations is due to NADPH oxidase.

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

Free Radical Biology and Medicine, 2013

Glutathionylation of the Na(+)-K(+) pump&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;... more Glutathionylation of the Na(+)-K(+) pump&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s 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…