Alvaro Garcia

Reversible Oxidative Modification. A Key Mechanism of Na+-K+ Pump Regulation

Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na+-K+ 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 β1, but not α1, subunits in rabbit ventricular myocytes at baseline. β1 Subunit glutathionylation was increased by peroxynitrite (ONOO−), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased α1/β1 subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with β1 subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na+-K+ pump current (Ip). Cysteine (Cys46) of the β1 subunit was the likely candidate for glutathionylation. We expressed Na+-K+ pump α1 subunits with wild-type or Cys46-mutated β1 subunits in Xenopus oocytes. ONOO− induced glutathionylation of β1 subunit and a decrease in Na+-K+ pump turnover number. This was eliminated by mutation of Cys46. ONOO− also induced glutathionylation of the Na+-K+ ATPase β1 subunit from pig kidney. This was associated with a ≈2-fold decrease in the rate-limiting E2→E1 conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na+-K+ pump occurs via glutathionylation of its β1 subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na+ levels.

Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase.

The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

FXYD proteins reverse inhibition of the Na-K pump mediated by glutathionylation of its β1 subunit

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 β1 subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β1 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 β1 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 β1 subunit, FXYD proteins reverse oxidative inhibition of the Na+-K+ pump and play a dynamic role in its regulation.

β3 Adrenergic Stimulation of the Cardiac Na+-K+ Pump by Reversal of an Inhibitory Oxidative Modification

Background— Inhibition of L-type Ca2+ current contributes to negative inotropy of &bgr;3 adrenergic receptor (&bgr;3 AR) activation, but effects on other determinants of excitation-contraction coupling are not known. Of these, the Na+-K+ pump is of particular interest because of adverse effects attributed to high cardiac myocyte Na+ levels and upregulation of the &bgr;3 AR in heart failure. Methods and Results— We voltage clamped rabbit ventricular myocytes and identified electrogenic Na+-K+ pump current (Ip) as the shift in holding current induced by ouabain. The synthetic &bgr;3 AR agonists BRL37344 and CL316,243 and the natural agonist norepinephrine increased Ip. Pump stimulation was insensitive to the &bgr;1/&bgr;2 AR antagonist nadolol and the protein kinase A inhibitor H-89 but sensitive to the &bgr;3 AR antagonist L-748,337. Blockade of nitric oxide synthase abolished pump stimulation and an increase in fluorescence of myocytes loaded with a nitric oxide–sensitive dye. Exposure of myocytes to &bgr;3 AR agonists decreased &bgr;1 Na+-K+ pump subunit glutathionylation, an oxidative modification that causes pump inhibition. The in vivo relevance of this was indicated by an increase in myocardial &bgr;1 pump subunit glutathionylation with elimination of &bgr;3 AR–mediated signaling in &bgr;3 AR−/− mice. The in vivo effect of BRL37344 on contractility of the nonfailing and failing heart in sheep was consistent with a beneficial effect of Na+-K+ pump stimulation in heart failure. Conclusions— The &bgr;3 AR mediates decreased &bgr;1 subunit glutathionylation and Na+-K+ pump stimulation in the heart. Upregulation of the receptor in heart failure may be a beneficial mechanism that facilitates the export of excess Na+.

The nitric oxide donor sodium nitroprusside stimulates the Na+–K+ pump in isolated rabbit cardiac myocytes

Nitric oxide (NO) affects the membrane Na+–K+ pump in a tissue‐dependent manner. Stimulation of intrinsic pump activity, stimulation secondary to NO‐induced Na+ influx into cells or inhibition has been reported. We used the whole‐cell patch clamp technique to measure electrogenic Na+–K+ pump current (Ip) in rabbit ventricular myocytes. Myocytes were voltage clamped with wide‐tipped patch pipettes to achieve optimal perfusion of the intracellular compartment, and Ip was identified as the shift in holding current induced by 100 μm ouabain. The NO donor sodium nitroprusside (SNP) in concentrations of 1, 10, 50 or 100 μm induced a significant increase in Ip when the intracellular compartment was perfused with pipette solutions containing 10 mm Na+, a concentration near physiological levels. SNP had no effect when the pump was near‐maximally activated by 80 mm Na+ in pipette solutions. Stimulation persisted in the absence of extracellular Na+, indicating its independence of transmembrane Na+ influx. The SNP‐induced pump stimulation was abolished by inhibition of soluble guanylyl cyclase (sGC) with 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one, by inhibition of protein kinase G (PKG) with KT‐5823 or by inhibition of protein phosphatase with okadaic acid. Inclusion of the non‐hydrolysable cGMP analogue 8pCPT‐cGMP, activated recombinant PKG or the sGC‐activator YC‐1 in patch pipette filling solutions reproduced the SNP‐induced pump stimulation. Pump stimulation induced by YC‐1 was dependent on the Na+ concentration but not the K+ concentration in pipette filling solutions, suggesting an altered sensitivity of the Na+–K+ pump to intracellular Na+.

Activation of cAMP-dependent signaling induces oxidative modification of the cardiac Na+-K+ pump and inhibits its activity

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 β1 subunit of the pump α/β 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 p47phox and p22phox, required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na+-K+ pump β1 subunit and decreased its co-immunoprecipitation with the α1 subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the α1/β1 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 ϵ-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 cAMP- and 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 β1 subunit.

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.