cGMP-Dependent Activation of Protein Kinase G Precludes Disulfide ActivationNovelty and Significance
Implications for Blood Pressure Control
Protein kinase G (PKG) is activated by nitric oxide (NO)-induced cGMP binding or alternatively by oxidant-induced interprotein disulfide formation. We found preactivation with cGMP attenuated PKG oxidation. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) blockade of cGMP production increased disulfide PKG to 13±2% and 29±4% of total in aorta and mesenteries, respectively. This was potentially anomalous, because we observed 2.7-fold higher NO levels in aorta than mesenteries; consequently, we had anticipated that ODQ would induce more disulfide in the conduit vessel. ODQ also constricted aorta, whereas it had no effect on mesenteries. Thus, mesenteries, but not aorta, can compensate for loss of NO-cGMP by recruiting disulfide activation of PKG. Mechanistically, this is explained by loss of cGMP allowing disulfide formation in response to basal oxidant production. Why aorta treated with ODQ generated less PKG disulfide that is insufficient to induce vasoconstriction was unclear. One potential explanation, especially because aorta were much less sensitive than mesenteries to exogenous H2O2-induced relaxation (EC50=205±24 and 33±2 µmol/L, respectively) was that conduit vessels may have higher peroxidase capacity. Indeed, we found that aorta express 49±22% and 80±25% more peroxiredoxin and thioredoxin, respectively, than mesenteries, and their 2-Cys peroxiredoxin peroxidatic cysteines were also less sensitive to hyperoxidation. The higher peroxidase capacity of aortas would explain their constriction during cGMP removal and their insensitivity to H2O2-induced relaxation compared with mesenteries. In summary, cGMP binding to PKG induces a state that is resistant to disulfide formation. Consequently, cGMP depletion sensitizes PKG to oxidation; this happens to a lesser extent in aortas than in mesenteries, because the conduit vessels generate more NO and express more peroxiredoxin.
A complete elucidation of blood pressure homeostasis is important because its dysregulation commonly results in hypertension, increasing the risk of kidney injury, myocardial infarction, heart failure, and stroke. Three principal pathways control vasodilation and blood pressure lowering, including nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). EDHF is largely absent in conduit vessels, but in resistance vessels, which are the principal regulators of blood pressure, it is a prevalent and perhaps the predominant mechanism controlling vasodilation.1–4
NO formation is stimulated by shear stress and circulating factors such as bradykinin, acetylcholine, and adenosine. The ability of NO to stimulate vessel relaxation is extensively characterized and involves its interaction with the heme center of guanylate cyclase, stimulating the catalytic ability of the enzyme to convert guanosine-5′-triphosphate to the second messenger cGMP. cGMP transduces many of the biological effects of NO by directly binding to and stimulating the activity of cGMP-dependent protein kinase, also known as protein kinase G (PKG). PKG activation induces substrate phosphorylation in vascular smooth muscle cells, resulting in blood vessel vasodilation by decreasing intracellular Ca2+ and myofilament Ca2+ sensitivity, thereby attenuating myosin actin crossbridge cycling.
In addition to the classic NO-cGMP pathway, PKG can also be activated by an oxidation mechanism during which the homodimer complex forms an interprotein disulfide.5 The disulfide forms in the N-terminus of PKG1α, which is held together by a leucine zipper, with structural studies confirming that Cys42 on each chain closely aligns to explain the susceptibility to oxidation. Oxidation to the disulfide state is sufficient in itself to enable PKG catalytic activity. Classic activation increases PKG Vmax, whereas disulfide activation increases the kinase affinity for substrate. H2O2 or related oxidants contribute to EDHF-dependent vasodilation of resistance vessel.6–11 This is at least, in part, attributed to EDHF-induced oxidation of PKG1α. PKG oxidation contributes to basal blood pressure as transgenic “redox-dead” Cys42Ser PKG1α knock-in mice have higher mean arterial blood pressure than wild-type littermates. Furthermore, resistance vessels isolated from knock-in mice have attenuated H2O2- and EDHF-dependent vasodilation responses compared with wild types.12
In this study we explored the functional significance of our novel primary observation that cGMP-stimulated PKG1α is resistant to H2O2-induced disulfide formation. Because vessels are bathed in NO-cGMP, which would block PKG oxidation to disulfide, this is potentially at odds with recent studies demonstrating that basal kinase oxidation contributes to blood pressure homeostasis. We previously showed knock-in mice expressing Cys42Ser redox-dead PKG1α that cannot form a disulfide and so cannot be activated by oxidants and are hypertensive compared with wild-type littermate controls. Thus, in the presence of NO-cGMP signaling the wild-type mice should not be susceptible to disulfide oxidation and should therefore perhaps have identical blood pressure to Cys42Ser redox-dead PKG1α mice. Here, we show that this potential anomaly is explained by resistance vessel having less NO and also less antioxidant peroxiredoxin peroxidase capacity than conduit vessels. Thus, resistance vessels are more sensitive to oxidant-induced relaxation because of their lower NO-cGMP and peroxiredoxin levels, which synergize to sensitize their PKG1α to oxidation. In contrast, PKG1α in conduit vessels is less susceptible to oxidation because of protection by peroxiredoxin being in greater abundance, as well as being intrinsically resistant because of higher cGMP levels.
See the online-only Data Supplement for detailed methods.
Human embryonic kidney (HEK) cells or rat primary smooth muscle cells were treated with 3 µmol/L of auranofin, 100 µmol/L of spermineNONOate, 100 µmol/L of 8-CPT-cGMP, or 100 µm of CPT-cAMP for 20 minutes before being treated for 10 minutes with 100 µmol/L of H2O2.
Vasotone measurements of aortic rings were made essentially as before,12 determining the responses of phenylephrine (EC80)-contracted vessels to H2O2 (0–1000 µmol/L) in the presence or absence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In these studies, vessels were preincubated for 30 minutes with 20 µmol/L of ODQ before treatment with increasing doses of H2O2 while being maintained in inhibitor. Vasotone measurements of mesenteric vessels were made by determining the responses of U46619 (EC80)-contracted vessels to H2O2 in the presence or absence of ODQ as above.
Proteins were resolved using SDS-PAGE, Western blotted, and then immunostained using antibodies to PKG1, PKARI, 2-Cys peroxiredoxins, thioredoxin, MnSOD, glutathione peroxidase 1, catalase, actin, hemoglobin α-subunit, Erk, Akt, or hyperoxidized 2-Cys peroxiredoxin.
Measurement of NO
The amount of nitrite was determined by measuring fluorescence (Molecular Devices, Spectra Max GeminiXS) using a 96-well plate reader. The amount of nitrite generated by each vessel was standardized per milligram of tissue.
Results are presented as mean±SEM. Differences between groups were assessed using ANOVA followed by a t test. In addition, where >2 groups were compared, they were assessed using a Bonferroni correction. Differences were considered significant at the 95% confidence level.
PKG1α Oxidation Is Attenuated by cGMP
The ability of PKG1α to form a disulfide in HEK cells is attenuated by spermineNONOate treatment, both under basal conditions and after pretreatment and followed by application of H2O2 (Figure 1A). These effects of spermineNONOate on PKG1α oxidation did not occur for PKARIα, another kinase that also forms a disulfide dimer in response to H2O2.13 In addition, the preincubation of HEK cells with the PKARI activator cAMP did not attenuate oxidation of PKG1α or PKARIα after H2O2 treatment (Figure 1B). However, the thioredoxin reductase inhibitor auranofin increased the oxidation of both kinases in HEK cells, with spermineNONOate treatment further increasing the oxidation of PKARIα but not PKG1α (Figure 1C). In addition, 8-CPT-cGMP decreased the sensitivity of PKG1α to oxidation but not PKARI in smooth muscle cells treated with H2O2, consistent with the effect observed in HEK cells treated with spermineNONOate (Figure 1D).
cGMP Attenuates PKG1α Oxidation Independent of Kinase Autophosphorylation
Figure 2A shows that mutation of the serine 64 autophosphorylation site of PKG1α to an alanine does not alter the ability of spermineNONOate to inhibit H2O2-induced kinase disulfide formation. The same also occurred when S64 was converted to the phosphomimetic aspartic acid. In addition, mutation of lysine 393 to a methionine that renders PKG1α catalytically inactive also did not prevent spermineNONOate attenuating H2O2-induced PKG1α disulfide formation. Although autophosphorylation was not required to prevent PKG1α disulfide formation by spermineNONOate, it alone was sufficient to decrease the sensitivity of PKG1α to oxidation by H2O2 (Figure 2B). This is evident for the phosphomimetic mutant, which formed less disulfide dimer compared with wild-type or S64A mutant PKG1α in HEK cells treated with H2O2. In addition, mutation of the redox-sensitive cysteine to a serine (C42S) completely prevented disulfide formation in response to H2O2.
Guanylate Cyclase Inhibition Increases the Sensitivity of PKG1α to Oxidation
Treatment of mesenteries or aorta with H2O2 promoted oxidation of both PKG1α and PKARIα, but the extent (ie, the proportion forming a disulfide dimer) was markedly higher in the mesenteries (Figure 3A). In addition, inhibition of guanylate cyclase with ODQ in unstimulated mesenteries increased PKG1α, but not PKARIα, oxidation 3.8-fold. The same observation was also made in the aorta, albeit to a lesser extent, generating a 2.9-fold increase in disulfide PKG1α. Furthermore, ODQ enhanced PKG1α oxidation in aortas, resulting in shift of the EC50 from 179.7±23.5 µmol/L in response to H2O2 to an EC50 of 57.5±14.4 µmol/L in the presence of both ODQ and H2O2, without affecting PKARIα oxidation.
In these studies, the amount of phenylephrine was lowered in ODQ-treated aorta to achieve EC80 of constriction with or without the soluble guanylate cyclase inhibitor, making the relaxation responses to H2O2 readily comparable. Thus 1 µmol/L of phenylephrine alone generated a constriction force of 4.16±0.29 mN in aorta, whereas in the presence of ODQ, 0.1 µmol/L of phenylephrine was sufficient to generate 4.62±0.36 mN. In mesenteries, 100 nmol/L of u46619 generated a constriction force of 8.25±1.17 and 8.60±0.41 mN in the presence or absence of ODQ, respectively. These data are consistent with the NO-cGMP pathway being a major regulator of vasotone in aorta but not mesenteries.
Measurement of nitrite formation using a nitrite assay showed significantly increased formation in unstimulated aortic vessels (2.70±0.53-fold) compared with mesenteries (Figure 3B). In addition, ODQ treatment increased basal constriction of aorta (2.7±0.6 mN) without affecting the mesenteries (Figure 3C).
Peroxiredoxin Levels Regulates Vessel Sensitivity to PKG1α Oxidative Activation
The inhibition of guanylate cyclase by ODQ increased the sensitivity of aorta to H2O2-induced vessel relaxation (control EC50 205±24 µmol/L, ODQ EC50 151±6 µmol/L) but had no effect on mesenteries (Figure 4A and 4B). This observation is consistent with a difference in peroxidase capacity between vessels with mesenterics expressing significantly lower 2-Cys peroxiredoxin (48.59±22.30% less) and thioredoxin (79.88±25.03% less) levels than aorta (Figure 4C). This is despite 91.46±36.70% higher catalase expression in mesenteries than in aortas. In addition, mesenteries also expressed 37.66±21.00% more MnSOD than aortas. The expression of the hemoglobin α-subunit is also 76.57±39.60% higher in mesenteries compared with aortas. Further support for a deficit in peroxide-decomposing activity of resistance vessels comes from studies showing that, in response to 100 µmol/L of H2O2, 2-Cys peroxiredoxin peroxidatic thiol hyperoxidation was 82.38±39.00% greater in mesenteries compared with aortas.
Two disparate mechanisms have evolved for activating PKG1α; one relies on binding of the second messenger cGMP, the other involves thiol oxidation inducing a disulfide homodimer. In this study, we found that these 2 mechanisms of activating PKG1α are intricately linked with the binding of cGMP preventing oxidation to the disulfide state. The N-terminus of PKG1α, which contains the redox-sensitive cysteine from each monomer of the dimer, have been mapped using NMR.14 This structural information shows that the redox cysteines in PKG1α are in close proximity and orientated to allow the formation of a disulfide bond when oxidants are present. Our observations are consistent with cGMP binding to PKG1α causing an allosteric structural change that reorientates the redox cysteines. This reorientation presumably moves the thiols too far apart or changes their molecular environment such that their pKa is increased to lower their reactivity with oxidants, either of which would attenuate disulfide formation. This effect of cGMP on PKG1α oxidation is specific to this cyclic nucleotide, because the same did not occur when cAMP levels were elevated. It was possible that PKG autophosphorylation was requisite for cGMP-mediated prevention of disulfide formation, because this site is in close proximity to the redox-sensitive cysteines and potentially may induce a conformational change because of the additional negative charge.15 However, mutation of the autophosphorylation site or preventing catalytic activity (K393M PKG1α) had no effect on the ability of cGMP to attenuate PKG1α oxidation. Conversely, the phosphomimetic mutant (S64D) form of PKG1α had significantly decreased sensitivity to oxidation, suggesting that sustained increases in cGMP (or other interventions that induce autophosphorylation) may enhance the inhibition of disulfide formation. We suggest that C42 and S64 may be close in space, and when the serine is phosphorylated this then donates protons to the thiol. This would increase the thiol pKa resulting in decreased oxidant sensitivity, as discussed earlier. Phosphoregulation of cysteine oxidant reactivity by altering thiol pKa has been reported previously for peroxiredoxin I.16
Our findings suggest that thioredoxin is capable of reducing disulfide PKG1α, because inhibition of this reductase with auranofin increased kinase oxidation. In addition, auranofin also increased the oxidation of PKARIα, which was potentiated by spermineNONOate. This NO donor may directly promote nitrosothiol formation, which can be an intermediate leading to disulfide.17 A nitrosylated PKA will rapidly transition to the disulfide state via reduction by the thiol on the opposite chain, it is likely that thioredoxin primarily reduces the disulfide kinase and not the short-lived nitrosylated intermediate, which it can also reduce.18 Because thioredoxin can also be nitrosylated to potentially induce inhibitory intradisulfide oxidation,19 this may also provide a mechanism promoting kinase oxidation. Furthermore, we found that the transnitrosylating agent nitrosocysteine can oxidize PKARIα.20 The increase in PKARIα oxidation with combined auranofin and spermineNONOate treatment did not occur for PKG1α, likely because of increased formation of cGMP, which prevents disulfide formation.
Previous studies with transgenic Cys42Ser PKG1α knock-in mice demonstrated that kinase oxidation to disulfide plays a crucial role in regulating physiological blood pressure and the EDHF response in small resistance vessels.12 However this is incompatible with the perceived view that blood vessels are bathed in NO. This is because NO stimulates cGMP synthesis, which we have shown here desensitizes PKG1α to oxidant-induced disulfide formation. To understand this potential disparity, we compared the relative sensitivity of PKG1α in resistance and conduit vessels with oxidant-induced disulfide formation. These studies showed that mesenteries were considerably more sensitive than aortas to H2O2-induced oxidation, which was potentiated by pharmacological inhibition of guanylate cyclase. This ODQ-induced potentiation of oxidation is explained by loss of cGMP, which otherwise binds PKG to induce a conformation that impedes kinase oxidation.
Surprisingly, guanylate cyclase inhibition increased PKG 1α oxidation to a greater extent in mesenteries despite these vessels generating less NO than aortas. In addition, treatment with ODQ induced contraction in aortas and improved the sensitivity of these vessels to H2O2-induced vasodilation. In contrast, both of these effects were absent in mesenteries. These findings suggest that aortas are dependent to a greater extent on NO for maintaining basal vasotone compared with mesenteries. These finding are supported by previous studies showing that vasodilation of conduit arteries is principally dependent on NO formation, whereas EDHF-mediated relaxation of small resistance vessels relies on oxidants derived from uncoupled NO synthase.21 In addition, we also found increased expression of the α-subunit of hemoglobin in mesenteries compared with aortas, corroborating the findings of a recent study showing that α-hemoglobin regulates NO bioavailability in arterioles. This may suggest that the lower amount of NO detected in mesenteries may be partly because it is scavenged by the α-subunit of hemoglobin, which is expressed in the vessel independent of red blood cells.22 Mesenteries compensate for lower NO by having cellular conditions that enhance PKG1α oxidation, which provides an alternate mechanism of vasodilation. We postulated that the increased sensitivity of PKG1α to oxidation in mesenteries (which is potentiated by ODQ) compared with aortas was because of a difference in the oxidant-decomposing reducing system between these vessels. An alternate explanation taking into account just the enhanced sensitivity of mesenteries to an EDHF protocol compared with aortas is that they produce more oxidants. However, the former hypothesis is supported by PKARIα in mesenteries being much more sensitive to exogenously applied H2O2 in terms of disulfide formation than in aortas. Furthermore, it is evidently clear that mesenteries are markedly more sensitive to exogenously applied oxidant-induced vasodilation than aortas (≈6-fold difference in EC50 for H2O2). Because the H2O2 is exogenously applied but clearly induces less PKG1α and PKARIα oxidation in aortas than in mesenteries, a logical possibility is that this is attributed to differential ability to decompose peroxide. Consistent with these ideas is evidence showing increased cellular catalase, an enzyme that decomposes H2O2 to H2O, attenuates EDHF-mediated dilation of resistance vessels.7,10 In contrast, we found mesenteries actually expressed significantly higher levels of catalase than aortas. However, catalase is unlikely to lower H2O2 concentration relevant to vasorelaxation because it is principally located in peroxisomes. Consequently, catalase may not compete with the highly abundant, ubiquitously expressed peroxiredoxin proteins, which also have a much higher affinity for H2O2, making them the principal enzymes that degrade peroxide at physiological concentrations. In many experimental models examining the mechanism of EDHF, pegylated catalase is exogenously added to vessels and, therefore, this enzyme is no longer compartmentalized, allowing it to more efficiently catalyse cytosolic H2O2. Thus, despite elevated catalase levels in mesenteries, it is unlikely to contribute to the decomposition of H2O2 at the physiological concentrations relevant to control of vasotone.
In support of decreased ability of mesenteries to decompose peroxide, we found considerably less 2-Cys peroxiredoxins and thioredoxin protein expression in these vessels compared with aortas. These enzymes play a crucial role in regulating cell redox state by decomposing H2O2. 2-Cys peroxiredoxins directly react with H2O2 to form an intramolecular disulfide, which is then reduced back to the basal reduced state by thioredoxin, which is also expressed at higher levels in the conduit vessel to provide greater peroxidase capacity. Endogenously derived oxidants, such as H2O2 generated by an EDHF protocol,21 would also probably be decomposed to a greater extent in aorta. This would help explain, along with the higher levels of NO-cGMP in aorta, why resistance but not conduit vessels have a significant EDHF response.12 Indeed, a typical EDHF protocol pharmacology inhibits NO synthesis using NG-nitro-l-arginine methyl ester or this can be achieved genetically using NO synthase null mice.23 Because this would lower cGMP levels, this would sensitize PKG1α to oxidation that would occur particularly efficiently in mesenteries because their lower relative peroxiredoxin and thioredoxin levels. A deficit in the overall peroxidase capacity of mesenteries is further supported by the prominent sensitivity of their intracellular 2-Cys peroxiredoxin to peroxidatic thiol hyperoxidation compared with aortas. The 2-Cys peroxiredoxins have a very low pKa peroxidatic cysteine thiol making them highly effective in reacting with and quenching intracellular H2O2. When this thiol hyperoxidizes, this inactivates the peroxidase activity of the peroxiredoxin and so the H2O2 concentration rises. This elevation in cellular H2O2 secondary to 2-Cys peroxiredoxin inactivation, a process that has been termed the “flood-gate effect”,24 allows less reactive higher pKa cysteine thiols to then be targeted for oxidation, such as those found on PKG1α.
In conclusion, here we provide 2 molecular-level mechanisms that synergize and help explain the differential sensitivity of resistance and conduit vessels to oxidant- or EDHF-induced vasodilation. Aortas have higher levels of NO-cGMP, as well as antioxidant peroxiredoxin and thioredoxin proteins, compared with mesenteries. The elevated cGMP promotes a PKG1α conformation that is resistant to oxidant-induced disulfide formation. In addition, aortas are better equipped to decompose oxidants such as H2O2, because they express more peroxiredoxin and thioredoxin protein than mesenteries, and this further limits PKG1α oxidation in the conduit vessel. Overall these studies help provide a mechanistic basis for the commonly held paradigm that conduit vessel vasodilation is primarily mediated by NO, whereas other mechanisms such as EDHF predominate in resistance vessels.
Sources of Funding
This work was supported by the Medical Research Council, the British Heart Foundation, the Leducq Foundation, and the Department of Health via the National Institute for Health Research Cambridge Biomedical Research Centre award to Guy’s and St Thomas’ National Health Service Foundation Trust. Also, J.R.B. is supported by a Sir Henry Wellcome postdoctoral fellowship from the Wellcome Trust (sponsor reference 085483/Z/08/Z).
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.112.198754/-/DC1.
- Received May 12, 2012.
- Revision received May 28, 2012.
- Accepted August 19, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is New?
Mesenteries are more sensitive to oxidant-induced PKG1α oxidation and vessel relaxation compared with aortas.
cGMP impairs PKG1α oxidation; this inhibitory process occurs to a greater extent in aortas because they generate more NO-cGMP.
Aortas also have higher peroxidase levels than mesenteries, also contributing to the elevated sensitivity of resistance vessels to oxidants.
What Is Relevant
Blood pressure lowering by resistance vessel dilation may be more dependent on oxidant-induced PKG1α activation than on stimulation by the NO-cGMP pathway.
Dysregulation of cGMP-dependent inhibition of PKG1α oxidation may result in hypertension.
Aortas produce more NO-cGMP and have greater peroxidase enzyme expression than resistance vessels, which together limits PKG1α oxidative activation in conduit vessels. These observations explain in part why resistance vessels are more sensitive to H2O2-induced PKG1α oxidation and vasodilation.