Roles for soluble guanylate cyclase and a thiol oxidation-elicited subunit dimerization of protein kinase G in pulmonary artery relaxation to hydrogen peroxide

Abstract

We have previously provided evidence that hydrogen peroxide (H₂O₂) stimulates soluble guanylate cyclase (sGC) under conditions where it relaxes isolated endothelium-removed bovine pulmonary arteries (BPAs). Since it was recently reported that H₂O₂ induces coronary vasorelaxation associated with a nitric oxide/cGMP-independent thiol oxidation/subunit dimerization-elicited activation of protein kinase G (PKG), we investigated whether this mechanism participates in the relaxation of BPAs to H₂O₂. BPAs precontracted with serotonin (incubated under hypoxia to lower endogenous H₂O₂) were exposed to increasing concentrations of H₂O₂. It was observed that 0.1–1 mM H₂O₂ caused increased PKG dimerization and relaxation. These responses were associated with increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at the serine-239 site known to be mediated by PKG. Treatment of BPAs with 1 mM DTT attenuated PKG dimerization, VASP phosphorylation, and relaxation to H₂O₂. An organoid culture of BPAs for 48 h with 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a heme oxidant inhibitor of sGC activation, depleted sGC expression by 85%, associated with a 67% attenuation of VASP phosphorylation and 48% inhibition of relaxation elicited by 100 μM H₂O₂. Thus both a sGC activation/cGMP-dependent and a thiol oxidation subunit dimerization/cGMP-independent activation of PKG appear to contribute to the relaxation of BPAs elicited by H₂O₂.

early studies on what appeared to be an autooxidation-mediated increase in soluble guanylate cyclase (sGC) activity in homogenates from rat lungs led to evidence that hydrogen peroxide (H₂O₂) could be a stimulator of this system (20). We had found that H₂O₂-elicited relaxation of bovine pulmonary arteries (BPAs) was associated with increases in tissue levels of cGMP (3–5). In addition, sGC activity in the 100,000 g supernatant fraction obtained from homogenates of BPAs or sGC purified from bovine lungs was stimulated by the metabolism of H₂O₂ by catalase, and superoxide appeared to function as an inhibitor of this mechanism of sGC activation (3–5, 7, 8). It was subsequently reported that endothelium-derived H₂O₂, potentially originating from an uncoupling of nitric oxide (NO) synthase, elicits the relaxation of canine coronary arteries associated with increases in tissue cGMP levels (9). Recently, a new mechanism for vasodilation of the coronary circulation of rat hearts and relaxation of isolated rat aorta by H₂O₂ has been described (2a), associated with peroxide causing a thiol oxidation-mediated dimerization of protein kinase G-1α (PKG-1α) subunits. This subunit dimerization results in a cGMP-independent activation of PKG (2a). Evidence for this PKG dimerization mechanism has also been detected in a H₂O₂-mediated vasodilator response of human coronary arterioles (21). Thus both cGMP-dependent and cGMP-independent mechanisms of PKG activation, and other processes such as thiol oxidation elicited opening of potassium channels (16, 19), potentially contribute to peroxide-elicited vasodilator mechanisms in vascular smooth muscle.

The objective of the present study was to examine the relationships and potential roles of cGMP-dependent and cGMP-independent mechanisms of PKG activation in the relaxation of BPAs to H₂O₂. Since the heme oxidant inhibitor of sGC activation 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) does not attenuate the stimulation of sGC by peroxide (12), alternative methods were adapted to examine the role of this mechanism of sGC activation in the relaxation of BPAs to H₂O₂. We developed a method for the depletion of sGC based on observations that an oxidation of the heme of sGC by ODQ promotes a ubiquitination and proteosomal degradation-depletion of this enzyme (14). This method was then used for an examination of the role of cGMP-independent vasodilator mechanisms in the response of BPAs to H₂O₂. The role of thiol oxidation-mediated subunit dimerization of PKG in the mechanism of relaxation to H₂O₂ was examined by detecting the subunit dimerization by Western blot analysis and by evaluating the effects of the reversal of dimerization by the thiol-reducing agent dithiothreitol (DTT) (2a). Changes in both the cGMP-dependent and cGMP-independent mechanisms of PKG activation were evaluated based on changes in PKG-mediated phosphorylation on the serine-239 of VASP (18).

EXPERIMENTAL METHODS

Materials.

Analyzed reagent-grade salts from Baker Chemical were used for making all physiological solutions, and all other chemicals were obtained from Sigma Chemical (St. Louis, MO) unless mentioned. cGMP-dependent protein kinase-1α (PKG-1α) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and sGC 1β-subunit and β-actin antibodies were purchased from Sigma Chemical. Vasodilator-stimulated phosphoprotein (VASP) antibodies were purchased from Cell Signaling (Beverly, MA). All gases were purchased from Tech Air (White Plains, NY).

Tissue preparation.

Bovine lungs were obtained from a slaughterhouse and maintained in ice-cold oxygenated PBS solution during transport to our laboratory. Briefly, the first and second order branches of the main lobar pulmonary arteries were cleaned and cut into rings (4 mm diameter and width), and the endothelium was removed by a gentle rubbing of the lumen (3). Fresh or organoid cultured vessels were used for vascular reactivity studies. Organoid cultured vessels were incubated in the presence and absence of 10 μM ODQ in DMEM with 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin, and amphotericin B) for 48 h at 37°C under a room air atmosphere containing 5% CO₂.

Vascular reactivity.

Freshly isolated and organoid cultured rings described in Tissue preparation were used for recording isometric force development with either Grass (FT-03) or Coulborne Instruments force displacement transducers through a PowerLab data acquisition system from ADInstruments (3, 15). Arterial rings were initially incubated at a passive tension of 5 g for 1 to 2 h in Krebs-bicarbonate buffer solution containing (in mM) 118 NaCl, 4.7 KCl, 1.5 CaCl₂, 25 NaHCO₃, 1.1 MgSO₄, 1.2 KH₂PO₄, and 5.6 glucose, gassed with 21% O₂-5% CO₂-74% N₂. The temperature was maintained at 37°C in the individually thermostated baths containing the rings. Following this 1 h of incubation, the arterial rings were depolarized with 123 mM KCl containing Krebs-bicarbonate buffer, and the rings were again reequilibrated with normal Krebs-bicarbonate buffer for another 30 min. In the initial studies investigating vascular reactivity to H₂O₂, fresh vessels were exposed to 10 min of hypoxia by changing the gassing in the baths from 21% O₂-5% CO₂-74% N₂ to 5% CO₂-95% N₂ (Po₂, ∼8–10 Torr), followed by contraction with 100 nM serotonin (5-HT) and exposure to increasing cumulative doses of H₂O₂ (1 μM–1 mM). In reducing studies, fresh vessels were incubated with 1 mM DTT and 5 mM deferoxamine for 20 min before exposure to hypoxia, 5-HT, and H₂O₂ (0.1 mM). In the subsequent studies, vessels (fresh or organoid culture) were precontracted with 25 mM KCl (25K), followed by 20 min of hypoxia. Arteries were then exposed to H₂O₂ (0.1 mM), 8-bromo-3′,5′-cGMP (300 μM), or isoproterenol (10 nM–1 μM) under these hypoxic conditions. Studies examining the effects of acute ODQ treatment were conducted in fresh or 48-h organoid-cultured arteries preincubated with 10 μM ODQ for 30 min before contraction with 5-HT or K⁺, exposure to hypoxia, and an addition of H₂O₂.

Western blot analysis.

Pulmonary arteries were rapidly frozen in liquid nitrogen under the hypoxic incubation conditions as described in results, which were used to study vascular responses (4, 5). Frozen arteries were subsequently pulverized and then homogenized in lysis buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, protease cocktail (Sigma Chemical), and phosphatase cocktail (Sigma Chemical). Protein assays were performed, and samples were prepared in electrophoresis sample buffer and separated using a 10% SDS-polyacrylamide gel. In this study, protein samples for PKG-1α were not exposed to the thiol-reducing conditions normally employed for the electrophoresis of proteins. Membranes were blocked for 1 h in Tris-buffered saline with Tween 20 plus 5% milk and incubated overnight with primary antibody. Membranes were exposed to a secondary horseradish peroxidase-linked antibody and visualized with an enhanced chemiluminescence kit (Pierce). The membranes were subsequently exposed to X-OMAT autoradiography paper (Kodak). The percentage of PKG-1α (dimer and monomer) and sGC 1β-subunit were normalized to β-actin, whereas VASP phosphorylation was obtained by normalizing phosphorylated VASP-S239 with total VASP. Protein levels were measured by densitometric analysis using the UN-SCAN-IT gel software by Silk Scientific.

Statistical analysis.

Values are means ± SE of the number of arterial segments (n) from different animals. Statistical analyses were performed with paired and unpaired Student's t-test, and a one-way ANOVA with Newman-Keuls correction was used for comparison between multiple groups. A value of P < 0.05 was used to establish statistical significance.

RESULTS

Increased dimerization and activity of PKG in BPAs exposed to 0.1–1 mM H₂O₂.

BPAs were incubated under hypoxia to lower endogenous H₂O₂ for the subsequent determination of whether H₂O₂ promotes activation of PKG as a result of a thiol oxidation-mediated subunit dimerization. Arteries were then precontracted with 100 nM 5-HT and then exposed to various doses of H₂O₂ (1 μM–1 mM). The 0.1–1 mM concentrations of H₂O₂ caused both relaxation (Fig. 1A) and enhanced dimerization of PKG (Fig. 1, B and C). PKG was dimerized by a disulfide linkage because when the gels were run under thiol-reducing conditions, only the monomer form of PKG could be detected (even under conditions of film overexposure) (Fig. 1B). The phosphorylation of VASP was also greatly increased at these concentrations of peroxide (Fig. 1D), consistent with peroxide increasing the activity of PKG.

Fig. 1.

Effects of various doses of H₂O₂ in stimulating relaxation of bovine pulmonary arteries (BPAs) associated with disulfide-mediated dimerization of PKG and stimulation of PKG-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP). A: relaxations seen at 0.1 and 1 mM doses of H₂O₂ reported as the percent relaxation of the force elicited by 100 nM serotonin (5-HT) under hypoxia (n = 6). B: Western blot showing 0.1–1 mM H₂O₂ increases dimerization of PKG, which is not seen when gels are run under thiol-reducing conditions. C: summary data for H₂O₂ increasing disulfide-mediated dimerization of PKG (n = 8). D: summary data for H₂O₂ increasing PKG-mediated VASP phosphorylation at 0.1–1 mM H₂O₂ concentrations (n = 6). *P < 0.05 vs. control (Ctrl); #P < 0.05 vs. Ctrl (dimer).

Thiol-reducing conditions attenuate BPA relaxation, disulfide dimerization, and PKG activation by H₂O₂.

The exposure of BPAs to 1 mM DTT (with 5 mM deferoxamine) 20 min before hypoxia and a subsequent contraction with 100 nM 5-HT was used as an approach to enhance thiol-reducing conditions. Deferoxamine was included to bind traces of iron that are present in physiological buffers to attenuate iron-mediated oxidation of DTT and H₂O₂. DTT-treated BPAs showed an attenuated relaxation to 0.1 mM H₂O₂ compared with H₂O₂-treated BPAs (Fig. 2A). The presence of DTT also attenuated H₂O₂-elicited increases in dimerization of PKG compared with increased dimerization caused by H₂O₂ in the absence or presence of deferoxamine (Fig. 2B). PKG activity was also reduced in DTT-treated BPAs exposed to 0.1 mM H₂O₂, based on detecting the lower levels of VASP phosphorylation under the thiol-reducing conditions examined (Fig. 2C).

Fig. 2.

Treatment of BPAs with the thiol-reductant DTT attenuates relaxation, disulfide-mediated dimerization of PKG, and VASP phosphorylation elicited by 0.1 mM H₂O₂. A: relaxation to 0.1 mM H₂O₂, reported as the percent relaxation of the force elicited by 100 nM 5-HT under hypoxia, is attenuated in the presence of 1 mM DTT (n = 7). B: typical Western blot analysis and summary data showing 1 mM DTT inhibits 0.1 mM H₂O₂-elicited dimerization of PKG (n = 6). C: summary data showing 1 mM DTT decreases 0.1 mM H₂O₂-elicited VASP phosphorylation by PKG (n = 9). Deferoxamine (DFO, 5 mM) was included in experiments with DTT to suppress its oxidation by iron impurities in buffers, and DFO did not alter the effects of 0.1 mM H₂O₂. *P < 0.05 vs. Ctrl; #P < 0.05 vs. Ctrl (dimer).

NO stimulation of sGC is not involved in H₂O₂-mediated relaxation in fresh BPAs.

To rule out the involvement of NO in H₂O₂-mediated relaxation in fresh BPAs, the vessels were incubated with 10 μM ODQ for 30 min before contracting with 5-HT. This acute treatment with ODQ did not alter force generation to 5-HT under hypoxic conditions (not shown). The relaxation of 72.6 ± 6.2% of the 0.1 μM 5-HT-induced force elicited by 0.1 mM H₂O₂ was not significantly altered (n = 9) in the presence of 10 μM ODQ (63.6 ± 6.0% relaxation). Since ODQ inhibits increases in BPA cGMP levels elicited by NO under hypoxic conditions (15), NO-mediated activation of sGC does not have a role in H₂O₂-mediated relaxation under the conditions examined.

Effects of depleting sGC in BPAs on relaxation to NO and H₂O₂.

The treatment of BPAs with the oxidant of sGC heme, 10 μM ODQ for 48 h under organoid culture conditions, was found to promote a marked (85%) depletion of sGC without altering the expression of the contractile apparatus protein α-actin (Fig. 3A). When sGC was depleted by organoid culture with ODQ, the relaxation to NO generated by exposing BPAs precontracted with 5-HT to cumulative increasing concentrations of 50 nM to 1 μM N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) was essentially eliminated, except for a small relaxation observed at the highest dose examined (Fig. 3B). While sGC depletion increased force development to 25 mM KCl (Fig. 3C), presumably by eliminating the cGMP-mediated relaxation to endogenous peroxide seen under aerobic conditions (1, 4, 5), this increase in force was relatively less in magnitude than the marked loss of relaxation to NO. These studies were conducted under aerobic conditions because hypoxia shifts the mechanism of relaxation to NO to a cGMP-independent mechanism (15). The depletion of sGC caused by 48 h of exposure to ODQ did not alter the time-dependent relaxation elicited by the activator of PKG 0.3 mM 8-bromo-3′,5′-cGMP (Fig. 4A) or cAMP-associated relaxation to isoproterenol (Fig. 4B) under the conditions where it markedly attenuated relaxation to spermine NONOate. ODQ-treated BPAs showed a decreased relaxation to H₂O₂ (Fig. 5A, top) under conditions where it markedly decreased the expression of sGC and did not alter relaxation to 8-bromo-3′,5′-cGMP or isoproterenol. The depletion of sGC by organ culture with ODQ did not alter (Fig. 5B) the relationship between PKG monomer and dimer formation seen in the presence of 0.1 mM H₂O₂. However, sGC depletion attenuated H₂O₂-elicited increases in PKG activity detected by VASP phosphorylation (Fig. 5C).

Fig. 3.

Effects of chronic 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) treatment on degradation of soluble guanylate cyclase (sGC). A: Western blot analysis showing chronic ODQ treatment-decreased expression of the sGC β₁-subunit (n = 10). With chronic ODQ treatment, relaxation to an nitric oxide donor is markedly attenuated (n = 7) (B), and force generation to 25 mM potassium is also enhanced (n = 11) (C). Spermine NONOate, N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine. *P < 0.05 vs. Ctrl.

Fig. 4.

Effects of sGC depletion on relaxation of BPAs under hypoxia to 8-bromo-3′,5′-cGMP (A) and isoproterenol (B). The absence or presence of 10 μM ODQ during 48 h of organoid culture did not alter the time dependence of relaxation to 300 μM 8-bromo-3′,5′-cGMP or relaxation to 10 nM–1 μM isoproterenol in BPAs precontracted with 25 mM KCl under hypoxia (n = 8).

Fig. 5.

Effects of sGC depletion with chronic ODQ treatment on H₂O₂-mediated BPA relaxation, PKG dimerization, and VASP phosphorylation. A, top: relaxation to 0.1 mM H₂O₂ is attenuated by the presence of 10 μM ODQ during 48 h of organoid culture in BPAs precontracted with 25 mM KCl under hypoxia (n = 7). A, bottom: acute treatment with 10 μM ODQ does not alter relaxation to 0.1 mM H₂O₂ in organ cultured arteries (n = 5). Western blot analysis showing dimerization of PKG in the presence of 0.1 mM H₂O₂ is not altered with chronic treatment with ODQ (n = 8) (B), whereas increased PKG-mediated VASP phosphorylation in the presence of 0.1 mM H₂O₂ is attenuated by ODQ-mediated depletion of sGC (n = 8) (C). *P < 0.05 vs. Ctrl.

NO stimulation of sGC is not involved in H₂O₂-mediated relaxation of organoid cultured BPAs.

To rule out a role for NO stimulation of sGC in H₂O₂-mediated relaxation of organoid cultured BPAs, arteries were incubated with 10 μM ODQ for 30 min before contracting with 25 mM KCl. An acute treatment with ODQ did not alter the force generated by 25 mM KCl or the increase in force elicited by hypoxia (not shown). The relaxation elicited by 0.1 mM H₂O₂ under hypoxic conditions was not altered by an acute treatment with ODQ in the organ-cultured arteries contracted with 25 mM KCl (Fig. 5A, bottom). Thus stimulation of sGC by endogenous NO does not appear to be upregulated during organoid culture, and it does not seem to contribute to H₂O₂-mediated relaxation.

DISCUSSION

The data in this study provide evidence that both cGMP-dependent and cGMP-independent mechanisms of PKG activation shown in Fig. 6 appear to participate in the relaxation of BPAs to H₂O₂. Since the depletion of sGC-attenuated relaxation and VASP phosphorylation, the stimulation of sGC by peroxide (3, 5) appears to be contributing to a cGMP-dependent stimulation of PKG, which is mediating vascular relaxation, whereas H₂O₂ also promoted a thiol oxidation-mediated dimerization of PKG, which was associated with increased VASP phosphorylation, suggesting that this cGMP-independent mechanism of PKG activation (2a) could also be participating in the relaxation elicited by peroxide.

Fig. 6.

Model showing hypothesized roles for both cGMP-dependent and cGMP-independent mechanisms of PKG activation in the mechanism of BPA relaxation to H₂O₂. Depletion of sGC by organ culture for 48 h with 10 μM ODQ provided evidence for the important role of a cGMP-dependent mechanism of PKG activation thought (3, 5) to result from peroxide metabolism by catalase-stimulating sGC, whereas the detection of a thiol oxidation-mediated dimerization of PKG (RSSR) provided evidence for this cGMP-independent mechanism of PKG activation also participating in the relaxation of BPAs to H₂O₂. VASP-Pi, VASP phosphorylation.

Our previous work provided evidence that H₂O₂ metabolism by catalase stimulated sGC activity and that H₂O₂ promoted increases in cGMP in BPAs in a manner that suggested that this mechanism participated in the relaxation that was observed (3, 5). Since sGC appears to be degraded after its heme has been oxidized by agents such as ODQ (14), we investigated whether organ culture with ODQ could be used as a method of depleting sGC. The data in Fig. 3A indicate that 48-h organoid culture with 10 μM ODQ depletes ∼85% of the sGC present in BPAs. When sGC has been depleted, the data in Fig. 5A, top, show that the relaxation and increase in VASP phosphorylation in response to 0.1 mM H₂O₂ was attenuated by ∼60%. Since this occurred without a detectable change in PKG subunit dimerization, it appears that the depletion of sGC is not altering this cGMP-independent mechanism of PKG activation. The data in Fig. 5A, bottom, showing the absence of an inhibitory effect of an acute addition of ODQ on the relaxation to H₂O₂, confirm previous reports (2a, 12) that the peroxide-mediated stimulation of both sGC activity and dimerization activation of PKG are not altered by this inhibitor of sGC activation by NO. In addition, the absence of an effect of acute exposure to ODQ on relaxation to H₂O₂ also provides evidence that NO is not involved in the response that is observed. Thus the previously reported (3, 5) mechanism of sGC stimulation by peroxide appears to have a prominent role in the mechanism of relaxation of BPAs to 0.1 mM H₂O₂ under the conditions examined in this study.

The detection of increased PKG dimerization at concentrations of H₂O₂ that elicit BPA relaxation suggests that this cGMP-independent mechanism of PKG activation may also contribute to the relaxation that is observed. The data in Fig. 2 provide evidence that 1 mM of the thiol-reductant DTT eliminated the relaxation to 0.1 mM H₂O₂ and any increases in PKG dimerization or VASP phosphorylation caused by this dose of peroxide. This effect of DTT supports the previously reported (2a) concept of peroxide, promoting a dimerization of PKG in BPAs through a thiol oxidation-mediated mechanism seen in saline-perfused rat hearts and isolated rat aortas. However, based on the effects of sGC depletion and the smaller extent of subunit dimerization observed in the present study, this cGMP-independent mechanism of PKG activation appears to be a less dominant factor in the mechanism of BPA relaxation compared with the studies previously reported (2a) in rat vascular tissue. A contributing factor to the prominent effects of DTT on inhibiting relaxation and VASP phosphorylation may result from it also impairing the metabolism of H₂O₂ by catalase associated with sGC activation. This could potentially occur as a result of DTT enhancing the reduction of thiols used for the metabolism of H₂O₂ by competing enzymes (10), such as glutathione peroxidase and/or peroxiredoxins. Since an acute treatment with ODQ does not attenuate relaxation associated with H₂O₂ stimulation of sGC, it is also possible that cGMP-dependent activation of PKG is an important factor in the previously reported (2a) rat vascular tissue study. This is because the previous study interpreted the absence of the effects of acute treatments with ODQ as evidence for sGC not being involved in relaxation to peroxide. Thus, while our study detects H₂O₂ promoting vascular relaxation associated with a thiol oxidation-mediated activation of PKG, the relatively small amount of dimerized PKG and prominent effects of sGC depletion seen in BPAs suggest the cGMP-independent mechanism of PKG activation may be a less dominant factor than its role reported in the previous study (2a), examining relaxation to H₂O₂ in rat vascular tissue.

This study provides evidence that PKG activation through both cGMP-dependent and cGMP-independent mechanisms appear to be prominent factors in the mechanism of BPA relaxation to peroxide. While peroxide could promote relaxation through more direct effects on relaxing mechanisms such as the opening of potassium channels (16, 19), PKG has effects on multiple relaxing mechanisms including the opening of potassium channels (13). The oxidation of cytosolic NADPH as a result of the metabolism of peroxide by thiol-dependent pathways is potentially an alternative mechanism that could be hypothesized to promote relaxation, because cytosolic NADPH oxidation appears to function as a coordinator of multiple mechanisms of relaxation (11). However, since our recent studies suggest that the oxidation of cytosolic NADPH resulting from the inhibition of its generation by glucose-6-phosphate dehydrogenase results in PKG dimerization and VASP phosphorylation in BPAs (17), PKG may also have a role in coordinating NADPH oxidation-regulated processes mediating relaxation. Since changes in H₂O₂ are thought to be contributing factors to vascular regulatory mechanisms involved in the sensing of hypoxia (4, 16, 19) and in promoting the development of pulmonary hypertension (2), the function of both cGMP-dependent and cGMP-independent mechanisms regulating PKG is likely to be an important factor in both physiological and pathophysiological processes influencing the pulmonary circulation.

GRANTS

This study was support by National Heart, Lung, and Blood Institute Grants HL-31069, HL-43023, and HL-66331.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).