NADPH and heme redox modulate pulmonary artery relaxation and guanylate cyclase activation by NO

Sachin A. Gupte, Tasneem Rupawalla, Donald Phillibert Jr., and Michael S. Wolin

Department of Physiology, New York Medical College, Valhalla, New York 10595


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hemoprotein oxidant ferricyanide (FeCN) converts the iron of the heme on soluble guanylate cyclase (sGC) from Fe2+ to Fe3+, which prevents nitric oxide (NO) from binding the heme and stimulating sGC activity. This study uses FeCN to examine whether modulation of the redox status of the heme on sGC influences the relaxation of endothelium-removed bovine pulmonary arteries (BPA) to NO. Pretreatment of the homogenate of BPA with 50 µM FeCN resulted in a loss of stimulation of sGC activity by the NO donor 10 µM S-nitroso-N-acetylpenicillamine (SNAP). In the FeCN-treated homogenate reconcentrated to the enzyme levels in BPA, 100 µM NADPH restored NO stimulation of sGC, and this effect of NADPH was prevented by an inhibitor of flavoprotein electron transport, 1 µM diphenyliodonium (DPI). In BPA the relaxation to SNAP was not altered by FeCN, inhibitors of NADPH generation by the pentose phosphate pathway [250 µM 6-aminonicotinamide (6-AN) and 100 µM epiandrosterone (Epi)], or 1 µM DPI. However, the combination of FeCN with 6-AN, Epi, or DPI inhibited (P < 0.05) relaxation to SNAP without significantly altering the relaxation of BPA to forskolin. The inhibitory effects of 1 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (a probe that appears to convert NO-heme of sGC to its Fe3+-heme form) on relaxation to SNAP were also enhanced by DPI. These observations suggest that a flavoprotein containing NADPH oxidoreductase may influence cGMP-mediated relaxation of BPA to NO by maintaining the heme of sGC in its Fe2+ oxidation state.

NADPH oxidoreductase; nitric oxide; pentose phosphate pathway


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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MULTIPLE REDOX-RELATED processes influence the activity of the cytosolic or soluble form of guanylate cyclase (sGC), and many of these mechanisms appear to control vascular function through cGMP. It is well established that nitric oxide (NO) produces relaxation by stimulating sGC and that superoxide anion is an extremely potent inhibitor of this mechanism as a result of the efficient conversion of NO to peroxynitrite (14, 17). Higher levels of NO have been observed to cause a prolonged cGMP-mediated relaxation by generating peroxynitrite in amounts that promote a thiol-dependent formation of NO donors (9). H2O2 has been shown to cause relaxation of endothelium-removed bovine pulmonary arteries (BPA) through a mechanism that appears to involve the stimulation of sGC, and superoxide anion is also a potent inhibitor of these responses (4, 5). Recent studies have provided evidence that when the activity of the cytosolic Cu-Zn form of superoxide dismutase (SOD) is impaired, cytosolic NAD(H) redox influences the inhibitory effect of superoxide anion on NO-elicited relaxation of BPA by modulating NADH oxidase activity (13). Thus cytosolic NAD(H) redox seems to have a major influence on vascular relaxant responses that are mediated through the stimulation of sGC as a result of its control over the production of superoxide anion and H2O2.

When sGC has been purified from tissues, it has been shown to be isolated with a bound Fe2+ heme group, and NO activates sGC by binding to the iron of this heme group (8, 11, 19, 29, 32, 33). The purified heme-containing form of sGC has also been reported to be activated by carbon monoxide (29), carbon-centered free radicals (19), and catalase while it is metabolizing H2O2 (6). A common mechanism for enzyme stimulation was proposed to involve modifying the heme group in a manner that breaks the bonding of Fe2+ to an amino acid on sGC (19, 32). Recent spectroscopic studies have demonstrated that when NO interacts with the heme of sGC, it causes the loss of a histidine-Fe2+ binding interaction (29, 33). When the heme of sGC is in its Fe3+ oxidation state, it does not readily bind NO, and NO becomes a markedly less potent stimulator of cGMP production than the Fe2+ form of this enzyme (8, 28, 29). Interestingly, some of the agents that are employed as probes to inhibit vascular relaxation mediated by the activation of sGC by NO, including methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), have recently been shown to cause a conversion of the NO heme form of sGC to its Fe3+ oxidation state (10, 28). Thus the redox state of the heme of sGC could potentially be an additional important regulator of vascular responses to NO that are mediated through cGMP.

Ferricyanide (FeCN) was initially observed to be an inhibitor of NO stimulation of sGC in tissue extracts (1). Recent studies have shown that the well-established action of FeCN as an oxidant of the iron of hemoproteins from Fe2+ to Fe3+ is the mechanism through which this agent inhibits the stimulation of sGC by NO (28, 29). Our preliminary observations that NADPH restored the ability of NO to stimulate the activity of sGC in an FeCN-pretreated BPA homogenate preparation resulted in the development of approaches to examine whether cytosolic NADPH and heme redox could influence the actions of NO as a relaxant agent and stimulator of sGC in BPA. Because the pentose phosphate pathway is likely to be the major source of cytosolic NADPH generation in BPA, agents that inhibit NADPH generation by this pathway [250 µM 6-aminonicotinamide (6-AN) and 100 µM epiandrosterone (Epi)] were employed together with FeCN to evaluate whether the modulation of cytosolic NADPH and heme redox could influence the relaxant response of BPA to NO. Both 6-AN (21, 27) and Epi (12) have been reported to attenuate the metabolism of glucose through the pentose phosphate pathway in intact cells and tissues as a result of inhibiting the activity of glucose-6-phosphate dehydrogenase. Because the flavoprotein probe diphenyliodonium (DPI; 1 µM) was also observed in preliminary experiments to attenuate the restoration of NO stimulation of sGC by NADPH in an FeCN-treated BPA homogenate preparation, DPI was used in studies examining the actions of FeCN on BPA relaxation to NO. Thus the purpose of this study was to examine whether cytosolic NADPH and heme redox could influence the actions of NO as a relaxant agent and stimulator of sGC in BPA.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. GTP, 3-isobutyl-1-methylxanthine, phosphocreatine, creatine phosphokinase, NG-nitro-L-arginine, 6-AN, Epi, FeCN, SOD from bovine blood (3,500 U/mg), catalase from Aspergillus niger (6,660 U/mg protein), DPI, dithiothreitol, collagenase (type 4), soybean trypsin inhibitor (type 1-S), elastase (type VI), EDTA, 3-(N-morpholino)propanesulfonic acid (MOPS), forskolin, NAD(P)H, and NAD(P) were purchased from Sigma (St. Louis, MO). ODQ and enzyme immunoassay kits for measurement of cGMP were purchased from Cayman Chemical (Ann Arbor, MI). S-nitroso-N-acetylpenicillamine (SNAP) was synthesized by methods previously published (18). All other chemicals were analyzed reagent grade from Baker Chemical (Phillipsburg, NJ).

Determination of changes in force in BPA. As previously described (4), the second and third branches of the main pulmonary artery of bovine calf lungs were isolated and cut into rings ~4 mm diameter and width, and the endothelium was removed by gentle rubbing. The arterial rings were mounted on wire hooks attached to Grass (FT03) force displacement transducers for measurement of changes in isometric force. Tension was adjusted to 5 g, which is the optimal passive force for maximal contraction. Changes in force were recorded on a Grass polygraph (model 7). Vessels were incubated in 10-ml baths (Metro Scientific), which were individually thermostated (37°C) in Krebs buffer gassed with 95% air-5% CO2. Krebs buffer contained (in mM) 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose. Arteries were incubated for 2 h, during which passive tension was adjusted to maintain 5 g. The vessels were then depolarized with Krebs buffer containing KCl (123 mM) in place of NaCl before experiments were conducted. Pulmonary arterial rings were incubated with various probes described in RESULTS for 30 min before the BPA were contracted with 40 mM KCl. The combinations of the probes (FeCN, ODQ, 6-AN, Epi, and DPI) employed in this study did not alter force generated by 40 mM KCl (data not shown). The vessels were allowed to stabilize, then they were relaxed with increasing cumulative concentrations of a stimulus of sGC (SNAP) or adenylate cyclase (forskolin) in the absence and presence of probes described in the experiments reported in RESULTS. Separate arterial rings were used to examine each experimental condition.

Determination of NADPH levels in BPA. The levels of NAD(P)H in BPA were determined by HPLC by use of adaptations of previously published methods (20, 25). Briefly, BPA were pretreated with and without 6-AN or Epi in a manner similar to the studies on vascular force and immediately frozen in liquid nitrogen. The frozen tissues were crushed and homogenized in an extraction medium consisting of 0.02 N NaOH containing 0.5 mM cysteine at 0°C. The extracts were then heated at 60°C for 10 min and neutralized with 2 ml of 0.25 M glycylglycine buffer, pH 7.6. The neutralized extracts were centrifuged at 10,000 g for 10 min, the supernatnants were passed through 0.45-µm Millipore filters, and the filtered solutions were used for measurement of NAD(P)H by HPLC. NAD(P)H was eluted on a reverse-phase HPLC column (4.6 × 250 mm; Bondapak C18, Shiseido) at 40°C by the Tosoh HPLC system with slight modifications of a previously reported (20) buffer system consisting of 100 mM potassium phosphate, pH 6.0 (buffer A), and 100 mM potassium phosphate, pH 6.0, containing 5% methanol (buffer B). The column was eluted with 100% buffer A from 0 to 8.5 min, 80% buffer A plus 20% buffer B from 8.5 to 14.5 min, and 100% buffer B from 14.5 to 40 min. The flow rate was 1.0 ml/min, and the ultraviolet absorbance was monitored at 260 nm. NADPH standards were used to calibrate the HPLC. Internal standards containing 2 nmol of NADPH were used to verify the quantitative recovery of the extraction procedure and HPLC retention time in the presence and absence of tissue samples.

Determination of glutathione levels in BPA. Frozen BPA pretreated with or without 6-AN or Epi were crushed and homogenized in 0.1 N HCl and then centrifuged at 10,000 g. The supernatant was used for measurement of glutathione (GSH) after separation by ion exchange with a Hitachi L-8500 amino acid analyzer according to the method of Murayama and Sugawara (24). Although this method is also suitable for measurement of the levels of oxidized GSH, the presence of other substances derived from BPA that chromatographed with oxidized GSH prevented the detection of the presumably very low levels of this metabolite.

Preparation of the homogenate fraction of BPA. The homogenate was prepared by a previously described method (23). Briefly, after isolation of the major lobar pulmonary arteries from four animals and removal of the endothelium, the medial layer of the artery was finely minced with a commercial meat grinder and then digested with a collagenase (91 mg/ml) solution containing soybean trypsin inhibitor (0.25 mg/ml) and elastase (0.125 mg/ml) in 20 mM MOPS-KOH buffer (pH 7.4) containing 250 mM sucrose (1 g tissue/2 ml buffer) at 37°C for 15 min. After the addition of GSH to a final concentration of 2 mM, the tissue was homogenized at 0-5°C in an Eberbach homogenizer at maximum speed with five 20-s treatments. The material retained on a stainless steel sieve was rehomogenized in 50% of the original volume of MOPS-sucrose buffer, the pooled vessel homogenate was filtered through four layers of cheesecloth, and the homogenate was reconcentrated to approximate tissue enzyme levels in the assay of sGC activity. Briefly, homogenate (15 ml) was reconcentrated eightfold by removal of the homogenization buffer with an Ultrafree-45 centrifugal filter with a pore size of 5,000 Da by centrifugation at 3,000 rpm over a period of 10-12 h at 4°C. It was found that the presence of GSH was essential for the observation of reproducible effects of probes on the activity of sGC in this homogenate preparation.

Treatment of homogenates with FeCN. To study heme-reducing mechanisms that maintain the heme of sGC in the Fe2+ form, which is required for NO stimulation, BPA homogenates were initially pretreated with FeCN to oxidize the heme on sGC (28, 29). Then the FeCN was removed by ultrafiltration dialysis-concentration before the assay of sGC activity in the presence of enzyme levels that approximated the amounts present in the intact vascular tissue. In each experiment, a homogenate preparation obtained from BPA of four animals was usually divided into two aliquots: one aliquot was incubated with 50 µM FeCN for 30 min at 37°C to oxidize the heme group on sGC, and the other was used as control. The FeCN-treated and control homogenate preparations were washed with MOPS-sucrose-GSH buffer five to seven times by employing an Ultrafree-45 centrifugal filter for ultrafiltration dialysis-concentration of the homogenate to remove FeCN. All the data shown in the figures reporting FeCN and control homogenate sGC activity are from assays conducted in the presence of catalase from A. niger to remove the complicating effects of peroxide stimulation of sGC activity (4, 13). Because of differences in the recovery of sGC activity after the washing-ultrafiltration dialysis-concentration procedures, all data shown in the figures reporting sGC activity were normalized to the basal activity observed in the absence of catalase from A. niger.

Determination of sGC activity in homogenate. Guanylate cyclase activity in the arterial homogenate was determined by enzyme immunoassay measurement of cGMP formation by an adaptation of previously described methods (4). Briefly, each reaction mixture (0.2 ml final volume) contained 20 mM MOPS-KOH (pH 7.4), 0.1 mM GTP, 2 mM MgCl2, 0.3 mM 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), a GTP-regenerating system consisting of 10 mM phosphocreatine and 150 U/ml creatine phosphokinase, 0.1 ml of concentrated homogenate, and test agents as indicated. Assays of sGC activity were initiated by the addition of arterial protein. Most assays of sGC activity in the homogenate were examined in the presence of 1 µM catalase from A. niger to remove the interference of peroxide-dependent stimulation of the enzyme (4, 13). Incubations were conducted for 10 min at 37°C, and they were terminated by addition of 0.1 ml of preheated 12 mM EDTA. Then the assay mixtures were boiled for 10-15 min. Each tube was centrifuged at 15,000 rpm, and the supernatant, which was subsequently diluted fivefold, was used to estimate cGMP by enzyme immunoassay. The 10-min incubation for the assay of sGC activity was chosen to optimize the detection of cGMP under the wide variety of conditions examined. In preliminary experiments for the present study and in our previous work, it was confirmed that the activity of sGC in the presence of NO donors including SNAP is linear with time for 10 min.

Statistical analysis. Values are means ± SE. The number (n) of animals employed or determinations made in separate preparations of pooled homogenates derived from arteries of four animals are reported. Comparisons between groups were made with an ANOVA, and a Student's t-test employing a Bonferroni correction was used to determine statistical significance between groups. P < 0.05 was considered to be statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of FeCN and inhibitors of NADPH generation on the relaxation of BPA to NO. To examine whether NO stimulation of cGMP-mediated vascular relaxation is inhibited by oxidizing the heme on sGC, 100 µM FeCN was employed to convert the iron of the heme on sGC from Fe2+ to Fe3+. As shown in Fig. 1A, FeCN did not significantly alter the relaxation of 40 mM K+-precontracted BPA to the NO donor SNAP. Inhibitors of the biosynthesis of NADPH by the pentose phosphate pathway, 6-AN and Epi, were employed to lower the levels of cytosolic NADPH. These probes were also observed to not have significant effects on the relaxation of BPA to SNAP. However, the combination of FeCN with 6-AN (Fig. 1A) or Epi (Fig. 2A) significantly inhibited the relaxation of BPA to SNAP.



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Fig. 1.   Effects of combinations of 100 µM ferricyanide (FeCN) and 250 µM 6-aminonicotinamide (6-AN) on relaxation of bovine pulmonary arteries to nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP; n = 9-11; A) and to stimulus of adenylate cyclase forskolin (n = 6; B). [SNAP] and [forskolin], SNAP and forskolin concentrations, respectively.




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Fig. 2.   Effects of combinations of 100 µM FeCN and 100 µM epiandrosterone (Epi) on relaxation of bovine pulmonary arteries to NO donor SNAP (n = 6-7; A) and to stimulus of adenylate cyclase forskolin (n = 5; B). Experiments were conducted in presence of vehicle used to dissolve Epi, i.e., 0.1% ethanol.

Effects of FeCN and an inhibitor of flavoprotein-mediated electron transport on the relaxation of BPA to NO. As shown in Fig. 3A, the inhibitor of flavoprotein electron transport, DPI (1 µM), did not alter the response to SNAP. However, the combination of FeCN with DPI (Fig. 3A) significantly inhibited the relaxation of BPA to SNAP.



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Fig. 3.   Effects of combinations of 100 µM FeCN and 100 µM diphenyliodonium (DPI) on relaxation of bovine pulmonary arteries to NO donor SNAP (n = 15; A) and to stimulus of adenylate cyclase forskolin (n = 6-8; B).

Effects of FeCN and inhibitors of NADPH generation and flavoprotein-mediated electron transport on the relaxation of BPA to forskolin. The effects of FeCN, 6-AN, Epi, and DPI on the response of endothelium-removed BPA to forskolin, an agent thought to cause a cAMP-mediated relaxation through the stimulation of adenylate cyclase, were examined to confirm that these treatments were selective in their inhibitory effects on relaxation to NO. Relaxation of BPA to forskolin was not significantly altered by FeCN in the absence and presence of inhibitors of the biosynthesis of NADPH, 6-AN (Fig. 1B), and Epi (Fig. 2B). As shown by the data in Fig. 3B, the response to forskolin was not altered by FeCN in the absence and presence of the inhibitor of flavoprotein electron transport DPI.

Effects of inhibitors of NADPH generation on NADPH and GSH levels in BPA. The tissue levels of NADPH were measured to confirm that treatment with 6-AN and Epi lowered the levels of NADPH. As shown in Table 1, NADPH levels were lowered by 6-AN and Epi. The levels of GSH were measured to determine whether the lowering of NADPH by 6-AN and Epi caused alterations in the levels of GSH. Interestingly, as shown in Table 1, incubation with 6-AN and Epi actually caused a significant increase in BPA levels of GSH.

                              
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Table 1.   Effects of 6-AN and Epi on BPA levels of NADPH and GSH

Effects of the heme oxidant FeCN on the stimulation of sGC in the homogenate of BPA in the absence and presence of NADPH. To identify heme-reducing mechanisms that maintain the heme of sGC in the Fe2+ form, which is required for NO stimulation, BPA homogenates were initially pretreated with FeCN, and then the FeCN was removed by ultrafiltration dialysis-concentration before the assay of sGC activity. As shown in Fig. 4A, the NO donor SNAP did not stimulate the activity of sGC in FeCN-pretreated homogenate. NADPH was observed to have a stimulatory effect on the activity of sGC. In the presence of NADPH, SNAP stimulated the activity of sGC in the FeCN-pretreated homogenates. As shown by the data in Fig. 4B, SNAP increased the activity of sGC by 1.4 ± 0.4-fold of the basal activity in the homogenate that was a control for the FeCN-treated preparation, and the effect of SNAP was further enhanced (P < 0.05) to an increase of 2.2 ± 1.0-fold of the basal activity by the presence of NADPH in the sGC assay. NADPH also caused a stimulation of sGC activity (Fig. 5) and an enhancement of SNAP activation (data not shown) in homogenates that were reconcentrated in the absence of the washing-ultrafiltration dialysis-concentration procedure that was used for the removal of FeCN.



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Fig. 4.   Effect of 100 µM NADPH on soluble guanylate cyclase (sGC) activity in reconcentrated pulmonary arterial homogenates that were pretreated with FeCN (n = 13-15; A) or prepared as a control (n = 25-30; B) on response to NO donor SNAP (10 µM) in absence and presence of 100 µM DPI. FeCN was removed by ultrafiltration dialysis-concentration before assay of sGC activity. Measurements were conducted in presence of 1 µM fungal catalase (control). Basal sGC activities in absence of fungal catalase of 142 ± 28 pmol cGMP · min-1 · mg protein-1 in FeCN-pretreated preparation and 161 ± 32 pmol cGMP · min-1 · mg protein-1 in control preparation were used for normalization of data.



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Fig. 5.   Effect on sGC activity in reconcentrated pulmonary arterial homogenates of 100 µM NADH and 100 µM NADPH in absence and presence of 0.3 µM superoxide dismutase (SOD), 1 µM catalase (Cat) from Aspergillus niger, 1 mM dithiothreitol (DTT), and 100 µM NG-nitro-L-arginine (L-NNA). Basal sGC activity was 173 ± 23 pmol · min-1 · mg protein-1 (n = 20-25).

Effects of inhibition of flavoprotein-mediated electron transport on the NADPH-dependent enhancement of NO stimulation of sGC activity. The inhibitor of flavoprotein electron transport, DPI (1 µM), did not alter the stimulation of sGC activity by SNAP in the homogenate that was a control for the FeCN-treated preparation (Fig. 4B). However, the activity of sGC in the presence of SNAP plus NADPH plus DPI was the same as that in the presence of SNAP alone, indicating that DPI eliminated the effects of NADPH. In the FeCN-treated preparation, DPI attenuated the NADPH-mediated potentiation of the stimulation of sGC activity by SNAP without altering sGC activity observed in the presence of SNAP (Fig. 4A).

Effects of probes for reactive oxygen species, thiol redox, and NO on the stimulation of sGC activity in the homogenate by NADPH. The effect of probes on the enhancement of sGC activity by NADPH in the reconcentrated homogenates obtained from BPA was employed to determine whether superoxide anion, H2O2 metabolism by catalase, thiol redox, and the endogenous formation of NO participated in the mechanism of sGC stimulation. As shown by the data in Fig. 5, the stimulation of sGC activity by NADPH occurred in the presence of a scavenger of superoxide anion (SOD), inhibitors (4) of sGC stimulation by H2O2 metabolism by catalase (catalase from A. niger and formate, data not shown), and an inhibitor of the biosynthesis of NO from NG-nitro-L-arginine. As previously reported (13), catalase from A. niger and formate (data not shown) had an inhibitory effect on basal sGC activity, suggesting that endogenously formed H2O2 was stimulating the activity of sGC under basal conditions. Thiol reductants (dithiothreitol and beta -mercaptoethanol; data not shown) stimulated sGC activity to the level caused by NADPH. The stimulation of sGC activity by NADPH was not observed in the presence of thiol reductants. NADH inhibited sGC activity from its basal state, and it has previously been demonstrated that this response is mediated by an NADH-dependent increase in superoxide anion (13).

Effects of inhibition of flavoprotein-mediated electron transport on the attenuation of cGMP-mediated relaxation of BPA to NO by ODQ. Because relaxation of the BPA to NO donors including SNAP is essentially eliminated by 10 µM ODQ (3, 16), the stimulation of sGC appears to be the primary mechanism that mediates relaxation of this vascular segment to NO donors. As shown in Fig. 6A, the inhibitory effects of a dose of ODQ (1 µM) that causes a partial attenuation of NO-mediated relaxation of BPA (3) were enhanced by inhibition of flavoprotein electron transport with 1 µM DPI. The presence of 1 µM DPI also prevented the observed recovery of BPA relaxation to SNAP after washout of 1 µM ODQ (Fig. 6B). Whereas incubation with 1 µM DPI for >1.5 h appeared to slightly depress the time control recovery response to SNAP (Fig. 6B), this effect was not statistically significant by the ANOVA employed.



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Fig. 6.   Effects of combinations of 1 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 100 µM DPI on relaxation of bovine pulmonary arteries to NO donor SNAP (n = 8; A). ODQ was washed out of BPA, and responses were reexamined in absence of ODQ after a 30-min equilibration with Krebs buffer in absence and presence of 1 µM DPI (n = 8; B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The systems in tissues that control the redox status of the heme of sGC required for activation by NO do not appear to have been previously investigated. Evidence for a flavoprotein-containing NADPH oxidoreductase with the ability to restore NO stimulation of sGC after its heme has been oxidized has been detected. Data reported in the present study suggest that the redox state of the heme of sGC could potentially influence the relaxant response of BPA to NO. Examination of the effects of preventing cytosolic NADPH generation with inhibitors of the pentose phosphate pathway or blockade of flavoprotein-mediated electron transport together with the hemoprotein oxidant FeCN has provided evidence for the hypothesis shown in Fig. 7 that a flavoprotein-containing NADPH oxidoreductase could also have an important role in maintaining the iron of the heme of sGC in its Fe2+ state, which is thought to be required for NO-mediated relaxation through the stimulation of sGC.


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Fig. 7.   Model summarizing potential sites of action of probes employed in this study on hypothesized role of an NADPH-dependent flavoprotein containing oxidoreductase, which reduces iron of heme on sGC to Fe2+ form required for expression of NO stimulation of cGMP production and vascular relaxation.

Heme groups of certain proteins, including myoglobin and Hb, require methemoprotein reductases to maintain their Fe2+ oxidation states, and NADH appears to be a key cofactor for enzymes with this activity (15). Characterization of methemoglobin reductases in erythrocytes has resulted in evidence that these enzymes seem to be flavoproteins (15). Because stimulation of sGC activity by the NO donor SNAP in the FeCN-treated homogenate required the presence of NADPH, BPA may contain methemoprotein reductase(s), which is able to transfer electrons from NADPH to the Fe3+ form of sGC. NADPH also enhanced the actions of SNAP in the untreated homogenate, suggesting that the heme of sGC could be undergoing a partial oxidation during the preparation of the homogenate or that it is not completely reduced in the intact BPA. The inhibitory effects of DPI on the restoration of NO stimulation of sGC activity after treatment with FeCN by NADPH are consistent with the methemoprotein reductase being a flavoprotein. The role of cytosolic NADH as a cofactor for methemoprotein reductase(s) that reduces the heme of sGC is difficult to evaluate because of the high level of NADH oxidase activity present in the homogenate (23). This oxidase has been shown to generate superoxide anion in amounts that are sufficient to cause an inhibition of the stimulation of sGC activity in the homogenate by NO derived from SNAP or endogenous H2O2 (13), and the latter interaction appears to be the origin of the NADH-elicited decrease in sGC activity shown in Fig. 5. The efficiency of NADPH in restoring NO-elicited stimulation of sGC in the FeCN-treated homogenate suggests that BPA contains NADPH oxidoreductase(s) with methemoprotein reductase activity in amounts sufficient to maintain the redox state of the heme on sGC in its Fe2+ form.

NADPH was observed to have a stimulatory effect on sGC activity in the BPA homogenate in the absence of NO. Because NADPH activated sGC in FeCN-treated and control homogenate preparations, the stimulation of sGC activity by NADPH does not appear to originate from an alteration in heme redox involving a reduction of heme to its Fe2+ form (Fig. 4). On the basis of the absence of effects of scavengers of superoxide anion (SOD) and H2O2 (fungal catalase) and an inhibitor of the biosynthesis of NO, it is likely that these species do not participate in the effects of NADPH on sGC activity. The actions of NADPH also do not appear to be mediated through stimulation of sGC activity by peroxide metabolism by catalase, since the effects of NADPH were observed in the presence of fungal catalase [a form of catalase that does not stimulate sGC activity (4)] or formate [the cooxidation of which by catalase prevents the stimulation of sGC by H2O2 (4)]. Interestingly, thiol reductants such as dithiothreitol stimulated sGC activity to the level caused by NADPH, and further stimulation of sGC activity by NADPH was not observed in the presence of the thiol reductants. Because sGC can be inhibited by modification of a key thiol group through disulfide formation with low-molecular-weight thiols (S-thiolation) such as GSH (2), these preliminary observations on the stimulatory actions of NADPH and dithiothreitol in the absence of NO could result from a common mechanism mediated by the reduction of a disulfide bond that is causing an inhibition of sGC activity.

Thiol redox mechanisms may have an important role in cellular processes that control the activity of sGC. In studies on purified sGC, thiol redox-altering agents appear to have effects on cGMP production through mechanisms that seem to be dominated by processes that directly inhibit the catalytic reaction of this enzyme (2). Although this could be hypothesized to occur through an alteration in the redox state of the heme of sGC, we are not aware of any studies on purified sGC that have evaluated these interactions. Modification of a thiol on sGC could result from chemical oxidation reactions or enzymatic processes occurring during the preparation of the homogenates. Alternatively, thiol modification could be a process that is regulating sGC activity in the BPA. Tissues typically contain enzymes, such as NADPH-dependent thioredoxin reductase (30), that could be participating in an NADPH-dependent reversal of a disulfide modification that inhibits the activity of sGC. In addition, the redox status of thiols such as GSH could influence the activity of sGC through alteration of the levels of reactive O2- and NO-derived species that are present. Because 6-AN and Epi increased GSH levels and the inhibitory effects of 6-AN and Epi on relaxation to NO were dependent on pretreatment with FeCN, the data appear to be more consistent with an inhibition of the reduction of the Fe3+ heme on sGC by NADPH oxidoreductases by these agents that lower NADPH levels than with processes that are known to affect the activity of sGC through alterations in thiol redox. Although thiol redox changes are potentially processes through which sGC activity could be controlled in tissues, the increases in GSH and decreases in NADPH caused by 6-AN and Epi were not associated with statistically significant alterations in responses to the NO donor SNAP. Thus additional studies are needed to evaluate how thiol redox changes influence the function of sGC and whether the thiol redox changes occurring under many of the conditions of the present study are contributing to the results that are observed.

Studies of various tissues have suggested that cytosolic NADP(H) appears to be maintained primarily in its reduced form by the pentose phosphate pathway and perhaps other metabolic systems. In contrast, cytosolic NAD(H) is thought to be primarily in its oxidized form (26). Previous studies in the BPA have provided evidence that cytosolic NAD(H) redox appears to control an H2O2- and cGMP-mediated relaxation under normoxic conditions (31). When SOD activity in BPA is inhibited, cytosolic NAD(H) redox appears to regulate a superoxide anion-mediated attenuation of NO-stimulated cGMP-mediated relaxation as a result of modulating the activity of NADH oxidase (13). Thus these other mechanisms of control of sGC activity may dominate the effects of redox systems linked to cytosolic NAD(H) redox in the BPA. Previous studies have employed FeCN as an extracellular electron acceptor for a membrane-bound NADH-dependent electron transport chain (7), which seems to be involved in the metabolic activation of NO release by BPA from nitroprusside (22). Although it is assumed that FeCN is not readily transported into cells, observations in the present study suggest that, on prolonged (30-min) incubation, enough FeCN may enter the BPA to permit the expression of its previously observed (28, 29) potent effects on oxidizing the heme of sGC. Detection of the effects of FeCN on BPA relaxation to NO required the presence of probes employed to inhibit the function of the NADPH oxidoreductase hypothesized to maintain the heme of sGC in its reduced form. In this study, 6-AN and Epi were employed to inhibit the biosynthesis of cytosolic NADPH by the pentose phosphate pathway. These probes were observed to lower the levels of NADPH in BPA by ~30-35%. Thus the limited effects of these probes used to inhibit the function of the NADPH oxidoreductase on NO-mediated relaxation could be explained by an incomplete loss of cytosolic NADPH. Interestingly, these probes were observed to increase the levels of GSH in BPA, suggesting that it is unlikely that the observed lowering of NADPH is causing a depletion of GSH through a loss of the maintenance of GSH redox by the NADPH-dependent GSH reductase reaction. Alternatively, other systems could influence the redox status of the heme on sGC. Because none of these probes when examined individually altered the response to SNAP, these agents do not appear to have detectable nonspecific effects or significant chemical interactions with the generation and action of NO. Interestingly, the combination of FeCN with the inhibitors of the pentose phosphate pathway (6-AN and Epi) or flavoprotein-mediated electron transport (DPI) caused a significant attenuation of the relaxation to SNAP without altering the cAMP-mediated relaxation to forskolin. Because DPI would be expected to attenuate effects of FeCN that originated from its function as an extracellular electron acceptor (22), it is likely that this mechanism is not involved in the actions of FeCN on the response to SNAP. Thus the actions of the probes employed in studies on BPA relaxation to SNAP potentially originate from FeCN oxidizing the heme on sGC and by prevention of the reduction of the heme of sGC by the inhibitors of NADPH production and flavoprotein-mediated electron transport.

The influence of DPI on the inhibitory actions of ODQ on relaxation of BPA to NO were examined because this agent appears to be a rather selective oxidant of the NO heme form of sGC (28). Because 10 µM ODQ essentially eliminates relaxation of BPA to the NO donor SNAP (16), stimulation of sGC appears to be the primary mechanism through which this NO donor causes relaxation of BPA. Although ODQ has been described as an irreversible inhibitor of purified sGC as a result of oxidation of its heme to the Fe3+ form (28), observations in the present study are consistent with BPA containing a DPI-sensitive flavoprotein that is able to reverse the inhibitory effects of ODQ on relaxation to NO, presumably through reducing the heme of sGC. These observations with ODQ are important because the heme oxidant FeCN is potentially interacting with multiple additional redox systems present in the BPA as a result of its heme oxidant and electron-accepting properties. Although FeCN is probably interacting with other redox systems, observations in the present study suggest that these actions of FeCN do not appear to significantly influence the signaling mechanisms that control force generation in the BPA under the experimental conditions examined. A similar concern exists for interpreting the actions of DPI because it is likely to be having an inhibitory effect on the function of multiple flavoprotein-containing systems. The observation that DPI had minimal effects on relaxation to NO, except under conditions where FeCN or ODQ was present, suggests that the present study appeared to employ a dose of DPI that attenuated the rate of reduction of the heme of sGC sufficiently to permit the detection of its effects under conditions where sGC heme oxidation was occurring, without inhibiting other flavoprotein systems to a degree that significantly influences the vascular responses examined.

Many redox systems potentially influence the activity of sGC, and most of the probes examined in the present study could have multiple interactions with redox systems. Nevertheless, the observed actions of these probes under the conditions used are most consistent with the detection of evidence for a hypothesized mechanism shown in Fig. 7 involving the control of the redox status of the heme on sGC by NADPH oxidoreductase activity in regulating the sensitivity of BPA to relaxation by NO. The ability of NADPH to restore activation of sGC by NO in a BPA homogenate that was pretreated with FeCN suggests that an NADPH-dependent methemoprotein reductase that has not yet been characterized is present in amounts sufficient to control the redox status of the heme on sGC. Because changes in NADP(H) and heme redox are likely to occur in vascular pathophysiology associated with altered redox states or oxidative stress, these processes could participate in the attenuated responses to NO that are typically seen in diseases that affect vascular function.


    ACKNOWLEDGEMENTS

We thank Drs. Kimie Murayama and Tsutomu Fujimura (Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, Tokyo, Japan) and Dr. Hiroko Kimura (Department of Forensic Medicine, Juntendo University) for helping us measure bovine pulmonary artery levels of NADPH and GSH.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-31069 and HL-43023.

Part of this work was presented at the 71st Scientific Sessions of the American Heart Association, Dallas, TX, November 1998 and has been published in abstract form (Circulation 98: I-342, 1998).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. S. Wolin, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (E-mail: mike_wolin{at}nymc.edu).

Received 23 March 1999; accepted in final form 27 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(6):L1124-L1132
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