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J. Biol. Chem., Vol. 277, Issue 21, 18253-18256, May 24, 2002

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ACCELERATED PUBLICATION
Effects of Nitroglycerin on Soluble Guanylate Cyclase

IMPLICATIONS FOR NITRATE TOLERANCE^*

Jennifer D. Artz, Bryan Schmidt^§, John L. McCracken^§, and Michael A. Marletta^¶

From the Departments of  Chemistry and ^¶ Molecular and Cell Biology, University of California, Berkeley, California 94720-1460 and the ^§ Department of Chemistry, Michigan State University, East Lansing, Michigan 48824

Received for publication, March 22, 2002, and in revised form, March 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Soluble guanylate cyclase (sGC) is a heterodimeric hemoprotein that catalyzes the conversion of GTP to cGMP. Upon binding NO to its heme cofactor, purified sGC was activated 300-fold. sGC was only activated 67-fold by nitroglycerin (GTN) and Cys; and in the absence of Cys, GTN did not activate sGC. Electronic absorption spectroscopy studies showed that upon NO binding, the Soret of ferrous sGC shifted from 431 to 399 nm. The data also revealed that activation of sGC by GTN/Cys was not via the expected ferrous heme-NO species as indicated by the absence of the 399 nm heme Soret. Furthermore, EPR studies of the reaction of GTN/Cys with sGC confirmed that no ferrous heme-NO species was formed but that there was heme oxidation. Potassium ferricyanide is known to oxidize ferrous sGC to the ferric oxidation state. Spectroscopic and activity data for the reactions of sGC with GTN alone or with K₃Fe(CN)₆ were indistinguishable. These data suggest the following: 1) GTN/Cys do not activate sGC via GTN biotransformation to NO in vitro, and 2) in the absence of added thiol, GTN oxidizes sGC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Soluble guanylate cyclase (sGC)¹ is a hemoprotein that catalyzes the conversion of guanosine 5'-triphosphate (GTP) to the second messenger, cyclic guanosine 3':5'-monophosphate (cGMP) (1, 2). The 11 form of the enzyme, originally isolated from lung tissue, is a heterodimer comprising a 79-kDa -subunit and a 70.5-kDa -subunit (3, 4). The N-terminal region of the -subunit contains the heme binding region where the heme axial ligand has been identified as His-105 (5-7). The bound heme is a ferrous protoporphyrin IX, and sGC is activated 200-400-fold upon binding nitric oxide (NO) to this heme cofactor (4, 8, 9).

NO is a key signaling molecule in the cardiovascular system involved in smooth muscle relaxation. In endothelial cells, NO (produced from L-arginine by nitric-oxide synthase) diffuses into smooth muscle cells where it stimulates sGC, which ultimately leads to vasodilation. It has been accepted by many that nitrovasodilators, including the organic nitrates like nitroglycerin (GTN), cause vasodilation by their biotransformation to NO. Although many pathways invoking a variety of enzymes and different intermediates have been proposed to be involved in the biotransformation of GTN to NO, none have conclusively described the mechanism of action of GTN (10, 11). Even the evidence for NO production from GTN is confounded by assays not sufficiently specific for NO (12-14). Clearly the mechanism of action of the nitrovasodilators is not fully understood.

The problem of nitrate tolerance provides an additional impetus for the ongoing study of GTN. Tolerance, first documented in 1867 (15), is a clinical condition associated with a serious impairment of nitrovasodilator function as a result of its continuous administration. Mechanisms proposed to account for the development of nitrate tolerance include the depletion of essential thiols (16), decreased biotransformation of GTN to NO (17), desensitization of sGC (1), enhanced phosphodiesterase activity (18), and production of vascular superoxide (19). Despite extensive research conducted on nitrate tolerance, debate about its origin persists (20, 21). Certainly understanding the mechanism by which GTN leads to smooth muscle relaxation will help to elucidate the cause of nitrate tolerance. Herein we have examined the reactivity of GTN with purified sGC to understand the activation of sGC by GTN and gain valuable insights into the potential role of sGC in the mechanism of nitrate tolerance.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Unless otherwise indicated, all reagents were purchased from Sigma. 2-(N,N-Diethylamino)-diazenenolate-2-oxide (DEA/NO) was purchased from Cayman Chemical Company, Ann Arbor, MI. GTN was synthesized according to the method of Dunstan (22). GMPCPP was a gift from Jonathan Winger, University of California, Berkeley, CA. Heterodimeric sGC (rat 11) was expressed using the baculovirus/Sf9 expression system and purified according to previously described methods (23) except that dithiothreitol was excluded from the final gel filtration column.

sGC Activity Assay-- End point assays were performed in triplicate in a final volume of 100 µl as described previously (24) except that the GTP-regenerating system (phosphocreatine and creatine kinase) and the phosphodiesterase inhibitor (isobutyl-3-methylxanthine) were omitted. The assay mixture was comprised of 50 mM HEPES, pH 7.4, 1.5 mM GTP, 5.0 mM MgCl₂, 2.0 mM Cys (where indicated), and other components as described. Assays were incubated with either DEA/NO, GTN, or potassium ferricyanide (K₃Fe(CN)₆) for 1 min, then initiated with 0.2-0.4 µg of sGC, and quenched after 2 min with 400 µl of 125 mM Na₂CO₃ and 500 µl of 125 mM Zn(CH₃CO₂)₂. GTN was added in Me₂SO up to a final maximal concentration of 4% (v/v) Me₂SO, which was shown not to affect the enzyme activity. DEA/NO stocks were prepared in 10 mM NaOH. After centrifugation of the assay mixtures and appropriate dilution, cGMP was measured as directed using the cGMP enzyme immunoassay kit, format B (BioMol).

Electronic Absorption Spectroscopy-- All electronic absorbance spectra were collected using a Cary 3E spectrophotometer at 10 °C in 50 mM HEPES, pH 7.4. Anaerobic samples were prepared on a gas train with 10 cycles of N₂ and vacuum.

Electron Paramagnetic Resonance-- X-band EPR spectra were recorded at 13 K on a Bruker ESP-300E spectrometer with a Bruker ST9234 cavity resonator. All spectra were measured using 2.0 milliwatts of microwave power and a modulation amplitude of 9.5 G. Each spectrum is the signal average of 16 scans with the buffer spectrum subtracted. The sample volumes may have been small enough that positioning of the sample in the EPR cavity was important. Therefore, the GTN and GTN/Cys reactions were normalized to the adventitious ferric iron signal at g = 4.3 to correct for potential variation in signal. This normalization is justified because these samples came from the same enzyme stock solution. The reaction with K₃Fe(CN)₆ was not normalized because of additional free ferric iron introduced by K₃Fe(CN)₆. Anaerobic samples were prepared in an anaerobic chamber (Coy Laboratory Products, Inc., Grass Lake, MI) at room temperature and frozen in liquid N₂. The reactions were allowed to proceed at room temperature until complete as determined by the separate analysis of the change in the heme Soret maximum (~5 min for the reactions with K₃Fe(CN)₆ and GTN and 10 min for the reaction of GTN/Cys).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

sGC Activity-- At 37 °C and pH 7.4, DEA/NO is known to have a half-life of ~2 min releasing 1.5 eq of NO (25). Purified sGC was activated 313-fold by DEA/NO with a maximal specific activity of 13,763 ± 649 nmol/min/mg (Table I). The inclusion of 2.0 mM Cys in this assay did not affect the -fold activation (321-fold) or the maximal response of DEA/NO (14,081 ± 900 nmol/min/mg). sGC was activated 67-fold by GTN in the presence of 2.0 mM Cys with a maximal specific activity of 2927 ± 495 nmol/min/mg. The activity of sGC after treatment with GTN (up to 1.0 mM) and in the absence of Cys remained at approximately the basal activity (Table I). As has been previously shown (26), K₃Fe(CN)₆ oxidized sGC to the ferric oxidation state with activity not significantly different from the basal activity of the ferrous enzyme. Oxidation of sGC by K₃Fe(CN)₆ followed by removal of excess K₃Fe(CN)₆ and subsequent treatment with Cys resulted in basal level activity (data not shown). The added Cys eventually reduced the ferric heme to ferrous but again yielded the expected basal level activity (data not shown).

Electronic Absorption Spectroscopy-- sGC was isolated in the ferrous oxidation state with a heme Soret at 431 nm (Fig. 1C). A comparison of the electronic absorption spectra of sGC-NO and sGC + GTN/Cys is shown (Fig. 1A). Upon addition of DEA/NO, the enzyme rapidly binds NO giving a heme Soret at 399 nm (4). In contrast, the reaction of GTN/Cys with sGC caused a heme Soret shift of the ferrous enzyme from 431 to 393 nm. Even under anaerobic conditions, we observed no shift in the heme Soret to 399 nm upon addition of GTN/Cys to sGC. Cysteine alone had no effect on the spectrum of ferrous sGC (data not shown). When Cys was included with the reaction of DEA/NO and sGC, a decrease in the 399 nm heme Soret was observed over time, due to a decrease in the half-life of sGC-NO in the presence of thiol, in agreement with previously reported results (24).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   The sGC (300 nM) electronic absorption spectra were collected at 10 °C in 50 mM HEPES, pH 7.4. A, reaction of 100 µM DEA/NO with sGC (solid line) gives a max of 399 nm, while reaction of 1.0 mM GTN and 2.0 mM Cys with sGC (dotted line) gives a max of 393 nm. B, reaction of 100 µM K3Fe(CN)6 (solid line) or 1.0 mM GTN (dotted line) with sGC gives a max of 392 nm. C, upon reaction with 1.0 mM GTN and 2.0 mM Cys in the presence of 1.0 µM GMPCPP the heme Soret of ferrous sGC with a max of 431 nm (solid line) is shifted to 395 nm (dotted line).

The electronic absorption spectra of the reactions of GTN and K₃Fe(CN)₆ with sGC are shown in Fig. 1B. Similar to the reaction with K₃Fe(CN)₆, GTN reacted with sGC shifting the heme Soret of the ferrous enzyme from 431 to 392 nm, indicative of ferric sGC.

GMPCPP (a non-hydrolyzable analogue of GTP) is a competitive inhibitor of sGC.² The electronic absorption spectrum of sGC-NO was unaffected by the presence of GTP (or GMPCPP) and MgCl₂ (data not shown). In contrast, in the presence of 1.0 mM GMPCPP and 5.0 mM MgCl₂, the change in the ferrous heme Soret induced by GTN/Cys was slightly altered from that in their absence, shifting the heme Soret from 431 to 395 nm (Fig. 1C). Similar results were seen using GTP (data not shown).

Electron Paramagnetic Resonance-- sGC (10 µM) was treated anaerobically with 1.0 mM GTN and 2.0 mM Cys (Fig. 2A), 1.0 mM GTN (Fig. 2B), or 810 µM K₃Fe(CN)₆ (Fig. 2C), and the 13 K EPR spectra of these reactions are shown. In each of the experiments, there are distinct rhombic signals indicative of ferric heme and consistent with earlier results with g = 6.36, 5.16, and 2.0 and line widths of 3.3, 4.1, and 3.3 mT (26). The signals in each spectrum at g = 4.3 are due to adventitious free ferric iron. In Fig. 2C, the large signal associated with K₃Fe(CN)₆ is clearly visible at g = 2.5. For the reaction of GTN/Cys (Fig. 2A), where 67-fold activation was measured, the only signals observed are for ferric sGC. Interestingly there are no three-line signals, previously shown to be characteristic of five-coordinate ferrous nitrosyl complexes (g = 2.076, 2.029, and 2.005 and line widths of 5.5, 5.7, and 5.8 mT) and indicative of activated sGC (27). The intensity of the high spin ferric heme signal for the reaction of GTN/Cys with sGC was less than that of GTN or K₃Fe(CN)₆ with sGC. This observation is consistent with less enzyme being in the ferric oxidation state for the GTN/Cys-treated samples. Support for this interpretation comes from our optical data that show a more pronounced long wavelength shoulder for the GTN/Cys-treated samples (Fig. 1, A and C, dashed lines) as compared with GTN- or K₃Fe(CN)₆-treated samples (Fig. 1B).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   The 13 K EPR spectra of 10 µM sGC treated anaerobically with 1.0 mM GTN and 2.0 mM Cys (A), 1.0 mM GTN (B), or 810 µM K3Fe(CN)6 (C) are shown. In each of the experiments, there are distinct rhombic signals indicative of ferric heme. All spectra were measured using 2.0 milliwatts of microwave power and a modulation amplitude of 9.5 G. Each spectrum is the signal average of 16 scans with the buffer spectrum subtracted. The signal at g = 4.3 is from adventitious free ferric iron. In C the broad signal at g = 2.5 is due to K3Fe(CN)6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Oxidation of sGC by GTN-- The results presented herein clearly support the conclusion that the reaction of GTN alone with sGC leads to the 1-electron oxidation of ferrous sGC as depicted in Scheme 1. Potassium ferricyanide is known to oxidize sGC to the ferric oxidation state (26), and our results with GTN and sGC are essentially the same as what was observed with K₃Fe(CN)₆. First, the electronic absorption spectra of the reactions of sGC with K₃Fe(CN)₆ and with GTN are nearly identical. Slight differences in the signal intensities of the heme Soret maxima can be attributed to the subtraction of the relatively large background K₃Fe(CN)₆ signal in that reaction. Second, the EPR spectra of the reactions of sGC with GTN or K₃Fe(CN)₆ gave signals that were the same as those previously reported for ferric sGC. The slight differences in the ferric heme signal at g = 2.0 are still under investigation. Finally, as expected for a sGC oxidant, GTN alone did not activate the enzyme.

View larger version (16K): [in this window] [in a new window]   Scheme 1.   Reaction of GTN with sGC. The top reaction summarizes the results observed with K3Fe(CN)6 oxidation of the enzyme. The middle reaction shows that GTN treatment of sGC leads to complete oxidation to the ferric oxidation state. The expected co-product, GTN radical anion, was not observed. The bottom reaction summarizes the observations of sGC treatment with GTN/Cys. Although the resultant activity was 67-fold over the basal activity, no nitrosyl species was detected. Spectroscopic results show that most sGC has been oxidized to the ferric oxidation state (A). Since ferric sGC has basal level activity, this must react further to yield B whose properties are unknown especially with respect to the heme (noted here as X). Alternatively GTN/Cys or GTN/Cys may react directly with unreacted ferrous sGC to give an activated sGC species (B).

Although these results support a 1-electron oxidation of the ferrous heme of sGC by GTN, the expected GTN radical anion (GTN) co-product of sGC oxidation was not observed. Several approaches were taken to detect this radical. The temperature of the sample shown in Fig. 1B was increased from 13 K to 80 and 120 K without observation of an organic radical by EPR. In another set of anaerobic EPR experiments, attempts to trap the GTN radical anion with either 100 µM N-benzylidene-tert-butylamine-N-oxide or 100 µM 5,5-dimethyl-1-pyrroline-N-oxide were unsuccessful. Presumably the GTN radical anion intermediate must be short-lived; thus, rapid freeze-quench EPR experiments will be required to observe it.

Activation of sGC by GTN/Cys-- GTN alone did not activate sGC above basal levels as expected based on previous results with ferric sGC (26). However, in the presence of Cys, GTN activated sGC ~67-fold over the basal activity. DEA/NO, which activates sGC via NO, stimulated the enzyme greater than 300-fold in agreement with previous studies (4). The attenuated maximal response for GTN/Cys appears to be a result of sGC activation through a species other than sGC-NO. The spectroscopic evidence presented herein clearly supports this hypothesis. Upon addition of sGC to GTN/Cys under conditions where the enzyme has been shown to be activated, a 399 nm heme Soret was not observed in the electronic absorption spectrum. sGC is an excellent trap for NO, binding at a nearly diffusion-controlled rate (28, 29). If GTN/Cys stimulate sGC via NO, then trapping NO at the ferrous heme of sGC should have been spectroscopically observable by a shift of the heme Soret to 399 nm (the Soret maximum for sGC-NO). More convincingly the EPR spectra show no ferrous nitrosyl species, which is well characterized for sGC. If sGC is activated by GTN/Cys through a sGC-NO mechanism, then a three-line signal with g values of 2.076, 2.029, and 2.005 and line widths of 5.5, 5.7, and 5.8 mT would be present in the EPR spectrum (27). Close scrutiny of the electronic absorption spectra shows a Soret generated on reaction of GTN/Cys at 393 nm (or 395 nm when GMPCPP is bound) that could be interpreted as the result of a mixture of ferric sGC and sGC-NO species. However, the EPR spectra clearly show no evidence for formation of sGC-NO in the reaction of GTN/Cys with sGC. The possibility exists that the ferrous nitrosyl species in the EPR is not observed because the enzyme does not have substrate bound since it has been shown that the presence of GTP or cGMP affects the observed rate constants for NO dissociation from sGC-NO (30) and the wavelength of the heme Soret of sGC-CO with YC-1 (31). However, reactions of GTN/Cys with 5.0 µM sGC carried out with substrate (1.5 mM GTP and 5.0 mM MgCl₂) or a non-hydrolyzable substrate analogue (1.5 mM GMPCPP and 5.0 mM MgCl₂) gave the same EPR spectra as those without substrate or analogue (data not shown).

Since activation of sGC by GTN/Cys does not proceed by way of sGC-NO, then clearly an alternate mechanism of sGC stimulation must be possible. Although not directly related to what we have observed here, CO has been shown to activate the enzyme 4.8-fold over basal activity (32), showing that it is possible to activate sGC in heme-dependent but NO-independent fashion. Previous work also supports a NO-independent pathway. Using rat aortic homogenate as a source of sGC, it was shown that the rate of NO release from GTN/Cys under physiologically relevant conditions is not fast enough to account for the observed activation of sGC by GTN/Cys (33). An important observation is the lack of sGC activation by GTN in the absence of Cys. Addition of Cys to ferric sGC (where excess K₃Fe(CN)₆ has been removed) did not activate sGC (data not shown). Thus, oxidation of sGC with the subsequent addition of a thiol was not sufficient for sGC activation. To account for the activation of sGC by GTN/Cys, there must be an activated sGC species present that cannot be sGC-NO or the same sGC species as generated upon its oxidation by K₃Fe(CN)₆ or GTN alone. One possibility is that GTN/Cys-induced activation of sGC is through a ferrous sGC species not detected by conventional EPR at low temperatures. The active species could be either an EPR-silent, low spin Fe(II) heme or a high spin, S = 2, Fe(II) heme adduct. Experiments using parallel mode excitation at X-band and high frequency-high field EPR methods will help distinguish between these possibilities. A second possibility involves the reaction (or interaction) of GTN/Cys or GTN/Cys with ferric sGC generating an sGC species that is indistinguishable by conventional EPR from the sGC reaction with K₃Fe(CN)₆ or GTN alone (Scheme 1).

The mechanism of activation by GTN/Cys remains unclear, but some important details presented herein have given insight to the species responsible for activation. Direct activation of sGC by an organic nitrate seems unlikely because of the necessity for specific thiols (34). Cys (or possibly other thiols) are required for activation of the enzyme, so any proposed mechanism must account for their role. The two reasonable alternatives discussed include: 1) an EPR-silent, low spin Fe(II) heme or a high spin, S = 2, Fe(II) heme adduct generated by reaction of GTN/Cys with the sGC heme, and 2) a modified ferric sGC species derived from the reaction (or interaction) with GTN/Cys or GTN/Cys. Further spectroscopic studies are underway to differentiate between these proposed sGC active species as well as to observe the GTN radical anion.

Although the mechanism of sGC activation by GTN/Cys has not been determined, the oxidation of sGC by GTN provides an attractive explanation for a potential role of sGC in the development of nitrate tolerance. Nearly 30 years ago, tolerance to GTN was observed to be associated with a decrease in the concentration of tissue thiols. Thus, it was put forth that the oxidation of free thiols to disulfides was responsible for the development of nitrate tolerance (16). Consistent with this, it has also been shown that addition of dithiothreitol could reverse tolerance and prevent the development of tolerance when added concurrently with GTN (34). Interestingly no correlation between the concentration of free thiol and the state of tolerance was found (35). This observation contradicts a theory of tolerance involving the oxidation of free thiol but is not contradictory to a hypothesis involving the oxidation of ferrous sGC. To show that oxidized sGC is responsible for nitrate tolerance, a means to observe the oxidized species in tolerant tissue would be necessary. Nitrate tolerance is clearly a complex phenomenon with many factors ultimately contributing to the clinical end point (21). The results reported here are consistent with the direct effect of organic nitrates on sGC playing a role in the tolerance that develops from sustained nitrate therapy.

	ACKNOWLEDGEMENTS

We acknowledge members of the Marletta laboratory for discussion and critical evaluation of the manuscript and Robert Hilliard for technical assistance.

	FOOTNOTES

^* This work was supported by the Howard Hughes Medical Institute (to M. A. M.) and National Institutes of Health Grant GM54065 (to J. L. M.).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. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry, University of California, 211 Lewis Hall, Berkeley, CA 94720-1460. Tel.: 510-643-9325; Fax: 510-643-9388; E-mail: marletta@uclink.berkeley.edu.

Published, JBC Papers in Press, April 5, 2002, DOI 10.1074/jbc.C200170200

² J. A. Winger and M. A. Marletta, unpublished results.

	ABBREVIATIONS

The abbreviations used are: sGC, soluble guanylate cyclase; GTN, glycerol trinitrate (nitroglycerin); DEA/NO, 2-(N,N-diethylamino)-diazenenolate-2-oxide; GMPCPP, guanylyl ,-methylenediphosphonate; YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; mT, milliteslas.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/21/18253    most recent C200170200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Artz, J. D. Articles by Marletta, M. A. Search for Related Content PubMed PubMed Citation Articles by Artz, J. D. Articles by Marletta, M. A. Social Bookmarking                      What's this?