©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Peroxynitrite-induced Accumulation of Cyclic GMP in Endothelial Cells and Stimulation of Purified Soluble Guanylyl Cyclase
DEPENDENCE ON GLUTATHIONE AND POSSIBLE ROLE OF S-NITROSATION (*)

(Received for publication, February 27, 1995; and in revised form, May 1, 1995)

Bernd Mayer (§) , Astrid Schrammel , Peter Klatt , Doris Koesling (1), Kurt Schmidt

From the Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, A-8010 Graz, Austria and the Institut für Pharmakologie, Freie Universität Berlin, D-14195 Berlin, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Peroxynitrite (ONOO) is widely recognized as mediator of NO toxicity, but recent studies have indicated that this compound may also have physiological activity and induce vascular relaxation as well as inhibition of platelet aggregation. We found that ONOO induced a pronounced increase in endothelial cyclic GMP levels, and that this effect was significantly attenuated by pretreatment of the cells with GSH-depleting agents. In the presence of 2 mM GSH, ONOO stimulated purified soluble guanylyl cyclase with a half-maximally effective concentration of about 20 µM. In contrast to the NO donor 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), ONOO was completely inactive in the absence of GSH, indicating that thiol-mediated bioactivation of ONOO is involved in enzyme stimulation. Studies on the reaction between ONOO and GSH revealed that about 1% of ONOO was non-enzymatically converted to S-nitrosoglutathione. The authentic nitrosothiol was found to be stable in solution, but slowly decomposed in the presence of GSH. GSH-induced decomposition of S-nitrosoglutathione was apparently catalyzed by trace metals and was accompanied by a sustained release of NO and a 40-100-fold increase in its potency to stimulate purified soluble guanylyl cyclase. Our data suggest that the biologic activity of ONOO involves S-nitrosation of cellular thiols resulting in NO-mediated cyclic GMP accumulation.


INTRODUCTION

Stimulation of soluble guanylyl cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2; sGC)()by L-arginine-derived NO results in intracellular accumulation of the second messenger cyclic GMP (cGMP) and represents a widespread signal transduction mechanism involved in a variety of biological processes, such as endothelium-dependent relaxation, platelet aggregation, and neurotransmission(1, 2, 3) . Purification of sGC revealed that the enzyme is a heterodimer containing stoichiometric amounts of ferro-protoporphyrin IX(4, 5, 6, 7) . NO exhibits high affinity for ferrous heme(8) , and a heme-deficient mutant of sGC has recently been shown to be insensitive to NO(9) , supporting the hypothesis that NO activates the enzyme through binding to the prosthetic heme group resulting in formation of a ferrous-nitrosyl-heme complex and consequent change in protein conformation(10) .

As a free radical, NO reacts with several intracellular targets(11) . Reaction with molecular oxygen results in the generation of NO or other nitrogen oxides (NO)with potential cytotoxic properties(12, 13) . This reaction follows second order kinetics with respect to NO and is thus rather slow at low NO concentrations(14, 15, 16) , suggesting that additional reactions may account for the short half-life of NO in tissues. Enhanced biological activity of NO in the presence of SOD has indicated that superoxide contributes to the inactivation of NO (17) due to a fast reaction of NO with O to yield peroxynitrite (ONOO)(18) . ONOO is stable in alkaline solutions, but a species with hydroxyl radical-like properties is generated as intermediate upon protonation of ONOO to the corresponding peroxynitrous acid(19) . The intermediate appears to be highly reactive, inducing oxidation of various cellular targets including sulfhydryls (20) and lipids (21, 22, 23) . More recently, it was demonstrated that ONOO apparently mediates several effects previously attributed to NO, e.g. covalent modification of glyceraldehyde-phosphate dehydrogenase (24) and inhibition of aconitase(25, 26) . Together with the finding that activated macrophages release most of their NO as ONOO(27) , these results suggest that decomposition of ONOO at physiological pH may be a major component of NO cytotoxicity.

Recent reports indicate that ONOO also may have beneficial effects. Solutions of authentic ONOO were shown to induce relaxation of vascular smooth muscle (28, 29) and to inhibit platelet aggregation(30) . The molecular mechanisms underlying these physiological effects of ONOO are unclear. Liu et al.(28) speculated that relaxation may be due to small amounts of NO spontaneously released during ONOO decomposition, and Wu et al.(29) reported on pronounced increases in NO release when ONOO was incubated in the presence of GSH or tissue homogenates. Similarly, Moro et al.(30) found that ONOO inhibited aggregation of blood platelets only when serum albumin or GSH were present. It is conceivable, therefore, that the NO-like properties of ONOO are due to S-nitrosation of cellular proteins or GSH. However, the nitrosating potential of ONOO is being discussed controversially. Wink et al.(31) failed to detect an S-nitrosated species arising from the reaction of ONOO with GSH, whereas others have reported on formation of GSNO (30) or another, as yet unidentified S-nitroso compound(29) . The present study was designed to elucidate the molecular mechanisms underlying the biologic activity of ONOO.


EXPERIMENTAL PROCEDURES

Materials

Recombinant bovine lung soluble guanylyl cyclase was purified from baculovirus-infected Sf9 cells as described previously(5, 9) . ONOO was synthesized as described (32, 33, 34) . For inactivation, ONOO (10 mM) was incubated for 5 min in 0.5 M phosphate buffer, pH 7.5. GSNO was a kind gift from Dr. Harold F. Hodson, Wellcome Research Laboratories, Beckenham, United Kingdom. 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO) (35) was from NCI Chemical Carcinogen Repository, Kansas City, MO. 10-fold concentrated stock solutions of DEA/NO were prepared in 10 mM NaOH. [-P]GTP (400 Ci/mmol) was purchased from MedPro (Amersham), Vienna, Austria; the other chemicals were from Sigma, Vienna, Austria.

Culture of Endothelial Cells and Determination of Intracellular cGMP

Porcine aortic endothelial cells were cultured as described previously(36, 37) . Briefly, endothelial cells were isolated by enzymatic treatment (0.1% collagenase) and cultured up to 3 passages in Opti-MEM (Life Technologies, Inc., Vienna, Austria) containing 3% fetal calf serum and antibiotics. Prior to experiments, endothelial cells were subcultured in 24-well plastic plates and grown to confluence (2 10 cells/dish). For depletion of GSH, cells were incubated for 30 min at 37 °C in the presence of different concentrations of CDNB or DEM prior to experiments. Subsequently, the culture medium was removed, and the cells were washed once and equilibrated in incubation buffer (isotonic 50 mM HEPES buffer, pH 7.4, containing 2.5 mM CaCl, 1 mM MgCl, 1 mM 3-isobutyl-1-methylxanthine, and 1 µM indomethacin). After 15 min, ONOO or DEA/NO were added to give the initial final concentrations as indicated, and reactions were stopped 4 min later by removal of the incubation buffer and treatment for 1 h with 1 ml of 0.01 N HCl. Intracellular cGMP was measured in the supernatants of the lysed cells by radioimmunoassay.

Determination of Endothelial GSH Levels

The DTNB colorimetric method (38) was used for determination of GSH, which represents the only measurable soluble thiol in endothelial cells(39) . Cells were cultured in Petri dishes (diameter 90 mm), and confluent monolayers (5 10 cells/dish) were preincubated in culture medium at 37 °C for 30 min in the presence of increasing concentrations of thiol-depleting agents or vehicle (0.1% ethanol). Cells were washed and equilibrated for 15 min in incubation buffer (see above). Subsequent to aspiration of the supernatant, 1 ml of a 2% (w/v) solution of 5-sulfosalicylic acid was added for cell lysis and deproteinization. Samples were centrifuged and aliquots of 0.5 ml mixed with 0.5 ml of a 300 mM sodium phosphate buffer, pH 7.5, containing 10 mM EDTA and 0.2 mM DTNB. After 5 min, the absorbance at 412 nm was measured and the amount of soluble thiols quantitated by comparison with GSH standards. The extinction coefficient of the chromophore was 14.3 mM cm under these conditions, which is closely similar to the reported value of 13.6 mM cm(38) .

Determination of Guanylyl Cyclase Activity

Purified soluble guanylyl cyclase (0.15 µg) was incubated at 37 °C for 10 min in a total volume of 0.2 ml of a triethanolamine/HCl buffer (50 mM, pH 7.5) containing 0.5 mM [-P]GTP (200,000-300,000 cpm), 3 mM MgCl, and 1 mM cGMP in the absence and presence of 2 mM GSH. Reactions were started by addition of 20-fold stock solutions of ONOO, DEA/NO, or GSNO and stopped by ZnCO precipitation followed by isolation of [P]cGMP by column chromatography as described(40) . In some experiments, sGC was preincubated for 2 min at 37 °C in the presence of 2 mM GSH and different concentrations of ONOO prior to further stimulation of the enzyme for 8 min with 10 µM DEA/NO in a total incubation volume of 0.2 ml. Results were corrected for enzyme-deficient blanks and recovery of cGMP.

Reaction of ONOOwith GSH

For analysis of S-nitrosated compounds derived from the reaction of ONOO with GSH, the authentic compounds (1 mM each) were incubated for 2 min in 100 mM phosphate buffer at pH values ranging from 6.0 to 8.5, and 30-µl samples were injected onto a 250 4-mm C reversed-phase column equipped with a 4 4-mm C precolumn for HPLC analysis (LiChroGraph L 6200, LiChrospher 100 RP-18, 5-µm particle size, Merck). Elution was performed isocratically at a flow rate of 0.75 ml/min with 20 mM sodium phosphate buffer, pH 7.4, containing 5% (v/v) methanol. Absorbance was continuously monitored at 338 nm (LiChroGraph L 4250, Merck) to detect S-nitrosated products. The method was calibrated daily with freshly prepared solutions of authentic GSNO. Identification of the reaction product was based on coelution with authentic GSNO in the following solvents: (i) 20 mM sodium phosphate buffer, pH 3.0, 5% (v/v) methanol; (ii) 20 mM sodium phosphate buffer, pH 7.4; (iii) 20 mM sodium phosphate buffer, pH 7.4, 1% (v/v) methanol; (iv) 20 mM sodium phosphate buffer, pH 7.4, 5% (v/v) methanol; and (v) acetonitrile, HO, acetic acid (2.5:97.5:0.1, v/v), yielding retention times of 6.3, 4.4, 4.1, 3.3, and 2.2 min, respectively. Solvent v has been used by Wu et al. for analysis of S-nitrosated products of GSH(29) .

GSH-induced Release of NO from GSNO

Stability of GSNO was assessed spectroscopically with a diode array spectrophotometer (8452A, Hewlett Packard, Vienna, Austria) in the absence and presence of 1 mM GSH. Release of NO was measured aerobically with a commercially available Clark-type electrode (Iso-NO, World Precision Instruments, Mauer, Germany) as described previously(16) . Samples were injected through a septum into completely filled vials to avoid interphase mass transfer. Calibration of the electrode was performed daily and showed linear response from 10 nM to 1 µM NO with an average slope of 0.8 nM NO/pA output current.


RESULTS

Effects of ONOOand DEA/NO on Endothelial cGMP Accumulation

As shown in Fig. 1, ONOO increased cGMP levels in cultured endothelial cells in a concentration-dependent manner with an EC of about 0.2 mM. Maximal effects were comparable to those elicited by the NO donor DEA/NO, which raised intracellular cGMP levels up to 50 pmol/10 cells with an EC of 0.2 µM. Inactivated ONOO (1 mM) induced only marginal accumulation of cGMP (from 2.4 ± 0.8 to 4.7 ± 1.1 pmol/10 cells; n = 3). Control experiments performed with NaNO showed that 0.1 and 1 mM nitrite enhanced endothelial cGMP levels to 3.1 ± 0.28 and 8.1 ± 1.04 pmol/10 cells (n = 3), respectively, indicating that nitrite, which was present in the ONOO stock solutions at about equimolar concentration, was responsible for the marginal effect of inactivated ONOO.


Figure 1: Effects of ONOO and DEA/NO on cGMP accumulation in endothelial cells. Endothelial cells were washed, equlibrated for 15 min at 37 °C in incubation buffer (see ``Experimental Procedures''), and stimulated for 4 min with the indicated concentrations of ONOO (filled circles) or DEA/NO (unfilled circles). Subsequent to aspiration of the supernatant, cells were lysed with HCl, and intracellular cGMP accumulation was measured by radioimmunoassay. Data represent mean values ± S.E. of six separate experiments performed in triplicate.



To investigate whether intracellular GSH mediates ONOO-induced cGMP accumulation, cells were pretreated for 30 min with increasing concentrations of the GSH-depleting agents CDNB or DEM(41) . Fig. 2A shows that CDNB (1-100 µM) decreased endothelial GSH levels in a concentration-dependent manner and significantly reduced maximal peroxynitrite-induced cGMP accumulation, whereas the effect of the NO donor DEA/NO remained unaffected. As shown in Fig. 2B, DEM had very similar effects, albeit at higher concentrations. At the used concentrations, neither of the two agents did induce any detectable release of lactate dehydrogenase into the culture medium (not shown). The GSH levels measured under control conditions (10 nmol/10 cells) are in excellent accordance with values reported previously for cultured endothelial cells(42) . Based on an endothelial cell volume of 1 pl(43) , this corresponds to intracellular GSH concentrations of approximately 10 mM.


Figure 2: Effects of CDNB (A) and DEM (B) on cGMP accumulation and levels of soluble thiols in endothelial cells. Endothelial cells were preincubated in culture medium at 37 °C for 30 min in the presence of increasing concentrations of CDNB, DEM, or vehicle, washed, equilibrated for 15 min in incubation buffer (see ``Experimental Procedures''), and stimulated for 4 min with 1 mM ONOO (unfilledcircles) or 1 µM DEA/NO (filledcircles). Subsequent to aspiration of the supernatant, cells were lysed with HCl and intracellular cGMP accumulation was measured by radioimmunoassay. Soluble thiols (stripedcolumns) were determined subsequent to preincubation of the cells with the indicated drugs by the DTNB colorimetric method as described under ``Experimental Procedures.'' Data represent mean values ± S.E. of three separate experiments performed in triplicate.



Effects of GSH on Stimulation of sGC by ONOOand DEA/NO

Subsequently, we have performed experiments with purified sGC to get insights into the mechanisms accounting for ONOO-induced cGMP accumulation. Incubations have been performed in the absence and presence of 2 mM exogenously added GSH, but it should be noted that sGC preparations contained endogenous GSH for stabilization of the enzyme during purification(5) . However, final dilutions of sGC resulted in GSH concentrations of less then 10 µM during incubations. Basal sGC activities were 40.0 ± 13.2 and 78.8 ± 15.8 nmol of cGMP mg min (mean ± S.E.; n = 11) in the absence and presence of 2 mM GSH, respectively. As shown in Fig. 3A, ONOO had no effect on cGMP formation at concentrations of up to 1 mM in the absence of the added thiol, but markedly stimulated the enzyme in the presence of 2 mM GSH. The effect was biphasic with a sharp maximum at 0.1 mM and an EC of approximately 20 µM ONOO. The sGC activities maximally achievable with ONOO were about 650 nmol cGMP mg min and thus considerably lower than those observed in the presence of DEA/NO (4 µmol mg min) or GSNO (1 µmol mg min) (see below). The effect of ONOO was virtually abolished (1.5-fold stimulation) when it had been inactivated by incubation for 5 min at pH 7.5 prior to experiments. As observed with endothelial cells, this marginal stimulation of sGC was mimicked by authentic nitrite, which induced a 1.6-fold stimulation of the enzyme at a concentration of 0.1 mM (not shown).


Figure 3: Effect of GSH on stimulation of sGC by ONOO and DEA/NO. Stimulation of purified sGC by ONOO (A) and DEA/NO (B) was assayed in the absence (filled circles) and presence (unfilledcircles) of 2 mM GSH as described under ``Experimental Procedures.'' Data represent mean values ± S.E. of three separate experiments performed in duplicate.



It has been suggested that protein SH-groups may be critically involved in the regulation of sGC(44) , indicating that the effect of GSH on ONOO-induced enzyme stimulation could reflect reduction of protein sulfhydryls essential for sGC stimulation. To address this issue, we have additionally studied the effect of GSH on cGMP accumulation induced by the spontaneous NO donor DEA/NO. Fig. 3B shows that DEA/NO potently stimulated purified sGC with an EC of about 50 nM even in the absence of GSH. Addition of GSH (2 mM) did not potentiate the effect of DEA/NO but, instead, induced a rightward shift of the concentration response curve resulting in an EC of DEA/NO of about 0.3 µM without affecting maximal guanylyl cyclase activity. These data demonstrate that ONOO-induced but not NO-induced stimulation of sGC requires presence of GSH, suggesting that the effect of the thiol is related to bioactivation of ONOO.

The biphasic effect of ONOO and the incomplete activation of sGC indicated that the enzyme may be inhibited or inactivated by high concentrations of ONOO. To investigate this, we have preincubated sGC for 2 min with increasing concentrations of ONOO prior to addition of DEA/NO (10 µM). TableI shows that pretreatment of non-stimulated sGC with high concentrations of ONOO (0.1 mM) antagonized further activation of the enzyme by NO. Taking into account the short half-life (1 s) of ONOO at physiological pH(18) , 2 min of preincubation should have resulted in virtually complete inactivation of ONOO, excluding the possibility that the inhibitory effects were due to a reaction of ONOO with free NO, as we have observed recently using a Clark-type electrode for NO detection.()

Formation, Stability, and Biologic Activity of GSNO

Reaction of ONOO with GSH (1 mM each) was complete within 2 min and resulted in formation of a compound, which exhibited a light absorbance maximum at 338 nm and co-eluted with authentic GSNO from RP-18 columns in five different solvents. Fig. 4shows that formation of GSNO was increased at increasing pH; half-maximal efficiency was observed at pH 7.0, maximal yields required pH values 8.5. The reaction was not inhibited in the presence of EDTA (not shown).


Figure 4: Formation of GSNO from ONOO and GSH as a function of pH. Authentic ONOO (1 mM) was allowed to react with GSH (1 mM) for 2 min at ambient temperature in 100 mM phosphate buffer adjusted to pH values as indicated. Samples (30 µl) were analyzed for GSNO by HPLC as described under ``Experimental Procedures.'' Data represent mean values ± S.E. of three separate experiments.



If this non-enzymatic S-nitrosation of GSH was responsible for the cGMP accumulation induced by ONOO, GSNO should decompose and release NO under certain conditions. We have investigated this issue by both spectrophotometric analysis of authentic GSNO and electrochemical measurement of NO release. As revealed by recording the absorbance at 338 nm over time, GSNO was stable for at least 5 h at pH 2.0-9.0. However, presence of 1 mM GSH induced a time-dependent, EDTA-sensitive decomposition of the nitrosothiol (t 3 h at pH 7.5; not shown). Decomposition of GSNO was accompanied by release of NO as revealed by the electrochemical measurements shown in Fig. 5. GSNO (50 µM) alone induced a very slight transient response of a Clark-type NO electrode, corresponding to apparent NO concentrations of less than 20 nM, whereas a pronounced, long-lasting release of NO was observed upon addition of 50 µM GSH. GSH-induced NO release was markedly enhanced in the presence of 10 µM CuCl and completely blocked by 10 mM EDTA. The initial rates of NO release shown in Fig. 5were 0.46 and 2.00 µM min in the absence and presence of added CuCl, respectively, steady state concentrations of NO were reached about 2 min after addition of GSH.


Figure 5: GSH-induced release of NO from GSNO. NO was measured with a Clark-type NO-sensitive electrode at ambient temperature in 1.8 ml of a 100 mM sodium phosphate buffer, pH 7.5, in the absence (--) or presence(- - -) of 10 mM EDTA as described under ``Experimental Procedures.'' At time point zero, a stock solution of GSNO was injected through a septum to give a final concentration of 50 µM. After 2 min, GSH and, where indicated, CuCl where added to give final concentrations of 50 and 10 µM, respectively. An original tracing representative for three similar experiments is shown.



GSH-induced release of NO from GSNO was further confirmed in experiments with purified sGC. Fig. 6shows that GSNO stimulated the formation of cGMP in a concentration-dependent manner. Maximal sGC activities of about 1 µmol of cGMP mg min were observed with 0.3 mM GSNO, the EC of the nitrosothiol was 8.0 µM. In the presence of 2 mM GSH, the EC of GSNO was 0.18 µM and maximal effects were obtained at GSNO concentrations as low as 1 µM, further suggesting that GSH triggers release of NO from the nitrosothiol.


Figure 6: Effect of GSH on stimulation of sGC by GSNO. Stimulation of purified sGC by authentic GSNO was assayed in the absence (filledcircles) and presence (unfilledcircles) of 2 mM GSH as described under ``Experimental Procedures.'' Data represent mean values ± S.E. of five separate experiments performed in duplicate.




DISCUSSION

Previous reports have demonstrated that ONOO exhibits NO-like biologic activities, inducing relaxation of blood vessels (28, 29) and inhibiting platelet aggregation(30) . The present study strongly suggests that these effects of ONOO may be mediated by thiol-dependent stimulation of sGC. Authentic ONOO induced a pronounced accumulation of endothelial cGMP levels with a maximal effect comparable to that of the NO donor DEA/NO. Consistent with rather long diffusion distances, presumably resulting in significant decomposition of the added ONOO(18) , accumulation of cellular cGMP required about 10-fold higher concentrations of ONOO (EC 0.2 mM) than stimulation of purified soluble guanylyl cyclase (EC 0.02 mM; see below). The effect of ONOO on endothelial cGMP accumulation was significantly attenuated by depletion of intracellular GSH with the thiol-depleting agents CDNB and DEM, indicating that GSH is an important cellular target mediating the biologic activity of ONOO. However, the effects of the drugs were incomplete, and at higher concentrations these agents were toxic to the cells (not shown). Thus, the residual GSH (approximately 1-2 mM) could be sufficient to support GSNO formation or, alternatively, additional targets may be involved in ONOO action. Free sulfhydryl groups of cellular proteins, for instance, could react with ONOO to yield comparably stable S-nitroso derivatives that slowly release NO(45, 46) . Lack of effect of CDNB and DEM on cGMP accumulation induced by DEA/NO is in accordance with previous findings showing that the effects of several NO donors were not significantly affected by pretreatment of endothelial cells with thiol-depleting drugs(39, 47, 48) . Taken together, these data indicate that NO-induced accumulation of cGMP occurs in a thiol-independent manner, whereas the effect of ONOO may involve modification of intracellular sulfhydryls and perhaps additional as yet unknown targets.

In good accordance to the results obtained with intact cells, we found that addition of GSH was not required for direct stimulation of purified sGC with the NO donor DEA/NO but obligatory for the effect of ONOO. Although presence of low concentrations of endogenous GSH did not allow us to unambiguously demonstrate thiol-independent enzyme stimulation, our results are consistent with a previous study investigating this issue using thiol-free sGC that had been purified under anaerobic conditions(49) . With an EC of about 50 nM, DEA/NO is the most potent NO donor described so far. For unknown reasons, DEA/NO was somewhat less potent in the presence of GSH. Under these conditions, the drug exhibited an EC of about 0.3 µM, which is virtually the same as that found for DEA/NO-induced endothelial cGMP accumulation (0.2 µM; this study) and vascular relaxation (0.2 µM)(35) . In contrast to DEA/NO, ONOO stimulated purified sGC in a strictly GSH-dependent manner. The maximal effects of ONOO were much less pronounced than those of DEA/NO, apparently due to the inactivation of sGC by high concentrations of ONOO, revealed by the biphasic effect shown in Fig. 3A and inhibition of sGC by preincubation with high concentrations of ONOO (Table 1).



Our data suggest that the biologic activity of ONOO may be mediated by non-enzymatic nitrosation of GSH. A previous investigation of ONOO-induced sulfhydryl oxidation points to the formation of the corresponding sulfenic or sulfinic acids(20) , but formation of nitrosothiols has not been investigated in this earlier study. Recently, Wink et al.(31) have reported lack of GSNO formation from ONOO reacting with GSH, but the applied photometrical method is probably not sufficiently sensitive to allow detection of small amounts of S-nitrosothiols. Others have reported on formation of an S-nitroso derivative of GSH being distinct from GSNO(29) , which we have never observed in the course of product analysis by HPLC. However, in accordance with a recent study by Moro et al.(30) , we found that about 1% of the added ONOO reacted with GSH to yield an S-nitrosylated compound, which has been identified as GSNO based on co-elution with the authentic nitrosothiol from a reversed phase HPLC column in five different solvents. The reaction mechanisms involved in ONOO-induced S-nitrosation of thiols is still elusive. The pH dependence of GSNO formation is in good accordance with previous results on ONOO-induced sulfhydryl oxidation(20) , suggesting that the reactive species is peroxynitrite anion, rather than peroxynitrous acid. However, direct reaction of ONOO with GSH to yield GSNO would result in concomitant release of peroxide anion and seems unlikely. Since only 1% of the added ONOO was converted to GSNO, it is conceivable that other reactive metabolites occurring in the course of sulfhydryl oxidation by ONOO(20) are also involved in S-nitrosation. Even though EDTA did not block GSNO formation, it cannot be ruled out that the reaction is catalyzed by trace metals, since the catalytic activity of transition metals is not always prevented by chelating compounds(50) .

In spite of the rather low yield, GSNO formation appears to fully account for ONOO-induced stimulation of purified sGC. According to the data shown in Fig. 4, presence of 0.1 mM ONOO should have resulted in formation of 0.6 µM GSNO at pH 7.5, and this is apparently sufficient for the observed stimulation of sGC (see Figs. 3A and 6). Further stimulation of cGMP formation is probably antagonized by inactivation of the enzyme in the presence of high concentrations of ONOO. This inhibitory effect of 0.1 mM ONOO could either be due to oxidation of critical amino acid residues or involve oxidative modification of the prosthetic heme group by reactive ONOO metabolites, resulting in decreased sensitivity of sGC toward NO.

Pure GSNO was stable over a wide range of pH values, but GSH induced a decomposition of the nitrosothiol that was accompanied by release of NO. These findings may, at least partially, explain earlier reports on tissue-induced NO release from GSNO(51, 52) . The effect of GSH may be mediated by trace metals, since both GSNO decomposition and NO release were completely inhibited by EDTA, and accelerated upon addition of CuCl (see Fig. 5). The effect of copper ions may be direct or involve GSH peroxides as suggested previously for thiol-mediated oxidation of low density lipoprotein(53) . Interestingly, the effect of GSH was mimicked by ascorbate (not shown), indicating that GSNO decomposition may be catalyzed by copper(I) ions and that GSH or other reducing agents are required for reduction of the added copper(II) ions (50) . These results are in accordance with a previous report on metal ion-catalyzed decomposition of the nitrosothiol S-nitroso-N-acetyl-DL-penicillamine (54) and with a recent study demonstrating that chelation of copper(I) ions reduces the biologic activity of GSNO(55) .

In keeping with its pronounced effect on NO release from GSNO, GSH markedly enhanced the potency of the nitrosothiol to stimulate sGC. Maximal effects of GSNO were smaller than those of DEA/NO, hinting at secondary reactions limiting the steady state concentrations of free NO under these conditions. Several mechanisms may account for the apparent thiol-independent effect of the nitrosothiol. First, it is possible that the parent compound interacts directly with the prosthetic heme group of sGC, resulting in enzyme activation. Second, NO may be released subsequent to trans-nitrosation (56) of free SH-groups of sGC as recently reported to occur with serum albumin(57) . Third, the endogenous GSH content of the enzyme preparations (10 µM) may have been sufficiently high to induce release of NO. Finally, enzyme stimulation could be due to the very minor amounts of NO that are apparently released from GSNO even in the absence of GSH (see Fig. 5).

The biological relevance of these findings remains to be established. Although low yields of GSNO might indicate that ONOO-mediated nitrosation is insignificant in cells, it is conceivable that the reaction becomes more efficient when ONOO is continuously generated instead of being added as concentrated stock solution. Furthermore, in intact cells, cGMP levels were increased to the same maximal values by ONOO and DEA/NO, whereas stimulation of purified sGC by ONOO was only 15-20% of that achieved with the NO donor, indicating that activation of ONOO through S-nitrosation may be facilitated in intact cells by specific enzymes or other mechanisms. If ONOO should turn out to be the physiological product of the NO synthase reaction, as suggested by recent studies with the purified enzyme (16) and agonist-stimulated endothelial cells(58) , ONOO-induced S-nitrosation could, in fact, represent a key reaction in NO/cGMP-mediated signal transduction.


FOOTNOTES

*
This work was supported by Grants P 10098 (to B. M.) and P 10573 (to K. S.) from the Fonds zur Förderung der Wissenschaftlichen Forschung in sterreich and Grant SFB 366 from the Deutsche Forschungsgemeinschaft (to D. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria. Tel.: 43-316-380-5567; Fax: 43-316-323-54-14.

The abbreviations used are: sGC, soluble guanylyl cyclase; CDNB, 1-chloro-2,4-dinitrobenzene; DEA/NO, 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt; DEM, diethyl maleate; DTNB, 5,5`-dithiobis-(2-nitrobenzoic acid); GSNO, S-nitrosoglutathione; HPLC, high performance liquid chromatography.

K. Schmidt, P. Klatt, and B. Mayer, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Harold F. Hodson, Wellcome Research Laboratories, Beckenham, United Kingdom, for kindly furnishing S-nitrosoglutathione. We gratefully acknowledge the excellent technical assistance of Eva Leopold, Margit Rehn, and Jürgen Malkewitz.


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