Peroxynitrite-induced Accumulation of Cyclic GMP in Endothelial Cells and Stimulation of Purified Soluble Guanylyl Cyclase
1995; Elsevier BV; Volume: 270; Issue: 29 Linguagem: Inglês
10.1074/jbc.270.29.17355
ISSN1083-351X
AutoresBernd Mayer, Astrid Schrammel, Peter Klatt, Doris Koesling, Kurt Schmidt,
Tópico(s)Renin-Angiotensin System Studies
ResumoPeroxynitrite (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. 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. Stimulation of soluble guanylyl cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2; sGC)1 1The abbreviations used are: sGCsoluble guanylyl cyclaseCDNB1-chloro-2,4-dinitrobenzeneDEA/NO2,2-diethyl-1-nitroso-oxyhydrazine sodium saltDEMdiethyl maleateDTNB5,5′-dithiobis-(2-nitrobenzoic acid)GSNOS-nitrosoglutathioneHPLChigh performance liquid chromatography. 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(1Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 2Garthwaite J. Semin. Neurosci. 1993; 5: 171-180Crossref Scopus (60) Google Scholar, 3Mayer B. Semin. Neurosci. 1993; 5: 197-205Crossref Scopus (21) Google Scholar). Purification of sGC revealed that the enzyme is a heterodimer containing stoichiometric amounts of ferro-protoporphyrin IX(4Gerzer R. Böhme E. Hofmann F. Schultz G. FEBS Lett. 1981; 132: 71-74Crossref PubMed Scopus (264) Google Scholar, 5Humbert P. Niroomand F. Fischer G. Mayer B. Koesling D. Hinsch K.-D. Gausepohl H. Frank R. Schultz G. Böhme E. Eur. J. Biochem. 1990; 190: 273-278Crossref PubMed Scopus (143) Google Scholar, 6Yu A.E. Hu S.Z. Spiro T.G. Burstyn J.N. J. Am. Chem. Soc. 1994; 116: 4117-4118Crossref Scopus (123) Google Scholar, 7Stone J.R. Marletta M.A. Biochemistry. 1994; 33: 5636-5640Crossref PubMed Scopus (609) Google Scholar). NO exhibits high affinity for ferrous heme(8Traylor T.G. Sharma V.S. Biochemistry. 1992; 31: 2847-2849Crossref PubMed Scopus (349) Google Scholar), and a heme-deficient mutant of sGC has recently been shown to be insensitive to NO(9Wedel B. Humbert P. Harteneck C. Foerster J. Malkewitz J. Böhme E. Schultz G. Koesling D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2592-2596Crossref PubMed Scopus (230) Google Scholar), 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(10Ignarro L.J. Blood Vessels. 1991; 28: 67-73PubMed Google Scholar). soluble guanylyl cyclase 1-chloro-2,4-dinitrobenzene 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt diethyl maleate 5,5′-dithiobis-(2-nitrobenzoic acid) S-nitrosoglutathione high performance liquid chromatography. As a free radical, NO reacts with several intracellular targets(11Henry Y. Lepoivre M. Drapier J.-C. Ducrocq C. Boucher J.-L. Guissani A. FASEB J. 1993; 7: 1124-1134Crossref PubMed Scopus (352) Google Scholar). Reaction with molecular oxygen results in the generation of N2O3 or other nitrogen oxides (NO)twith potential cytotoxic properties(12Wink D.A. Kasprzak K.S. Maragos C.M. Elespuru R.K. Misra M. Dunams T.M. Cebula T.A. Koch W.H. Andrews A.W. Allen J.S. Keefer L.K. Science. 1991; 254: 1001-1003Crossref PubMed Scopus (1131) Google Scholar, 13Nguyen T. Brunson D. Crespi C.L. Penman B.W. Wishnok J.S. Tannenbaum S.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3030-3034Crossref PubMed Scopus (949) Google Scholar). This reaction follows second order kinetics with respect to NO and is thus rather slow at low NO concentrations(14Wink D.A. Darbyshire J.F. Nims R.W. Saavedra J.E. Ford P.C. Chem. Res. Toxicol. 1993; 6: 23-27Crossref PubMed Scopus (479) Google Scholar, 15Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1994; 269: 5881-5883Abstract Full Text PDF PubMed Google Scholar, 16Mayer B. Klatt P. Werner E.R. Schmidt K. J. Biol. Chem. 1995; 270: 655-659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), 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 (17Gryglewski R.J. Palmer R.M.J. Moncada S. Nature. 1986; 320: 454-456Crossref PubMed Scopus (2187) Google Scholar) due to a fast reaction of NO with O2− to yield peroxynitrite (ONOO−)(18Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6716) Google Scholar). 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(19Crow J.P. Spruell C. Chen J. Gunn C. Ischiropoulos H. Tsai M. Smith C.D. Radi R. Koppenol W.H. Beckman J.S. Free Radical Biol. Med. 1994; 16: 331-338Crossref PubMed Scopus (201) Google Scholar). The intermediate appears to be highly reactive, inducing oxidation of various cellular targets including sulfhydryls (20Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar) and lipids (21Radi R. Beckman J.S. Bush K.M. Freeman B.A. Arch. Biochem. Biophys. 1991; 288: 481-487Crossref PubMed Scopus (2050) Google Scholar, 22Hogg N. Darley-Usmar V.M. Wilson M.T. Moncada S. Biochem. J. 1992; 281: 419-424Crossref PubMed Scopus (621) Google Scholar, 23Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar). More recently, it was demonstrated that ONOO− apparently mediates several effects previously attributed to NO, e.g. covalent modification of glyceraldehyde-phosphate dehydrogenase (24Mohr S. Stamler J.S. Brüne B. FEBS Lett. 1994; 348: 223-227Crossref PubMed Scopus (197) Google Scholar) and inhibition of aconitase(25Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 26Castro L. Rodriguez M. Radi R. J. Biol. Chem. 1994; 269: 29409-29415Abstract Full Text PDF PubMed Google Scholar). Together with the finding that activated macrophages release most of their NO as ONOO−(27Ischiropoulos H. Zhu L. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 446-451Crossref PubMed Scopus (1092) Google Scholar), 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 (28Liu S. Beckman J.S. Ku D.D. J. Pharmacol. Exp. Ther. 1994; 268: 1114-1121PubMed Google Scholar, 29Wu M.D. Pritchard K.A. Kaminski P.M. Fayngersh R.P. Hintze T.H. Wolin M.S. Am. J. Physiol. 1994; 266: H2108-H2113PubMed Google Scholar) and to inhibit platelet aggregation(30Moro M.A. Darley-Usmar V.M. Goodwin D.A. Read N.G. Zamorapino R. Feelisch M. Radomski M.W. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6702-6706Crossref PubMed Scopus (337) Google Scholar). The molecular mechanisms underlying these physiological effects of ONOO− are unclear. Liu et al.(28Liu S. Beckman J.S. Ku D.D. J. Pharmacol. Exp. Ther. 1994; 268: 1114-1121PubMed Google Scholar) speculated that relaxation may be due to small amounts of NO spontaneously released during ONOO− decomposition, and Wu et al.(29Wu M.D. Pritchard K.A. Kaminski P.M. Fayngersh R.P. Hintze T.H. Wolin M.S. Am. J. Physiol. 1994; 266: H2108-H2113PubMed Google Scholar) reported on pronounced increases in NO release when ONOO− was incubated in the presence of GSH or tissue homogenates. Similarly, Moro et al.(30Moro M.A. Darley-Usmar V.M. Goodwin D.A. Read N.G. Zamorapino R. Feelisch M. Radomski M.W. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6702-6706Crossref PubMed Scopus (337) Google Scholar) 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.(31Wink D.A. Nims R.W. Darbyshire J.F. Christodoulou D. Hanbauer I. Cox G.W. Laval F. Laval J. Cook J.A. Krishna M.C. Degraff W.G. Mitchell J.B. Chem. Res. Toxicol. 1994; 7: 519-525Crossref PubMed Scopus (358) Google Scholar) failed to detect an S-nitrosated species arising from the reaction of ONOO− with GSH, whereas others have reported on formation of GSNO (30Moro M.A. Darley-Usmar V.M. Goodwin D.A. Read N.G. Zamorapino R. Feelisch M. Radomski M.W. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6702-6706Crossref PubMed Scopus (337) Google Scholar) or another, as yet unidentified S-nitroso compound(29Wu M.D. Pritchard K.A. Kaminski P.M. Fayngersh R.P. Hintze T.H. Wolin M.S. Am. J. Physiol. 1994; 266: H2108-H2113PubMed Google Scholar). The present study was designed to elucidate the molecular mechanisms underlying the biologic activity of ONOO−. Recombinant bovine lung soluble guanylyl cyclase was purified from baculovirus-infected Sf9 cells as described previously(5Humbert P. Niroomand F. Fischer G. Mayer B. Koesling D. Hinsch K.-D. Gausepohl H. Frank R. Schultz G. Böhme E. Eur. J. Biochem. 1990; 190: 273-278Crossref PubMed Scopus (143) Google Scholar, 9Wedel B. Humbert P. Harteneck C. Foerster J. Malkewitz J. Böhme E. Schultz G. Koesling D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2592-2596Crossref PubMed Scopus (230) Google Scholar). ONOO− was synthesized as described (32Papee H.M. Petriconi G.L. Nature. 1964; 204: 142-144Crossref Scopus (61) Google Scholar, 33Hughes M.N. Nicklin H.G. J. Chem. Soc. Abstr. 1968; : 450-452Crossref Google Scholar, 34Schmidt K. Klatt P. Mayer B. Biochem. J. 1994; 301: 645-647Crossref PubMed Scopus (65) Google Scholar). 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) (35Maragos C.M. Morley D. Wink D.A. Dunams T.M. Saavedra J.E. Hoffman A. Bove A.A. Isaac L. Hrabie J.A. Keefer L.K. J. Med. Chem. 1991; 34: 3242-3247Crossref PubMed Scopus (701) Google Scholar) was from NCI Chemical Carcinogen Repository, Kansas City, MO. 10-fold concentrated stock solutions of DEA/NO were prepared in 10 mM NaOH. [α-32P]GTP (400 Ci/mmol) was purchased from MedPro (Amersham), Vienna, Austria; the other chemicals were from Sigma, Vienna, Austria. Porcine aortic endothelial cells were cultured as described previously(36Schmidt K. Mayer B. Kukovetz W.R. Eur. J. Pharmacol. 1989; 170: 157-166Crossref PubMed Scopus (82) Google Scholar, 37Mayer B. Brunner F. Schmidt K. Biochem. Pharmacol. 1993; 45: 367-374Crossref PubMed Scopus (443) Google Scholar). 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 × 105 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 CaCl2, 1 mM MgCl2, 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. The DTNB colorimetric method (38Beutler E. Duron O. Kelly B.M. J. Lab. Clin. Med. 1963; 61: 882-888PubMed Google Scholar) was used for determination of GSH, which represents the only measurable soluble thiol in endothelial cells(39Ghigo D. Alessio P. Foco A. Bussolino F. Costamagna C. Heller R. Garbarino G. Pescarmona G.P. Bosia A. Am. J. Physiol. 1993; 265: C728-C732Crossref PubMed Google Scholar). Cells were cultured in Petri dishes (diameter 90 mm), and confluent monolayers (˜5 × 106 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-1 cm-1 under these conditions, which is closely similar to the reported value of 13.6 mM-1 cm-1(38Beutler E. Duron O. Kelly B.M. J. Lab. Clin. Med. 1963; 61: 882-888PubMed Google Scholar). 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 [α-32P]GTP (200,000-300,000 cpm), 3 mM MgCl2, 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 ZnCO3 precipitation followed by isolation of [32P]cGMP by column chromatography as described(40Schultz G. Böhme E. Bergmeyer H.U. Bergmeyer J. Gra M. Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, Germany1984: 379-389Google Scholar). 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. 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 C18 reversed-phase column equipped with a 4 × 4-mm C18 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, H2O, 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(29Wu M.D. Pritchard K.A. Kaminski P.M. Fayngersh R.P. Hintze T.H. Wolin M.S. Am. J. Physiol. 1994; 266: H2108-H2113PubMed Google Scholar). 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(16Mayer B. Klatt P. Werner E.R. Schmidt K. J. Biol. Chem. 1995; 270: 655-659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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. As shown in Fig. 1, ONOO− increased cGMP levels in cultured endothelial cells in a concentration-dependent manner with an EC50 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/106 cells with an EC50 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/106 cells; n = 3). Control experiments performed with NaNO2 showed that 0.1 and 1 mM nitrite enhanced endothelial cGMP levels to 3.1 ± 0.28 and 8.1 ± 1.04 pmol/106 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−. 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(41Meister A. Science. 1983; 220: 472-477Crossref PubMed Scopus (1093) Google Scholar). 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/106 cells) are in excellent accordance with values reported previously for cultured endothelial cells(42Murphy M.E. Piper H.M. Watanabe H. Sies H. J. Biol. Chem. 1991; 266: 19378-19383Abstract Full Text PDF PubMed Google Scholar). Based on an endothelial cell volume of 1 pl(43Baydoun A.R. Emery P.W. Pearson J.D. Mann G.E. Biochem. Biophys. Res. Commun. 1990; 173: 940-948Crossref PubMed Scopus (126) Google Scholar), this corresponds to intracellular GSH concentrations of approximately 10 mM. 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(5Humbert P. Niroomand F. Fischer G. Mayer B. Koesling D. Hinsch K.-D. Gausepohl H. Frank R. Schultz G. Böhme E. Eur. J. Biochem. 1990; 190: 273-278Crossref PubMed Scopus (143) Google Scholar). 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-1× min-1 (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 EC50 of approximately 20 μM ONOO−. The sGC activities maximally achievable with ONOO− were about 650 nmol cGMP × mg-1× min-1 and thus considerably lower than those observed in the presence of DEA/NO (˜4 μmol × mg-1× min-1) or GSNO (˜1 μmol × mg-1× min-1) (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). It has been suggested that protein SH-groups may be critically involved in the regulation of sGC(44Craven P.A. DeRubertis F.R. Biochim. Biophys. Acta. 1978; 524: 231-244Crossref PubMed Scopus (47) Google Scholar), 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 EC50 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 EC50 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(18Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6716) Google Scholar), 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.2 2K. Schmidt, P. Klatt, and B. Mayer, unpublished results. 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. 4 shows 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). 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 CuCl2 and completely blocked by 10 mM EDTA. The initial rates of NO release shown in Fig. 5 were 0.46 and 2.00 μM min-1 in the absence and presence of added CuCl2, respectively, steady state concentrations of NO were reached about 2 min after addition of GSH. GSH-induced release of NO from GSNO was further confirmed in experiments with purified sGC. Fig. 6 shows that GSNO stimulated the formation of cGMP in a concentration-dependent manner. Maximal sGC activities of about 1 μmol of cGMP × mg-1× min-1 were observed with 0.3 mM GSNO, the EC50 of the nitrosothiol was 8.0 μM. In the presence of 2 mM GSH, the EC50 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. Previous reports have demonstrated that ONOO− exhibits NO-like biologic activities, inducing relaxation of blood vessels (28Liu S. Beckman J.S. Ku D.D. J. Pharmacol. Exp. Ther. 1994; 268: 1114-1121PubMed Google Scholar, 29Wu M.D. Pritchard K.A. Kaminski P.M. Fayngersh R.P. Hintze T.H. Wolin M.S. Am. J. Physiol. 1994; 266: H2108-H2113PubMed Google Scholar) and inhibiting platelet aggregation(30Moro M.A. Darley-Usmar V.M. Goodwin D.A. Read N.G. Zamorapino R. Feelisch M. Radomski M.W. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6702-6706Crossref PubMed Scopus (337) Google Scholar). 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−(18Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6716) Google Scholar), accumulation of cellular cGMP required about 10-fold higher concentrations of ONOO− (EC50˜ 0.2 mM) than stimulation of purified soluble guanylyl cyclase (EC50˜ 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(45Stamler J.S. Simon D.I. Osborne J.A. Mullins M.E. Jaraki O. Michel T. Singel D.J. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 444-448Crossref PubMed Scopus (1305) Google Scholar, 46Stamler J.S. Jaraki O. Osborne J. Simon D.I. Keaney J. Vita J. Singel D. Valeri C.R. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7674-7677Crossref PubMed Scopus (1131) Google Scholar). 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(39Ghigo D. Alessio P. Foco A. Bussolino F. Costamagna C. Heller R. Garbarino G. Pescarmona G.P. Bosia A. Am. J. Physiol. 1993; 265: C728-C732Crossref PubMed Google Scholar, 47Hecker M. Siegle I. Macarthur H. Sessa W.C. Vane
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