Attenuation of NMDA Receptor Activity and Neurotoxicity by Nitroxyl Anion, NOϪ

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Neuron, Vol. 24, 461–469, October, 1999, Copyright 1999 by Cell Press Attenuation of NMDA Receptor Activity and Neurotoxicity by Nitroxyl Anion, NOϪ

§ § Won-Ki Kim,* k Yun-Beom Choi,* by hemoglobin (Gow and Stamler, 1998), neuronal nitric Posina V. Rayudu,* Prajnan Das,* Wael Asaad,* oxide synthase (NOS) (Hobb et al., 1994; Pufahl et al., Derrick R. Arnelle,† Jonathan S. Stamler,† 1995; Zhao et al., 1995; Schmidt et al., 1996), and endog- and Stuart A. Lipton*‡ enous S-nitrosothiols (SNOs) (Arnelle and Stamler, 1995; *Cerebrovascular and NeuroScience Hogg et al., 1996)—has remained unknown. Recently, Research Institute NOϪ chemistry in biological systems has begun to re- Brigham and Women’s Hospital and ceive consideration (Hughes, 1999) and has been impli- Program in Neuroscience cated in toxicity of human breast cancer (MCF-7) cells Harvard Medical School (Chazotte-Aubert et al., 1999). Here, we show that NOϪ Boston, Massachusetts 02115 reacts with NMDA receptor thiol to downregulate recep- † Howard Hughes Medical Institute tor activity and provide neuroprotection, and we verify Departments of Medicine and Cell Biology that NO· is ineffective. Our results thus expand the para- Duke University Medical Center digm of NO signaling by showing that NOϪ can poten- Durham, North Carolina 27710 tially provide negative feedback to curtail excessive NMDA receptor activity. We further show that NOϪ effects are mimicked by NOS activity, and we suggest a Summary new therapeutic approach, using NOϪ donors, to ameliorate NMDA receptor-mediated neuronal damage. Recent evidence indicates that the NO-related species, nitroxyl anion (NOϪ), is produced in physiological Results systems by several redox metal–containing proteins, including hemoglobin, nitric oxide synthase (NOS), su- Endogenous NOS Activity Can Decrease peroxide dismutase, and S-nitrosothiols (SNOs), which NMDA Responses have recently been identified in brain. However, the We sought evidence using digital Ca2ϩ imaging tech- chemical biology of NOϪ remains largely unknown. niques in our rat cerebrocortical cultures for NOS down- Here, we show that NOϪ—unlike NO·, but reminiscent regulation of NMDA responses. We found that addition of NO؉ transfer (or S-nitrosylation)—reacts mainly with of the NOS substrate L-arginine to the medium led to a Cys-399 in the NR2A subunit of the N-methyl-D-aspar- decrease in NMDA response amplitude (Figure 1) but .(tate (NMDA) receptor to curtail excessive Ca2؉ influx did not affect responses to Kϩ (50 mM) or kainate (50 ␮M and thus provide neuroprotection from excitotoxic in- This effect was blocked by the NOS inhibitor L-nitro- sults. This effect of NOϪ closely resembles that of arginine. Additional in vitro and in vivo evidence that NOS, which also downregulates NMDA receptor activ- physiological NOS activity can affect NMDA responses ity under similar conditions in culture. has been presented (Manzoni and Bockaert, 1993; Ken- drick et al., 1996), indicating that endogenous form(s) of Introduction NO-related species can downregulate NMDA receptor function. Despite the fact that nitric oxide (NO·) and its redox- related species are known to mediate a wide range of Endogenously Produced NOϪ or Exogenous NOϪ physiological activities, the chemical reactions and tar- Donors Decrease NMDA Receptor Responses get proteins underlying these regulatory processes are Next, we found that enzymatic generation of endoge- just beginning to be elucidated (Stamler et al., 1992b, nous NOϪ or application of exogenous NOϪ donors to 1997; Butler et al., 1995). The N-methyl-D-aspartate the cultured cortical neurons also decreased NMDA re- (NMDA) subtype of glutamate receptor has provided a ceptor activity, as assayed by digital Ca2ϩ imaging and useful physiological model to test the chemical biology whole-cell recording with patch electrodes (Figures 2 of NO-related species. In particular, S-nitrosylation and 3, respectively). Three donors of NOϪ (Oxi-NO, Sulfi- ϩ (transfer of NO [nitrosonium ion] to critical thiols) has NO, and Piloty’s acid) all exhibited qualitatively similar been shown to downregulate NMDA channel activity effects in this regard (at millimolar concentrations, these and thus confer neuroprotection, while under the same donors generate low micromolar levels of NOϪ) (Fukuto · conditions NO does not (Stamler et al., 1992a, 1992b; et al., 1992; Nagasawa et al., 1995; Feelisch and Stamler, Lipton et al., 1993; Lipton and Stamler, 1994). In contrast, 1996). For example, Oxi-NO inhibited the NMDA-evoked Ϫ the activity of NO (nitroxyl anion)—which can be formed 2ϩ [Ca ]i responses in a manner similar to the strong oxidizing agent 5–5Ј-dithiobis-(2-nitrobenzoic acid) (DTNB) ‡ To whom correspondence should be addressed at the following (Figure 2). Piloty’s acid, consistent with recent reports present address: Del E. Webb Center for Neuroscience and Aging that it is a better NO· than NOϪ donor at physiological Research, The Burnham Institute, 10901 North Torrey Pines Road, pH (Zamora et al., 1995; Feelisch and Stamler, 1996), La Jolla, CA 92037 (e-mail: [email protected]). exhibited less of an effect. In a series of experiments in § These authors contributed equally to this work. which the sulfhydryl reducing agent dithiothreitol (DTT) k Present address: Department of Pharmacology, Ewha Medical School, 911-1 Mok-6-dong, Yangchun-ku, Seoul 158-750, Republic was initially applied to maximize NMDA responses of Korea. (Aizenman et al., 1989), subsequent addition of Oxi-NO Neuron 462

Figure 3. NOϪ-generating Drugs Inhibit NMDA-Evoked Current Re- Figure 1. Endogenous NOS Activity Can Attenuate NMDA-Evoked sponses Responses Inhibition of NMDA-evoked whole-cell currents by Oxi-NO (5 mM) Addition of the NOS substrate, L-arginine (1 mM), to the bathing and reversal of effect after incubation in DTT (2 mM), as assessed solution for 20 min resulted in a dramatic decrease in NMDA-evoked in patch-clamp recordings. Note that the inhibitory effect of Oxi-NO responses, while preincubation in the NOS inhibitor, L-nitro-arginine on NMDA responses was observed on neurons that had not been (1 mM), prevented this effect, as assessed with digital quantitation previously exposed to DTT. 2ϩ of [Ca ]i with fura-2 in cultured rat cortical neurons. Control responses to NMDA (100 ␮M ϩ 2 ␮M glycine for 20 s) prior to the 2ϩ addition of L-arginine averaged 276 Ϯ 12 ␮M [Ca ]i (mean Ϯ SEM, n ϭ 60 neurons; *p Ͻ 0.01 by Student’s t test). This experiment was disulfide bonds and removes the NO group (Xu et al., replicated five times with similar results. 1998), reversed these inhibitory effects on NMDA re- Ϫ 2ϩ sponses. NO could conceivably inhibit [Ca ]i responses other than at the level of the NMDA receptor. 2ϩ decreased NMDA-evoked [Ca ]i responses by 46% Ϯ Therefore, to more directly assess the effects of NOϪ 5% (mean Ϯ SEM, n ϭ 6), Sulfi-NO by 32% Ϯ 8% (n ϭ on the NMDA receptor, electrophysiological recordings 6), and DTNB by 45% Ϯ 4% (n ϭ 8). DTT, which reduces were obtained using the patch-clamp technique. During whole-cell recording performed on neurons that had not been previously exposed to DTT, Oxi-NO inhibited NMDA-evoked activity by 28% Ϯ 10% (n ϭ 4; Figure 3). In contrast to the inhibitory effects of NOϪ on NMDA 2ϩ responses, kainate-evoked [Ca ]i responses in our culture system are not modulated by redox reagents or other NO-related species (Aizenman et al., 1989; Lei et al., 1992).

Nitroxyl Reactions with Thiol Groups The electrons in the outer molecular orbital of NOϪ are thought to exist in two distinct excitation states, a lower energy state, designated the triplet state, and a higher energy singlet state (Bonner and Stedman, 1996). Impor- tantly, the reactivities of these species are different. Singlet NOϪ tends to react with thiol groups (Doyle et al., 1988) to form disulfide and hydroxylamine, whereas Ϫ Ϫ 2ϩ triplet NO favors reaction with O2, producing peroxyni- Figure 2. NO -generating Drugs Inhibit NMDA-Evoked [Ca ]i Re- sponses trite (ONOOϪ) (Stamler et al., 1992b; Arnelle and Stamler, 2ϩ Digital quantitation of [Ca ]i with fura-2 in cultured rat cortical neu- 1995; Butler et al., 1995; Bonner and Stedman, 1996). rons. In the presence of glycine (1 ␮M) and tetrodotoxin (TTX; 0.5 The latter oxidizes thiols but shows less target selectiv- 2ϩ Ϫ ␮M), 30 ␮M NMDA elicited large increases in [Ca ]i. Oxi-NO or ity. Recent evidence suggests that the NO donor drugs Piloty’s acid at 5 mM were used to generate low micromolar concen- used here generate singlet NOϪ (Fukuto et al., 1992; Ϫ trations of NO (Fukuto et al., 1992; Nagasawa et al., 1995; Feelisch Nagasawa et al., 1995; Bonner and Stedman, 1996; Fee- and Stamler, 1996). These drugs decreased NMDA responses following exposure to 2 mM DTT, but Oxi-NO was much more potent lisch and Stamler, 1996; Wink and Feelisch, 1996). Under than Piloty’s acid, producing an effect equivalent to that of 0.5 mM physiological conditions, nitroxyl can also be produced DTNB; there was no further inhibition of NMDA-evoked responses (presumably in the singlet state), for example, during the by Oxi-NO after prior oxidation with DTNB. Values are mean Ϯ SEM generation of met hemoglobin from partially nitrosylated (*p Ͻ 0.05, **p Ͻ 0.01 compared to response after DTT, analyzed deoxy hemoglobin (Gow and Stamler, 1998). N-ethylma- with an analysis of variance [ANOVA] followed by a Scheffe´ multiple leimide (NEM, Յ1 mM), which irreversibly alkylates thiol comparison of means). Dissipation of NOϪ from these drugs prior to their use by incubating at room temperature for 1 day (labeled groups of the NMDA receptor, should prevent the inhibi- Ϫ “old Oxi-NO” and “old Piloty’s acid”) abrogated their effect. This tion of NMDA-evoked responses by singlet NO donors. experiment was repeated eight times with similar results. Indeed, in our experiments following treatment with Nitroxyl Attenuates NMDA Activity 463

(Figures 5C and 5D). Mutation of NR2A Cys-399 alone to alanine abrogated approximately 60% of the NOϪ effect (Figure 5D). One potential caveat in this approach is that mutation of cysteine residues can possibly affect the structural integrity of the receptor. Therefore, as controls, we tested the NMDA and glycine dose-response curves of these mutants, as well as sensitivity to Mg2ϩ block, to insure the integrity of the agonist site, coago- nist site, and channel pore. The mutants manifest only minor effects on these NMDA receptor properties. Taken together, these findings strongly suggest that NOϪ can react with multiple extracellular cysteine residues on the NR1 and NR2A subunits of the NMDA receptor.

Lack of Action of NO· The NOϪ donors also generate NO·, albeit in very small amounts (Fukuto et al., 1992; Nagasawa et al., 1995; Feelisch and Stamler, 1996). Therefore, to ensure that Ϫ Figure 4. Quantification of Digital Imaging to Show Irreversible Al- the effects of NO donors were not due to their produc- · kylation of Thiol Groups with NEM Prevents the Effect of NOϪ on tion of NO , we performed control experiments using the 2ϩ · NMDA-Evoked [Ca ]i Responses more specific NO donors, diethylamine-NO (DEA/NO) DTT increased NMDA-evoked responses, and NOϪ donors de- and spermine/NO (SPER/NO). With the use of a specific creased them. Alkylation of thiol groups with NEM (1 mM) prevented NO·-sensing electrode (WPI) (Lipton et al., 1993), we the subsequent inhibition of NMDA responses by the various oxidiz- calibrated the concentrations of these NO· donors to Ϫ ing agents, including DTNB or the NO donors. Values are mean Ϯ generate similar or slightly greater amounts of NO· over SEM (**p Ͻ 0.01 compared to NMDA response after DTT). a 2 min period compared to the NOϪ donors (Table 1). At these concentrations, neither DEA/NO nor SPER/NO influenced the NMDA-evoked responses (e.g., Figure 6), NEM, NMDA responses were not altered by Oxi-NO, consistent with our previous findings that NO· itself does Piloty’s acid, or DTNB (Figure 4). not downregulate NMDA receptor activity (Lipton et al., In additional experiments aimed at proving that NOϪ 1993). Thus, NOϪ and not NO· appears to be responsible reacted specifically with thiol groups on the NMDA re- for the inhibitory effect on the NMDA receptor–channel ceptor, we studied recombinant NR1/NR2A receptors complex described here. expressed in Xenopus oocytes. In this system, as in native cerebrocortical neurons, NOϪ decreased NMDA responses recorded under two-electrode voltage clamp Production of Nitroxyl Anion in Biological Tissues (Figure 5A, upper trace). We then used methanethiosul- As recently shown, hemoglobin can produce NOϪ under fonate derivatives that react specifically and rapidly with physiological conditions (Gow and Stamler, 1998). Addi- thiol groups to form mixed disulfides (Akabas et al., tionally, it was reported that NOϪ is a physiological prod- 1992). Prior incubation with the thiol-specific methane- uct of the enzyme neuronal nitric oxide synthase (nNOS) sulfonate, 2-(trimethlylammonium)ethyl methanethiosulfo- and S-nitrosoglutathione (GSNO) (Hobb et al., 1994; nate (MTSET), prevented inhibition of NMDA responses Zhao et al., 1995; Schmidt et al., 1996; Mayer et al., by the NOϪ-generating drugs (Figures 5A, lower trace, 1998), the latter being present in the brain at micromolar and 5B). These findings indicate that the predominant concentrations (Hogg et al., 1996; Kluge et al., 1997). mechanism of inhibition of NMDA responses by the NOϪ Endogenous NOϪ can also be generated from NO· by donors involves interaction with free thiol groups. More- other redox metal–containing enzymes, such as SOD over, compared to other methanethiosulfonate deriva- [Cu(I)] (Equation 1) (Murphy and Sies, 1991). tives, MTSET is a larger, charged agent that does not easily permeate cell membranes. The fact that bath ap- Enz[M] ϩ NO· Enz[Mϩ] ϩ NOϪ (1) plication of this charged compound prevented the effect →← of NOϪ suggests therefore that the critical thiol groups where Enz indicates enzyme and M indicates metal [for are located extracellularly on the NMDA receptor. copper, Cu(I) Cu(II); for iron, Fe(II) Fe(III)]. To study this possibility further, we performed site- We confirmed→← that SOD[Cu(I)] could→← support the re- directed mutagenesis of extracellular cysteine residues. duction of NO· by assaying the conversion of met myo- We found that the following cysteine residues were criti- globin to nitrosyl myoglobin (Murphy and Sies, 1991; cal for the response to NOϪ: NR2A Cys-87, Cys-320, and Arnelle and Stamler, 1995). We then took advantage of Cys-399; NR1 Cys-744 and Cys-798. When we mutated the reaction in Equation 1 to produce endogenous NOϪ these multiple cysteine residues on NR1 and NR2A sub- in our culture system by reacting reduced SOD (SOD units, the inhibition of NMDA-evoked currents by the [Cu(I)]) with NO·, using DEA/NO as the NO· donor (Mar- NOϪ-generating agent Piloty’s acid was virtually abol- agos et al., 1991). Under these conditions, NOϪ was ished [inhibition of 1% Ϯ 1%, n ϭ 6 for NR1(C744A, apparently generated in its triplet energy state since C798A)/NR2A(C87A,C320A,C399A) receptors versus we did not detect hydroxylamine, as would have been 19% Ϯ 3%, n ϭ 6 for wild-type NR1/NR2A receptors] expected had singlet NOϪ been produced (Arnelle and Neuron 464

Figure 5. NOϪ Inhibits NMDA Evoked Currents in NR1/NR2A Receptors, MTSET Prevents the NOϪ Effect, and Specific Extracellular Cysteine Residues on NR1/NR2A Underlie the NOϪ Effect (A) Upper trace: NMDA-evoked currents (by 200 ␮M NMDA plus 100 ␮M glycine) are inhibited by a 3 min application of the NOϪ-generating drug Oxi-NO (3 mM) in oocytes expressing NR1/NR2A receptors. Following washout of Oxi-NO, the decrease in NMDA-evoked currents persisted (greater than 30 min, the duration of the experiment). Lower trace: in a different oocyte, addition of MTSET (0.5 ␮M for 2 min) prevented inhibition of NMDA-evoked currents by Oxi-NO (3 mM for 3 min). Currents were recorded at room temperature at a holding potential of Ϫ80 mV (pH 7.5). (B) Inhibition of NR1/NR2A receptor currents by Oxi-NO, expressed as a percentage of the response to NMDA prior to Oxi-NO application (mean Ϯ SEM, n ϭ 4 for each group). Inhibition by Oxi-NO after MTSET exposure was significantly less than that without MTSET treatment (*p Ͻ 0.01 by Student’s t test). (C) Current tracings showing the absence of inhibition of NMDA-evoked currents by the NOϪ-generating drug Piloty’s acid (10 mM) in oocytes expressing NR1(C744A,C798A)/NR2A(C87A,C320A,C399A) mutant receptors. (D) Inhibition of NMDA-evoked currents by Piloty’s acid in wild-type or mutant NR1/NR2A receptors expressed as a percentage of the response to NMDA prior to Piloty’s acid application (mean Ϯ SEM, n ϭ 6 for each group). Inhibition by Piloty’s acid of NR1/NR2A(C399A) receptor responses was significantly less than that of wild-type NR1/NR2A receptors (*p Ͻ 0.01, ANOVA followed by Fisher’s PLSD test). Additionally, inhibition by Piloty’s acid of NR1(C744A,C798A)/NR1(C87A,C320A,C399A) receptor responses was significantly less than that of NR1/ NR2A(C399A) receptors (†p Ͻ 0.05).

Stamler, 1995; Gow and Stamler, 1998). NOϪ generated (Kim et al., 1996), whereas it is neurodestructive at higher 2ϩ in this manner decreased NMDA-evoked [Ca ]i re- concentrations (Beckman et al., 1990; Dawson et al., sponses by 25% Ϯ 5% (n ϭ 4, Figure 6), similar to 1993; Lipton et al., 1993). We therefore speculate that the NOϪ-generating drugs. As controls, neither DEA/NO triplet NOϪ is acting here via formation of low concentra- alone, SOD[Cu(I)] alone, nor the combination of SOD tions of ONOOϪ to downregulate NMDA receptor ac- [Cu(II)] plus DEA/NO affected NMDA-evoked responses. tivity. Triplet NOϪ does not react directly with thiol groups Ϫ but can react with O2, forming peroxynitrite (ONOO ) Nitroxyl Attenuates NMDA Receptor-Mediated (Stamler et al., 1992b; Arnelle and Stamler, 1995; Butler Excitotoxicity et al., 1995), which does react with thiol (Radi et al., In the following experiments, the NOϪ donors signifi- 1991). At low concentrations, peroxynitrite decreases cantly ameliorated NMDA receptor-mediated neurotox- NMDA receptor activity by oxidizing critical thiol groups icity, consistent with the finding that NOϪ downregulates Nitroxyl Attenuates NMDA Activity 465

Table 1. NO· Production from Various NO Group Donors

AUC (␮M * 2 min) Drug (Concentration) (mean Ϯ SEM) n Spermine/NO (200 ␮M) 1.28 Ϯ 0.14 9 DEA /NO (20 ␮M) 2.38 Ϯ 0.38 5 Piloty’s acid (5 mM) 0.82 Ϯ 0.18 3 Sulfi-NO (5 mM) 0.90 Ϯ 0.06 4 Oxi-NO (5 mM) 1.46 Ϯ 0.44 4 The concentrations of the NO· donors, spermine/NO and DEA /NO, were chosen to generate amounts of NO· that were approximately equal to or slightly greater than that the NO· production of the NOϪ- generating drugs, Oxi-NO, Sulfi-NO, and Piloty’s acid (the latter is a poor generator of NOϪ). Thus, the NO· donors serve to ensure that the effects of the NOϪ-generating drugs were not due to NO·. The total NO· production during a 2 min incubation was determined by the area under the curve (AUC) of NO· versus time, as read out by an NO·-sensitive electrode to a digital meter and graphed electronically. AUC values were calculated by the method of trapezoids and expressed as the mean Ϯ SEM for n experiments. excessive NMDA-evoked responses (Figure 7A). In 18 of 22 experiments, similar in design to that illustrated in Figure 7, the NOϪ donors Oxi-NO, Sulfi-NO, or Piloty’s Ϫ · acid (1–5 mM) afforded protection from NMDA-induced Figure 7. Oxi-NO, NO , but not DEA/NO, NO , Ameliorates NMDA Receptor–Mediated Toxicity of Rat Cortical Neurons in Culture neurotoxicity (p Ͻ 0.002 by the Sign test). To be certain Ϫ of the identity of the redox form of the NO group involved (A) To test the effects of an NO donor on neuronal survival, cerebrocortical cultures were placed in Earle’s balanced salt solution (EBSS) in this neuroprotective effect, we incubated the cortical and preincubated in 1 mM Oxi-NO (or control solution) for 2 min · cultures in the NO donor DEA/NO (20 ␮M), which pro- prior to the addition of NMDA. Cells were then exposed to 300 ␮M duces a greater amount of NO· than any of the NOϪ- NMDA (plus 10 ␮M glycine) for 10 min, rinsed, and placed back generating drugs (Table 1). At this concentration, DEA/ in EBSS in the incubator overnight prior to the determination of neurotoxicity. Neuronal damage was then assessed by measuring the release of lactate dehydrogenase (LDH) and verified by cell counts, as previously described (Koh and Choi, 1987; Lipton et al., 1993). Data are expressed as mean Ϯ SEM for an experiment performed in triplicate on sibling cultures. The asterisk indicates significantly different compared to the group exposed to NMDA plus Oxi-NO (p Ͻ 0.05 by Student’s t test). (B) Addition of DEA/NO (20 ␮M) prior to NMDA and glycine exposure did not rescue neurons from damage. Similar treatment with DEA/ NO alone had no effect on viability, as assessed after overnight incubation as in (A), above. The asterisk indicates significantly different compared to the group exposed to control or to DEA/NO (p Ͻ 0.01, ANOVA followed by a Scheffe´ multiple comparison of means).

NO had no effect on the viability of the neurons and did not protect from NMDA-induced toxicity (Figure 7B). Together, these findings suggest that NOϪ is associated with significant attenuation of NMDA receptor-mediated neurotoxicity. Figure 6. NOϪ, Generated from NO· by SOD[Cu(I)], Decreases 2ϩ NMDA-Evoked [Ca ]i Responses Discussion DEA/NO was used as the NO· donor (Maragos et al., 1991); NO· reacts with SOD[Cu(I)] (equivalent to reduced SOD, labeled Red- Our results suggest that NOϪ can react with critical thiol SOD in the figure) to generate NOϪ. Neither DEA/NO alone (20 ␮M), groups of the NMDA receptor to downregulate its activ- SOD[Cu(I)] alone (50 U/ml), nor SOD[Cu(II)] (50 U/ml) in the presence ity (Figure 8). The evidence includes the fact that the of DEA/NO affected NMDA-evoked responses (SOD[Cu(II)], equivalent to oxidized SOD or Oxi-SOD, does not react with DEA/NO). specific thiol-reacting agent MTSET as well as the thiol- Values in the calcium imaging panels are expressed as the mean Ϯ alkylating agent NEM prevent the effect of nitroxyl on SEM, normalized to the first NMDA response following a 2 min NMDAR activity, and site-directed mutagenesis of extra- exposure to DTT; this response was assigned a value of 100% cellular cysteine residues abrogates the effect of NOϪ. 2ϩ (indicated by the arrow). The maximum [Ca ]i response to NMDA We show, moreover, that multiple cysteine residues of -␮M. Re- the NMDA receptor, both on the NR1 and NR2A sub 1ف following incubation in DTT in any one neuron was sponses to NMDA that were statistically smaller than those obtained Ϫ immediately after the first exposure to DTT are indicated by asterisks units, may participate in NO reactions with a varying (*p Ͻ 0.01, analyzed with an ANOVA followed by a Scheffe´ multiple degree of effect. Involvement of several cysteines may comparison of means). Values are mean Ϯ SEM. be the general case in such receptors, whereby they Neuron 466

may provide a molecular basis for recognizing and dif- ferentiating oxidative from nitrosative signals (Xu et al., 1998). In this case, most of the NOϪ effect derives from modification of a single cysteine at position 399 of the NR2A subunit. The reaction mechanisms for singlet and triplet NOϪ are different, and the identity of the function-regulating species in biological fluids is likely to be system and source dependent (Wink and Feelisch, 1996; Bonner and Stedman, 1996). However, the resulting oxidation of thiol is reminiscent of the reactions of nitrosothiols, which act as nitrosyl (NOϩ) donors to downregulate NMDA channel activity. Such reactions with critical thiol group(s) of the NMDA receptor afford protection from excitotoxic damage to neurons (Lipton et al., 1993; Lipton and Stamler, 1994; Sullivan et al., 1994) and stand in stark contrast to the neurodestructive potential of NO·-related pathways, as delineated previously (Beckman et al., 1990; Dawson et al., 1991, 1993; Lipton et al., 1993; Bonfoco et al., 1995). Inasmuch as nNOS produces NOϩ and NOϪ activity (Stamler et al., 1992b; Stamler, 1994; Hobb et al., 1994; Zhao et al., 1995; Schmidt et al., 1996; Kluge et al., 1997), our results explain downregulation of NMDA responses by virtue of reaction with critical cysteines. Recently, we and others have demonstrated additional protein thiol targets for the NO group in addition to the NMDAR that may also contribute to neuroprotection. These targets include critical cysteine residues in p21ras and the active site of caspase enzymes (Dim- meler et al., 1997; Lander et al., 1997; Melino et al., 1997; Ogura et al., 1997; Tenneti et al., 1997; Yun et al., 1998; Mannick et al., 1999). nNOS has recently been colocalized with the NMDA Figure 8. Proposed Mechanism for the Action of NOϪ, Nitroxyl receptor through PDZ domain interactions (Brenman et Anion al., 1996)—whereby it can achieve local action. Indeed It has been proposed that NOϪ can be generated in two forms, NOH or HNO, both of which may exist in the triplet or singlet states the physical proximity of nNOS and the NMDA receptor, (Bonner and Stedman, 1996; Wink and Feelisch, 1996). For example, the cellular experiments by us and others showing NOS in the singlet state, two antibonding electrons occupy a single outer downregulation of NMDA receptor activity, and our ␲* (pi antibonding molecular orbital), with spins opposed. In the chemical data on NOϩ and NOϪ are all internally consis- triplet state, the two ␲* antibonding orbitals each contain an electron tent and indicate that NOϩ or NOϪ regulation of the with spins aligned (with a z component of electron spin of ϩ1, 0, NMDA receptor, as well as of other protein targets (Ten- or Ϫ1; hence, the designation “triplet state”). neti et al., 1997; Yun et al., 1998), is likely to be physiolog- (A) In the singlet state, NOϪ/HNO (pK 4.7) can react with critical a ically relevant. It is also intriguing to speculate on the thiols of the NMDA receptor to yield an R-SNH-OH derivative (A1) potential role of NOϪ in localizing “NO” signals in a way or disulfide (A2), in which case hydroxylamine (NH2OH) is also formed (Doyle et al., 1988; Arnelle and Stamler, 1995). These reactions would that is inherently resistant to neurotoxic mechanisms. downregulate NMDA receptor activity (Lipton et al., 1993; Sullivan et That NOϪ can ameliorate excitotoxic neuronal damage al., 1994). The presence of a disulfide bond was recently confirmed in raises the possibility that exogenous donor drugs may the crystal structure of GluR2 receptors, which revealed the forma- be used for therapeutic gain in a wide variety of neuro- tion of disulfide between cysteine residues homologous to NR1 Cys- logic disorders that are mediated, at least in part, by 744 and Cys-798 (Armstrong et al., 1998), and this may also be true excessive stimulation of NMDA receptors (Choi, 1988; for NR2A Cys-87 and Cys-320 (Y.-B. Choi et al., 1997, Soc. Neurosci., abstract). To confirm this type of reaction scheme here, we have Meldrum and Garthwaite, 1990; Dawson et al., 1991; shown that hydroxylamine forms when an NOϪ-generating drug re- Lipton and Rosenberg, 1994). acts with vicinal thiols. Thus, reactions of singlet NOϪ are likely to be neuroprotective, at least under our conditions. Experimental Procedures (B) Triplet NOϪ, rather than reacting directly with thiol, tends to react with molecular oxygen to form peroxynitrite (ONOOϪ) (Bonner and Cerebrocortical Cultures Stedman, 1996), which can also oxidize thiols to disulfide (B1) (Radi Cortical cultures of mixed neurons and glia were derived from em- et al., 1991); this reaction can be reversed with DTT. Alternatively, bryonic (fetal day 15 or 16) Sprague-Dawley rats as described pre- ONOOϪ can oxidize thiol to produce an -OH modification of sulfur viously (Lei et al., 1992; Lipton et al., 1993). Briefly, following dissoci- (a sulfenic acid or R-SOH, as shown in [B2]), which is also reversible ation in 0.027% trypsin, cerebral cortical cells were plated at a with DTT (Becker et al., 1998; Stamler and Hausladen, 1998). Indeed, we have demonstrated that at low concentrations peroxynitrite downregulates NMDA receptor activity, apparently reacting at a redox roprotective—are likely to be more tenuous in vivo, as peroxynitrite modulatory site(s) on the receptor since DTT can reverse this effect is a powerful oxidant that has been shown to account for NO-related Ϫ (Kim et al., 1996). Such effects of triplet NO /O2—while supportive neurotoxicity (Beckman et al., 1990; Dawson et al., 1993; Lipton et of the molecular mechanism entertained here and theoretically neu- al., 1993; Bonfoco et al., 1995). Nitroxyl Attenuates NMDA Activity 467

density of 4.5 ϫ 105 per 35 mm dish containing poly-L-lysine-coated Preparation of Xenopus Oocytes and Injection of cRNA glass cover slips in Dulbecco’s modified Eagle’s medium with Ham’s Frog oocytes of stages V or VI were surgically removed from the F12, and heat-inactivated iron-supplemented calf serum (HyClone) ovaries of Xenopus laevis. Lumps of about 100 oocytes were incu- at a ratio of 8:1:1. After 15 days in culture (when the astrocyte layer bated with 580 U/ml (2 mg/ml) collagenase type I (Sigma) for 2 hr had become confluent), the cultures were treated with cytosine in Ca2ϩ-free frog Ringer’s solution (82.5 mM NaCl, 2 mM KCl, 1 mM arabinoside for 72 hr. The culture medium was replenished three MgCl2, 5 mM HEPES [pH 7.5 with NaOH]). After slow agitation to times weekly. Cultures were incubated at 36ЊC in a 5% CO2/95% remove the follicular cell layer, the oocytes were washed extensively air humidified atmosphere. The cultures were used for experiments with Ca2ϩ-free frog Ringer’s solution. Oocytes were maintained at at room temperature (21ЊC–24ЊC) 3–4 weeks after plating. Neurons 18ЊC in frog Ringer’s solution (96 mM NaCl, 2 mM KCl, 1.8 mM could be reliably identified by morphological criteria under phase- CaCl2, 1 mM MgCl2, 5 mM HEPES [pH 7.5 with NaOH]) supplemented contrast optics, as later confirmed by patch-clamp recording. To with 550 mg/L sodium pyruvate as a carbon source and 100 ␮g/ml permit physiology experiments (calcium imaging or patch-clamping) gentamicin. Oocytes were injected with up to 10 ng of cRNA of each under normal room air conditions, just prior to a recording session subunit 24 hr later, using a 10 ␮l Drummond microdispenser under the culture medium was exchanged for a solution based on Hanks’ visual control with a dissecting microscope. balanced salts. The Hanks’ saline consisted of: 137.6 mM NaCl; 1 mM NaHCO3; 0.34 mM Na2HPO4; 5.36 mM KCl; 0.44 mM KH2PO4; Two-Electrode Voltage Clamp Recording of Oocytes 1.25–2.5 mM CaCl2; 5 mM HEPES; 22.2 mM dextrose (adjusted to pH 7.2 with 0.3 M NaOH). Two days after injection, oocytes were recorded in frog Ringer’s solution under two-electrode voltage clamp at Ϫ80 mV. Recordings were performed with an Oocyte Clamp OC-725b amplifier (Warner Nitroxyl-Producing Drugs, Calcium Imaging, Instrument) using MacLab v3.5 software (ADI Instruments). Voltage- and Patch-Clamp Recording sensing electrodes had a resistance of 1–4 M⍀ and current-injecting NOϪ-generating drugs included Oxi-NO (sodium trioxodinitrate, also electrodes, 0.5–1 M⍀. Both were filled with 3 M KCl. Oocytes were known as Angeli’s salt), Sulfi-NO (N-nitrosohydroxylamine-N-sulfo- continuously superfused with a solution containing 90 mM NaCl, 1 nate/ammonium salt), and Piloty’s acid (benzenesulfohydroxamic mM KCl, 10 mM HEPES, 1.5 mM BaCl2, and 100 ␮M glycine (pH acid) (Fukuto et al., 1992; Nagasawa et al., 1995; Zamora et al., 7.5). Barium was used as the divalent cation, rather than calcium, 2ϩ in order to minimize secondary activation of calcium-activated ClϪ 1995; Feelisch and Stamler, 1996). Digital [Ca ]i imaging was performed with standard methods, as previously described in this prep- current (Leonard and Kelso, 1990). Drugs dissolved in frog Ringer’s ml/min in 2ف aration (Lei et al., 1992; Lipton et al., 1993). All redox reagents, solution were applied by superfusion at a flow rate of ␮l chamber with an array of pipettes similar to the sewer 100ف including DTT (2 mM), DTNB (0.5 mM), NEM (1 mM), and the NOϪ- a generating drugs, were superfused for 2 min, washed away with pipe system used in patch-clamp recording in order to achieve nominally Mg2ϩ-free Hanks’ buffered saline solution for 3–4 min, relatively rapid solution exchange. Using a concentration jump para- 2ϩ digm with Mg2ϩ, we measured a time constant of 3 s for complete ف ,and then the NMDA-elicited [Ca ]i responses were monitored. DTT DTNB, and NOϪ donors, either during incubation or after thorough solution exchange in the recording chamber. 2ϩ washout, did not alter basal [Ca ]i. NMDA-evoked whole-cell currents were recorded using standard Physiological Generation of Nitroxyl Anion patch-clamp techniques, as previously described in this preparation in Cerebrocortical Cultures (Lei et al., 1992; Lipton et al., 1993). Cortical neurons were voltage- Cortical neurons were incubated for 2 min in a mixture of 20 ␮M clamped at Ϫ60 mV and continuously superfused in Mg2ϩ-free DEA/NO (an NO· generator) and 50 U/ml SOD[Cu(I)] to mimic the Hanks’ balanced salt solution with 1.25 mM Ca2ϩ,1␮M TTX, and 1 physiological reduction of NO· to NOϪ that can be achieved by ␮M glycine (pH 7.2). The pipette-filling solution contained 120 mM several redox enzymes including nNOS and SOD. Endogenous NOϪ CsCl, 20 mM tetraethylammonium chloride, 2 mM MgCl ,1mM 2 can be generated from NO· by reduced SOD, as described previously CaCl , 2.25 mM EGTA, and 10 mM Cs-HEPES (pH 7.2). NMDA (200 2 (Murphy and Sies, 1991) and confirmed by us. After thorough rinsing ␮M) was puffed onto neurons with a pneumatic pipette. Under our in Mg2ϩ-free Hanks’ buffered salt solution, NMDA-elicited [Ca2ϩ] conditions, NO· donors alone did not affect NMDA-evoked re- i responses were assessed. The concentration of NO· was measured sponses (for further discussions of this point, see Manzoni et al., with an NO·-selective electrode (WPI), as previously detailed (Lipton 1992, and Fagni et al., 1995, vis-a´ -vis Lipton et al., 1993, 1996, and et al., 1993). These measurements were performed under the same Lipton, 1999). conditions as the electrophysiological recordings, that is, in Hanks’ solution in the presence of the cells in culture. The generation of hydroxylamine during the reaction of singlet NOϪ with thiol to form cRNA Synthesis disulfide was followed spectrophotometrically, as previously de- cDNA for NR1–1a (the exclusive splice variant of NR1 used in these scribed (Arnelle and Stamler, 1995; Gow and Stamler, 1998). studies) and for NR2A were the kind gifts of Shigetada Nakanishi, Stephen Heinemann, and Peter Seeburg. The template was pre- pared from a circular plasmid cDNA by linearizing in the 3Ј untrans- Neurotoxicity Assays lated region with NheI (for NR1) or EcoRV (for NR2A). cRNA that For the neurotoxicity experiments, the normal culture medium was 7 incorporates 5Ј cap analog (m G[5Ј]ppp[5Ј]G) was transcribed from exchanged at room temperature for Earle’s Balanced Salt Solution 1 ␮g of linearized template in vitro by T7 (for NR1) or T3 (for NR2A) (EBSS) without phenol red (GIBCO #310–4015AG). Some cultures RNA polymerase according to the Ambion (mMessage mMachine) were pretreated with NO-related species a couple of minutes prior protocol. cRNA concentrations were determined by measuring the to the addition of NMDA. The cultures were then incubated in 300 optical density at 260 nm, and the purity of the cRNA was assessed ␮M NMDA plus 10 ␮M glycine and assayed for neurotoxicity after by agarose gel electrophoresis. overnight incubation by monitoring leakage of lactate dehydrogenase (LDH) into the medium with verification by cell counts, as previously described (Koh and Choi, 1987; Lei et al., 1992; Lipton Site-Directed Mutagenesis et al., 1993; Bonfoco et al., 1995). Mutants were generated using the Chameleon double-stranded site- directed mutagenesis kit based on T7 DNA polymerase (Strata- gene). Mutants were verified by sequencing. The NR1 mutants, Acknowledgments NR1(C744A) and NR1(C798A), were available to us from J. M. Sulli- van. NR1 constructs were cloned into the high expression vector This work was supported in part by NIH grants from the National pGEMHE (Liman et al., 1992) to maximize the expression of NR1 Eye Institute (R01 EY05477 and R01 EY09024 to S. A. L.), the National receptor protein in Xenopus oocytes. Multiple cysteine mutants Institute of Child Health and Human Development (P01 HD29587 to were generated as necessary by restriction enzyme digestion and S. A. L.), and the National Heart, Lung, and Blood Institute (R01 subcloning of relevant fragments. HL52529 and HL59130 to J. S. S.). S. A. L. was a consultant to and Neuron 468

received sponsored research support from Neurobiological Tech- In Methods in Nitric Oxide Research. M. Feelisch and J.S. Stamler, nologies (Richmond, CA) and Allergan (Irvine, CA) in the field of eds. (Chichester, United Kingdom: Wiley), pp. 71–115. NMDA receptor antagonists. Fukuto, J.M., Wallace, G.C., Hszieh, R., and Chaudhuri, G. (1992). Chemical oxidation of N-hydroxyguanidine compounds. Release of Received October 21, 1998; revised August 31, 1999. nitric oxide, nitroxyl and possible relationship to the mechanism of biological nitric oxide generation. Biochem. Pharmacol. 43, 607–613. Gow, A.J., and Stamler, J.S. (1998). Reactions between nitric oxide References and haemoglobin under physiological conditions. Nature 391, 169–173. Aizenman, E., Lipton, S.A., and Loring, R.H. (1989). Selective modulation of NMDA responses by reduction and oxidation. Neuron 2, Hobb, A.J., Fukuto, J.M., and Ignarro, L.J. (1994). Formation of free 1257–1263. nitric oxide from L-arginine by nitric oxide synthase: direct enhance- ment of generation by superoxide dismutase. Proc. Natl. Acad. Sci. Akabas, M.H., Stauffer, D.A., Xu, M., and Karlin, A. (1992). Acetylcho- USA 91, 10992–10996. line receptor channel structure probed in cysteine-substitution mutants. Science 258, 307–310. Hogg, N., Singh, R.J., and Kalyanaraman, B. (1996). The role of glutathione in the transport and catabolism of nitric oxide. FEBS Armstrong, N., Sun, Y., Chen, G.-Q., and Gouaux, E. (1998). Structure Lett. 382, 223–228. of a glutamate-receptor ligand-binding core in complex with kainate. Hughes, M.N. (1999). Relationships between nitric oxide, nitroxyl Nature 395, 913–917. ion, nitrosonium cation and peroxynitrite. Biophys. Biochim. Acta ϩ · Ϫ Arnelle, D.R., and Stamler, J.S. (1995). NO ,NO, and NO donation 1411, 263–272. by S-nitrosothiols: implications for regulation of physiological func- Kendrick, K.M., Guevara-Guzman, R., de la Riva, C., Christensen, tions by S-nitrosylation and acceleration of disulfide formation. Arch. J., Østergaard, K., and Emson, P.C. (1996). NMDA and kainate- Biochem. Biophys. 318, 279–285. evoked release of nitric oxide and classical transmitters in the rat Becker, K., Savvides, S.N., Keese, M., Schirmer, F.H., and Karplus, striatum: in vivo evidence that nitric oxide may play a neuroprotec- P.A. (1998). Enzyme inactivation through sulfhydryl oxidation by tive role. Eur. J. Neurosci. 8, 1619–1634. physiologic NO-carriers. Nat. Struct. Biol. 5, 267–271. Kim, W.-K., Rayudu, P.V., Mullins, M.E., Stamler, J.S., and Lipton, Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., and Free- S.A. (1996). Down regulation of NMDA receptor activity in cortical man, B.A. (1990). Apparent hydroxyl radical production by peroxyni- neurons by peroxynitrite. In The Biology of Nitric Oxide, Part 5, S. trite: implications for endothelial injury from nitric oxide and super- Moncada, J.S. Stamler, S. Gross, and E.A. Higgs, eds. (London: oxide. Proc. Natl. Acad. Sci. USA 87, 1620–1624. Portland Press), p. 26. Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P., and Lipton, Kluge, I., Gutteck-Amsler, U., Zollinger, M., and Do, K.Q. (1997). S.A. (1995). Apoptosis and necrosis: two distinct events induced S-nitrosoglutathione in rat cerebellum: identification and quantita- respectively by mild and intense insults with NMDA or nitric oxide/ tion by liquid chromatography-mass spectrometry. J. Neurochem. superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. USA 92, 69, 2599–2607. 7162–7166. Koh, J.Y., and Choi, D.W. (1987). Quantitative determination of gluta- Bonner, F.T., and Stedman, G. (1996). The chemistry of nitric oxide mate mediated cortical neuronal injury in cell culture by lactate and redox-related species. In Methods in Nitric Oxide Research. M. dehydrogenase efflux assay. J. Neurosci. Methods 20, 83–90. Feelisch and J.S. Stamler, eds. (Chichester, United Kingdom: Wiley), Lander, H.M., Hajjar, D.P., Hempstead, B.L., Mirza, U.A., Chait, B.T., pp. 3–27. Campbell, S., and Quillam, L.A. (1997). A molecular redox switch on Brenman, J.E., Chao, D.S., Gee, S.H., McGee, A.W., Craven, S.E., p21(ras). Structural basis for the nitric oxide-p21(ras) interaction. J. Santilliano, D.R., Wu, Z., Huang, F., Xia, H., Peters, M.F., et al. (1996). Biol. Chem. 272, 4323–4326. Interaction of nitric oxide synthase with the postsynaptic density Lei, S.Z., Pan, Z.-H., Aggarwal, S.K., Chen, H.S., Hartman, J., Sucher, protein PSD-95 and ␣1-syntrophin mediated by PDZ domains. Cell N.J., and Lipton, S.A. (1992). Effect of nitric oxide production on 84, 757–767. the redox modulatory site of the NMDA receptor–channel complex. Butler, A.R., Flitney, F.W., and Williams, D.L. (1995). NO, nitrosonium Neuron 8, 1087–1099. ions, nitrosothiols and iron-nitrosyls in biology: a chemist’s perspec- Leonard, J.P., and Kelso, S.R. (1990). Apparent desensitization of tive. Trends Pharmacol. Sci. 16, 18–22. NMDA responses in Xenopus oocytes involves calcium-dependent Chazotte-Aubert, L., Oikawa, S., Gilibert, I., Bianchini, F., Sawanishi, chloride current. Neuron 4, 53–60. S., and Oshima, H. (1999). Cytotoxicity and site-specific DNA dam- Liman, E.R., Tygat, J., and Hess, P. (1992). Subunit stoichiometry of Ϫ age induced by nitroxyl anion (NO ) in the presence of hydrogen a mammalian Kϩ channel determined by construction of multimeric peroxide. J. Biol. Chem. 274, 20909–20915. cDNAs. Neuron 9, 861–871. Choi, D.W. (1988). Glutamate neurotoxicity and diseases of the ner- Lipton, S.A. (1999). Redox sensitivity of NMDA receptors. Methods vous system. Neuron 1, 623–634. Mol. Biol. 128, 121–130. Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S., and Snyder, Lipton, S.A., and Rosenberg, P.A. (1994). Mechanisms of disease: S.H. (1991). Nitric oxide mediates glutamate neurotoxicity in primary Excitatory amino acids as a final common pathway for neurologic cortical cultures. Proc. Natl. Acad. Sci. USA 88, 6368–6371. disorders. N. Engl. J. Med. 330, 613–622. Dawson, V.L., Dawson, T.M., Bartley, D.A., Uhl, G.R., and Snyder, Lipton, S.A., and Stamler, J.S. (1994). Actions of redox-related con- S.H. (1993). Mechanisms of nitric oxide-mediated neurotoxicity in geners of nitric oxide at the NMDA receptor. Neuropharmacology primary brain cultures. J. Neurosci. 13, 2651–2661. 33, 1229–1233. Dimmeler, S., Haendeler, J., Nehls, M., and Zeiher, A.M. (1997). Lipton, S.A., Choi, Y.-B., Pan, Z.-H., Lei, S.Z., Chen, H.-S.V., Sucher, Suppression of apoptosis by nitric oxide via inhibition of interleukin- N.J., Loscalzo, J., Singel, D.J., and Stamler, J.S. (1993). A redox- 1␤-converting enzyme (ICE)-like and cysteine protease protein based mechanism for the neuroprotective and neurodestructive ef- (CPP)-32-like proteases. J. Exp. Med. 185, 601–607. fects of nitric oxide and related nitroso-compounds. Nature 364, Doyle, M.P., Surendra, M.N., Broene, R.D., and Guy, J.K. (1988). 626–632. Oxidation and reduction of hemoproteins by trioxodinitrite(II). The Lipton, S.A., Choi, Y.-B., Sucher, N.J., Pan, Z.-H., and Stamler, J.S. role of nitrosyl hydride and nitrite. J. Am. Chem. Soc. 110, 593–599. (1996). Redox state, NMDA receptors, and NO-related species. Fagni, L., Olivier, M., Lafon-Cazal, M., and Bockaert, J. (1995). Trends Pharmacol. Sci. 17, 186–187. Involvement of divalent ions in the nitric oxide-induced blockade Mannick, J.B., Hausladen, A., Liu, L., Hess, D.T., Zeng, M., Miao, of N-methyl-D-aspartate receptors in cerebellar granule cells. Mol. Q.X., Kane, L.S., Gow, A.J., and Stamler, J.S. (1999). Fas-induced Pharmacol. 47, 1239–1247. caspase denitrosylation. Science 284, 651–654. Feelisch, M., and Stamler, J.S. (1996). Donors of nitrogen oxides. Manzoni, O., and Bockaert, J. (1993). Nitric oxide synthase activity Nitroxyl Attenuates NMDA Activity 469

endogenously modulates NMDA receptors. J. Neurochem. 61, Zamora, R., Grzesiok, A., Weber, H., and Feelisch, M. (1995). Oxida- 368–370. tive release of nitric oxide accounts for guanylyl cyclase stimulating, Manzoni, O., Prezeau, L., Marin, P., Deshager, S., Bockaert, J., and vasodilator and anti-platelet activity of Piloty’s acid: a comparison Fagni, L. (1992). Nitric oxide-induced blockade of NMDA receptors. with Angeli’s salt. Biochem. J. 312, 333–339. Neuron 8, 653–662. Zhao, X.-J., Sampath, V., and Caughey, W.S. (1995). Cytochrome c Maragos, C.M., Morley, D., Wink, D.A., Dunams, T.M., Saavedra, oxidase catalysis of the reduction of nitric oxide to nitrous oxide. J.E., Hoffman, A., Bove, A.A., Isaac, L., Hrabie, J.A., and Keefer, Biochem. Biophys. Res. Commun. 212, 1054–1060. L.K. (1991). Complexes of ·NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects. J. Med. Chem. 34, 3242–3247. Mayer, B., Pfeiffer, S., Schrammel, A., Koesling, D., Schmidt, K., and Brunner, F. (1998). A new pathway of nitric oxide/cyclic GMP signaling involving S-nitrosoglutathione. J. Biol. Chem. 273, 3264– 3270. Meldrum, B., and Garthwaite, J. (1990). Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11, 379–387. Melino, G., Bernassola, F., Knight, R.A., Corasaniti, M.T., Nistico` , G., and Finazzi-Agro` , A. (1997). S-nitrosylation regulates apoptosis. Nature 388, 432–433. Murphy, M.E., and Sies, H. (1991). Reversible conversion of nitroxyl anion to nitric oxide by superoxide dismutase. Proc. Natl. Acad. Sci. USA 88, 10860–10864. Nagasawa, H.T., Kawle, S.P., Elberling, J.A., DeMaster, E.O., and Fukuto, J.M. (1995). Prodrugs of nitroxyl as potential aldehyde dehydrogenase inhibitors vis-a´ -vis vascular smooth muscle. J. Med. Chem. 38, 1865–1871. Ogura, T., Tatemichi, M., and Esumi, H. (1997). Nitric oxide inhibits CPP32-like activity under redox regulation. Biochem. Biophys. Res. Commun. 236, 365–369. Pufahl, R.A., Wishnok, J.S., and Marletta, M.A. (1995). Hydrogen peroxide-supported oxidation of NG-hydroxy-L-arginine by nitric oxide synthase. Biochemistry 34, 1930–1941. Radi, R., Beckman, J.S., Bush, K.M., and Freeman, B.A. (1991). Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266, 4244–4250. Schmidt, H.H.H.W., Holman, H., Schindler, U., Shutenko, Z.S., Cun- ningham, D.D., and Feelisch, M. (1996). No ·NO from NO synthase. Proc. Natl. Acad. Sci. USA 93, 14492–14497. Stamler, J.S. (1994). Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78, 931–936. Stamler, J.S., and Hausladen, A. (1998). Oxidative modifications in nitrosative stress. Nat. Struc. Biol. 5, 247–249. Stamler, J.S., Simon, D.I., Osborne, J.A., Mullins, M.E., Jaraki, O., Michel, T., Singel, D.J., and Loscalzo, J. (1992a). S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologi- cally active compounds. Proc. Natl. Acad. Sci. USA 89, 444–448. Stamler, J.S., Singel, D.J., and Loscalzo, J. (1992b). Biochemistry of nitric oxide and its redox activated forms. Science 258, 1898–1902. Stamler, J.S., Toone, E.J., Lipton, S.A., and Sucher, N.J. (1997). (S)NO signals: translocation, regulation, and a consensus motif. Neuron 18, 691–696. Sullivan, J.M., Traynelis, S.F., Chen, H.S., Escobar, W., Heinemann, S.F., and Lipton, S.A. (1994). Identification of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor. Neuron 13, 929–936. Tenneti, L., D’Emilia, D.M., and Lipton, S.A. (1997). Suppression of neuronal apoptosis by S-nitrosylation of caspases. Neurosci. Lett. 236, 139–142. Wink, D.A., and Feelisch, M. (1996). Formation and detection of nitroxyl and nitrous oxide. In Methods in Nitric Oxide Research. M. Feelisch and J.S. Stamler, eds. (Chichester, United Kingdom: Wiley), pp. 403–412. Xu, L., Eu, J.P., Meissner, G., and Stamler, J.S. (1998). Activation of the cardiac calcium release channel (ryanodine receptor) by poly- S-nitrosylation. Science 279, 234–237. Yun, H.-Y., Gonzalez-Zulueta, M., Dawson, V.L., and Dawson, T.M. (1998). Nitric oxide mediates N-methyl-D-aspartate receptor-induced activation of p21ras. Proc. Natl. Acad. Sci. USA 95, 5773–5778.