Different Signalling Pathways Mediate the Opposite Effects ofEndogenous Versus Exogenous Nitric Oxide on Hydroperoxide

Toxicity in CHP100 Neuroblastoma Cells

Andrea Guidarelli, *†Emilio Clementi, †Clara Sciorati, and Orazio Cantoni

Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, Universita` di Urbino, Urbino;*Dipartimento Farmacobiologico, Universita` della Calabria, Cosenza; and†Consiglio Nazionale delle Ricerche, Molecular and

Cellular Pharmacology Centre, DIBIT—San Raffaele Scientific Institute, Milano, Italy

Abstract: The results presented in this study indicate thatthe toxic response brought about by increasing concentra-tions of tert-butylhydroperoxide in CHP100 cells was miti-gated significantly by exogenously added nitric oxide do-nors via a cyclic GMP-independent mechanism. In contrastwith these results, endogenous nitric oxide generated bythe Ca21-mobilizing agent caffeine was found to increasehydroperoxide toxicity. Under these conditions, nitric oxidewas not directly toxic to the cells. Rather, nitric oxide wasfound to promote the caffeine-mediated release of Ca21

from ryanodine-sensitive Ca21 stores via a cyclic GMP-independent mechanism. Release of the cation from ryan-odine-sensitive Ca21 stores was causally linked with thecaffeine/nitric oxide-mediated enhancement of tert-butylhy-droperoxide toxicity. It is concluded that endogenous andexogenous nitric oxide activate diverging signalling path-ways independent of cyclic GMP formation and causingopposite effects on the toxic response evoked by tert-butylhydroperoxide in CHP100 cells. Key Words: Nitric ox-ide —Nitric oxide donors—tert-Butylhydroperoxide —Cy-totoxicity—CHP100 neuroblastoma cells.J. Neurochem. 73, 1667–1673 (1999).

Nitric oxide (NO) is a free radical that is producedendogenously by the enzyme NO synthase (NOS), whichcatalyzes the oxidation ofL-arginine, yielding NO andL-citrulline (Bredt and Snyder, 1994; Knowles andMoncada, 1994). NOS exists in three different isoforms,two of which are constitutive, whereas the third is in-ducible. The constitutive enzymes, which were first char-acterized in neuronal and endothelial cells, are activatedby increases in the intracellular Ca21 concentration([Ca21]i) and produce low levels of NO (Fo¨rstermannand Kleinert, 1995). In contrast, the inducible NOS isCa21-independent and, once expressed following cellactivation with cytokines and bacterial products, sustainsthe continuous generation of high amounts of NO (Bredtand Snyder, 1994; Knowles and Moncada, 1994).

NO regulates various cell functions via cyclic GMP(cGMP)-dependent and -independent mechanisms

(Moncada et al., 1994; Stamler, 1994), and some of theseeffects may be beneficial in an array of pathologicalconditions (Moncada et al., 1991). Among these are theputative antioxidant effects that result in cytoprotectionof oxidatively injured cells (Lipton et al., 1993; Wink etal., 1993; Gorbunov et al., 1997). Whether NO behavesas a true antioxidant is still a matter of debate and,although experimental support for this mechanism hasbeen found in some investigations (Lipton et al., 1993;Wink et al., 1993), other authors have based the mech-anism of cytoprotection observed in cells exposed toorganic hydroperoxides on nitrosylation of heme andnon-heme iron (Gorbunov et al., 1997). Nitrosylationwould appear to be favored by oxidizing conditions, towhich NO can also contribute (Bolan˜os et al., 1996;Clementi et al., 1998).

The present report demonstrates for the first time thatexogenous and endogenous NO promote opposite effectsin oxidatively injured cells. Indeed, as previously re-ported by our laboratory (Guidarelli et al., 1997a) andothers (Gorbunov et al., 1997), exogenously added NOdonors afforded cytoprotection in CHP100 cells injuredby tert-butylhydroperoxide (tBu-OOH). Formation ofendogenous NO, however, was causally linked to thecaffeine (Cf)-mediated enhancement of the toxic re-

Received March 10, 1999; revised manuscript received June 14,1999; accepted June 14, 1999.

Address correspondence and reprint requests to Dr. O. Cantoni atIstituto di Farmacologia e Farmacognosia, Universita` di Urbino, Via S.Chiara 27, 61029 Urbino (PS), Italy.

Abbreviations used:[Ca21]i, intracellular Ca21 concentration; Cf, caf-feine; cGMP, cyclic GMP; DETA-NO, (Z )-1-[(2-aminoethyl)-N-(2-am-monioethyl)amino]diazen-1-ium-1,2-diolate; GSNO,S-nitrosoglutathione;IP3, inositol 1,4,5-trisphosphate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;L-NAME, NG-nitro-L-arginine methyl ester;L-NIO, L-N5-(1-iminoethyl)ornithine; NO, nitric oxide; NOS, nitric oxidesynthase; ODQ, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; PTIO,2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide; Ry, ryanodine;SERCA, sarcoplasmic/endoplasmic reticulum Ca21-ATPase; SNAP,S-nitroso-N-acetylpenicillamine; tBu-OOH,tert-butylhydroperoxide; Tg,thapsigargin.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 1999 International Society for Neurochemistry

sponse promoted by the hydroperoxide. NO was notdirectly toxic to the cells but, rather, it appeared to playa pivotal role in the Cf-induced release of Ca21 fromryanodine (Ry)-sensitive Ca21 stores, which eventuallyleads to cell damage.

MATERIALS AND METHODS

MaterialsDulbecco’s modified Eagle’s medium, Ham’s nutrient mixture

F-12 medium, fetal clone III, andL-glutamine were purchasedfrom HyClone Europe, Ltd. (Cramlington, U.K.). Tissue cultureflasks (Falcon 3024) and multiwell tissue culture plates (Falcon3046) were obtained from Becton–Dickinson (Lincoln Park, NJ,U.S.A.). tBu-OOH, Cf, NG-nitro-L-arginine methyl ester (L-NAME), L-N5-(1-iminoethyl)ornithine (L-NIO), S-nitroso-N-acetylpenicillamine (SNAP),S-nitrosoglutathione (GSNO), Ry,2-phenyl-4,4,5,5-tetramethylmidazolin-1-oxyl-3-oxide (PTIO),1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), gentamicin,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), and the remaining chemicals were from Sigma–Aldrich(Milano, Italy). (Z )-1-[(2-Aminoethyl)-N-(2-ammonioethyl)ami-no]diazen-1-ium-1,2-diolate (DETA-NO) was obtained fromAlexis Corp. (Laufelfigen, Switzerland).

Cell culture and treatmentsCHP100 human neuroblastoma cells were grown in a 1:1

mixture of Dulbecco’s modified Eagle’s medium and Ham’snutrient mixture F-12 medium supplemented with 15% heat-inactivated fetal clone III, gentamicin (0.1 mg/ml), andL-glutamine (2 mM), at 37°C in T-75 tissue culture flasks gassedwith an atmosphere of 95% air/5% CO2.

Stock solutions of tBu-OOH and DETA-NO were freshlyprepared in saline A (0.14M NaCl, 5 mM KCl, 4 mM NaHCO3,5 mM glucose). Cf,L-NAME, 8-bromo-cGMP, andL-NIO weredissolved in distilled water. SNAP, GSNO, Ry, PTIO, andODQ were dissolved in 95% ethanol. At the treatment stage,the final ethanol concentration was never higher than 0.05%and, under these conditions, was not toxic nor did it affect thecytotoxicity of tBu-OOH. A light-exposed solution of SNAP,which decomposes to penicillamine orN-acetylpenicillamine,was prepared by leaving SNAP in saline A at room temperatureunder a table lamp (100 W) for 1 day.

Cytotoxicity assayCells were subcultured from confluent 75-cm2 flasks and

seeded in 35-mm six-well plates (33 105 cells/well). Afterculturing for 16 h, the medium was removed and the cultureswere washed and treated for 30 min in saline A (2 ml). After thetreatments, the cells were washed with saline A and incubatedin complete medium at 37°C for 6 h. At the fifth hour ofincubation, MTT (25mg/ml) was added to each well, and theplate was incubated for a further 1 h at37°C in 5% CO2 untilblue formazan was visible. The medium was then removed andreplaced with 1 ml of dimethyl sulfoxide, and cell viability wasassessed by measuring MTT reductase activity. Absorbancewas read at 570 nm. Results are expressed as the percentage ofMTT-reducing activity (absorbance in treated versus controlcells).

[Ca21]i measurementsCells were harvested, washed three times by centrifugation,

and resuspended in Krebs–Ringer–HEPES medium containing125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4,2 mM CaCl2, 6 mM glucose, and 25 mM HEPES–NaOH (pH

7.4). Cell suspensions were loaded with the Ca21-sensitive dyefura-2 acetoxymethyl ester (3mM final concentration) for 30min at 25°C in Krebs–Ringer–HEPES medium and kept at37°C until use. Cell aliquots (43 106 cells) were washed threetimes and resuspended in saline A, transferred to a thermostat-ted cuvette in a Perkin–Elmer LS-50 fluorimeter, and main-tained at 37°C under continuous stirring.

Statistical analysisStatistical analysis of the data for multiple comparisons was

performed by ANOVA followed by Dunnett’s test. For singlecomparisons, the significance of differences between meanswas determined by Student’st test.

RESULTS

NO donors mitigate the toxic response of CHP100cells exposed to tBu-OOH

The effect of the NO donor SNAP on the toxicitymediated by tBu-OOH in CHP100 cells was investi-gated. In these experiments the cells were exposed for 30min in a glucose-containing saline to 3 mM tBu-OOH, inthe absence or presence of increasing concentrations(3–60mM) of SNAP (given to the cultures 5 min beforeaddition of the hydroperoxide), and the level of toxicitywas assessed after 6 h of posttreatment incubation infresh culture medium using the MTT assay. As illus-trated in Fig. 1A, the toxic response mediated by tBu-OOH, which caused a loss in MTT-metabolizing activityof ;50% (Fig. 1B), was mitigated significantly bySNAP. Cytoprotection was also afforded by 300mMGSNO and 1 mM DETA-NO (Fig. 1B). This effect of theNO donors appears to be due to release of NO, becausethe SNAP (30mM)-mediated protective effects wereprevented by the NO scavenger PTIO (50mM; Fig. 1B)and disappeared when the NO donor was decomposed byprolonged exposure to light (Fig. 1A). Furthermore,PTIO also abolished the protective effects afforded byGSNO or DETA-NO (Fig. 1B).

The effects of exogenous NO in oxidatively injuredcells appear to be mediated by cGMP-independent mech-anisms, because the guanylate cyclase inhibitor ODQ (1mM) did not modify the effects promoted by SNAP,GSNO, or DETA-NO (Fig. 1B). In addition, the toxicresponse evoked by tBu-OOH was not affected by 8-bromo-cGMP (1 mM; Fig. 1B). Taken together, theseresults indicate that exogenous NO mitigates tBu-OOH-induced toxicity in CHP100 cells via a cGMP-indepen-dent mechanism.

Cf enhances the toxic response of CHP100 cellsexposed to tBu-OOH via a mechanism involvingendogenous NO as well as release of Ca21 fromRy-sensitive Ca21 stores

CHP100 cells express a constitutive, Ca21-dependentNOS isoform that can be stimulated to generate NO byvarious agonists that increase the [Ca21]i, including Cf(Corasaniti et al., 1992; Clementi et al., 1996a; Gui-darelli et al., 1998), a drug known to release Ca21 fromRy-sensitive Ca21 stores (Meissner, 1994). We thereforeused Cf as a tool for assessing the role of endogenous NO

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1668 A. GUIDARELLI ET AL.

in the toxic response evoked by tBu-OOH. In theseexperiments, the cells were exposed for 30 min to in-creasing concentrations of tBu-OOH, in the absence orpresence of 10 mM Cf (given to the cultures 5 min beforeaddition of the hydroperoxide), and the level of toxicitywas assessed after 6 h of posttreatment incubation infresh culture medium by the MTT assay. As illustrated inFig. 2A, Cf significantly increased the toxicity inducedby tBu-OOH. The enhancing effect of Cf was concen-tration dependent (inset to Fig. 2A) and was not observedunder conditions in which this drug was given to thecultures only during the 6 h ofposttreatment incubation

(Fig. 2A). Importantly, Cf was not cytotoxic under any ofthe experimental conditions used in this study (inset toFig. 2A).

The effect of 20mM Ry, which prevents the Cf-mediated efflux of Ca21 from Ry-sensitive Ca21 stores(Meissner, 1994; see below), on the toxicity promoted bytBu-OOH alone or associated with Cf was investigatednext. In these experiments, the cells were exposed for 30min to 3 mM tBu-OOH, in the absence or presence of Cf(10 mM) and/or Ry (20mM). Cf was given to thecultures 5 min before tBu-OOH and Ry 5 min before Cf.The results illustrated in Fig. 2B indicate that Ry pre-

FIG. 2. Cf potentiates the toxicity mediated by tBu-OOH inCHP100 cells. A: The cells were exposed for 5 min to 0 (E) or 10mM Cf (■) and for an additional 30 min to increasing concen-trations of tBu-OOH, and then postincubated in fresh culturemedium for 6 h. In some experiments, the cells were first treatedwith the hydroperoxide and then exposed to 10 mM Cf duringthe 6-h recovery phase (h). Inset: The effect of increasing con-centrations of Cf in the absence (Œ) or presence (■) of 2 mMtBu-OOH. In these experiments, treatments were for 30 minfollowed by postincubation in fresh culture medium for 6 h. TheMTT-metabolizing activity was measured as detailed in Materialsand Methods. Results represent the means 6 SEM calculatedfrom three to nine separate experiments. The toxicity of tBu-OOH was enhanced significantly by Cf at *p , 0.05 or **p , 0.01(ANOVA followed by Dunnett’s test). B: The cells were exposedfor 5 min in saline A to 20 mM Ry, 200 mM L-NAME, 200 mML-NIO, 1 mM ODQ, or 50 mM PTIO and for an additional 5 min to0 or 10 mM Cf, and then treated for a further 30 min with 3 mMtBu-OOH. After the treatments, the cells were postincubated infresh culture medium for 6 h and processed as described in (A).Ry, the NOS inhibitors, PTIO, and ODQ alone in both the ab-sence and presence of Cf were not toxic. Results represent themeans 6 SEM calculated from three to 10 separate experi-ments. The toxicity of tBu-OOH/Cf was reduced significantly at*p , 0.01 (unpaired t test).

FIG. 1. Three different NO donors reduce the toxicity mediatedby tBu-OOH in CHP100 cells. A: The cells were exposed for 5min in saline A to increasing concentrations of freshly prepared(E) or light-decomposed (F) SNAP and then treated for a further30 min with 3 mM tBu-OOH. The MTT-reducing activity wasmeasured after 6 h of postchallenge incubation. Data representthe means 6 SEM of the percent inhibition of the toxic responseevoked by 3 mM tBu-OOH mediated by increasing concentra-tions of SNAP calculated from three to nine separate experi-ments. The toxicity of tBu-OOH was reduced significantly at *p, 0.05 and **p , 0.01 (ANOVA followed by Dunnett’s test). B:The cells were exposed for 5 min to 1 mM ODQ, 1 mM 8-bromo-cGMP, or 50 mM PTIO and for an additional 5 min to 30 mMSNAP, 300 mM GSNO, or 1 mM DETA-NO, and then treated fora further 30 min with 3 mM tBu-OOH. After the treatments, thecells were postincubated in fresh culture medium for 6 h andprocessed as described in (A). Results represent the means6 SEM calculated from three to seven separate experiments.The toxic response evoked by tBu-OOH was reduced signifi-cantly in the presence of the NO donors at *p , 0.01 (unpairedt test).

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vented the Cf-mediated enhancement of the tBu-OOH-induced cytotoxic response. Ry, however, did not affectthe toxicity brought about by the hydroperoxide alone.

The possible involvement of NO and cGMP in thetoxicity evoked by tBu-OOH alone or associated with Cfwas investigated by testing the effects of two differentNOS inhibitors (200mM L-NAME and 200mM L-NIO),a NO scavenger (50mM PTIO), and a guanylate cyclaseinhibitor (1 mM ODQ). As illustrated in Fig. 2B, each ofthese treatments, with the exception of ODQ, preventedthe enhancing effects mediated by Cf. However,L-NAME, L-NIO, or PTIO did not affect the toxic re-sponse brought about by tBu-OOH alone. Importantly,none of the compounds used to modulate the toxicity oftBu-OOH, with or without Cf, was toxic when givenalone to the cultures (data not shown).

The rate of disappearance of tBu-OOH from the ex-tracellular milieu of CHP100 cells was investigated next.For this purpose, the cells were treated with 3 mMtBu-OOH, in the absence or presence of Cf, in a glucose-containing saline, and the levels of the hydroperoxide inthe extracellular medium were measured immediatelyafter addition of the drug, as well as after various timeintervals (2.5, 5, and 10 min). We found that, under thetwo different treatment conditions, the rate of disappear-ance of tBu-OOH from the culture medium was linearand remarkably similar (data not shown), strongly sug-gesting a lack of relationship between cytotoxicity andrate and/or extent of hydroperoxide metabolism/activa-tion to toxic radical species.

Finally, the toxicity mediated by tBu-OOH was notaffected by the addition of nitrite (10–200mM) at thetime of peroxide exposure (data not shown).

Taken together, these results indicate that endogenousNO plays a critical role in the Cf-mediated enhancementof the tBu-OOH-induced toxicity in CHP100 cells. Thiseffect of Cf is mediated by release of Ca21 from Ry-sensitive Ca21 stores.

Release of Ca21 mediated by Cf, unlike thatpromoted by tBu-OOH, is NO-dependent

A number of studies had demonstrated previously thattBu-OOH enhances the [Ca21]i (Thor et al., 1984; Sa-kaida et al., 1991; Livingston et al., 1992; Guidarelli etal., 1997b). Similarly, we found that a short treatmentwith 3 mM tBu-OOH elevated the [Ca21]i in CHP100cells (Fig. 3). This increase in [Ca21]i was due to releasefrom internal stores, because the experiments were per-formed using nominally Ca21-free medium (saline A).Furthermore, similar results (data not shown) were ob-tained using 10mM EGTA-containing saline A (estimat-ed extracellular [Ca21] ' 1028 M). The Ca21 poolmobilized by tBu-OOH was insensitive to pretreatmentof the cells with the sarcoplasmic/endoplasmic reticulumCa21-ATPase (SERCA) blocker thapsigargin (Tg; Fig.3A), the inositol 1,4,5-trisphosphate (IP3)-generating ag-onist ATP (data not shown), or the Ry receptor agonistCf (Fig. 3A). In accordance with these results, the mo-bilization of Ca21 by either Cf (Fig. 3B) or Tg (Fig. 3C)

was not affected by pretreatment with tBu-OOH. TheCa21 pool mobilized by tBu-OOH, however, was dis-charged by ionomycin, an ion carrier specific for divalentcations that acts by exchanging Ca21 with H1 (data notshown). This indicates that tBu-OOH mobilizes Ca21

from intracellular compartments of neutral pH.Thus, tBu-OOH mobilizes Ca21 from pools different

from the one known to be affected by Cf. This conclu-sion is confirmed by the observation that Ry (20mM)prevented the increase in [Ca21]i elicited by Cf in theabsence of detectable effects on the increase in [Ca21]imediated by tBu-OOH (Fig. 4A). It is interesting thatunder conditions in which NO generation was abolishedusing L-NAME (300 mM; Fig. 4B), the Ca21 releaseresponse evoked by Cf was severely impaired. In addi-tion, both the Cf- and the tBu-OOH-induced increases in[Ca21]i were insensitive to ODQ (1mM; Fig. 4B). Fi-nally, SNAP (3–30mM) modified neither the basal[Ca21]i nor the increased [Ca21]i mediated by 3 mMtBu-OOH (data not shown).

Taken together, the above results indicate that toxiclevels of tBu-OOH mobilize Ca21 from pools that aredifferent from the IP3- and Ry-sensitive Ca21 stores.This response appears to be insensitive to endogenous as

FIG. 3. tBu-OOH elevates [Ca21]i in CHP100 cells. A: Fura-2-loaded cells were supplemented with saline A (continuous trace),Tg (30 nM, dotted line), or Cf (10 mM, dashed line), as indicated.tBu-OOH (3 mM ) was added to all samples 5 min later. Thenumbers on the left indicate [Ca21]i. Traces are representative ofeight consistent experiments. B and C: Fura-2-loaded cells weresupplemented with tBu-OOH (3 mM ), and either Cf (B, 10 mM )or Tg (C, 30 nM ) was added 5 min later. Traces are representa-tive of five consistent experiments.

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well as exogenous NO. In addition, these results providegrounds upon which the involvement of endogenous NOin the Cf-mediated enhancement of the tBu-OOH-in-duced toxicity in CHP100 cells can be explained. NOappears to be involved at the level of release of Ca21

mediated by Cf from Ry-sensitive Ca21 stores.

DISCUSSION

The results presented in this study demonstrate thatNO can either decrease or increase the toxic responseevoked by tBu-OOH in cultured CHP100 cells, depend-ing on whether this pleiotropic messenger is generatedexogenously or endogenously. The notion that exoge-nous NO is cytoprotective in oxidatively injured cells iswell established, because a number of laboratories havereported that different NO donors mitigate the lethalresponse evoked by tBu-OOH in an array of differentcell types, including K562 cells (Gorbunov et al., 1997),V79 cells (Wink et al., 1993, 1995), mesencephalic cells(Wink et al., 1993), cardiomyocytes (Gorbunov et al.,1998), and U937 cells (Guidarelli et al., 1997a). We nowreport results indicating that exogenously generated NOalso reduces the lethal response evoked by tBu-OOH incultured CHP100 cells. Three lines of evidence implicateNO as the cytoprotective species in these experiments,Firstly, very low levels of SNAP significantly reducedthe toxicity mediated by tBu-OOH and the light-decom-posed SNAP was inactive (Fig. 1A). Secondly, a protec-tive effect was also observed with two different NOdonors, namely, GSNO and DETA-NO (Fig. 1B). Fi-nally, the specific NO scavenger PTIO, although notaffecting the toxicity caused by tBu-OOH alone, fully

prevented cytoprotection mediated by SNAP, GSNO, orDETA-NO (Fig. 1B).

In principle, this effect of NO could be mediated byeither cGMP-dependent or -independent mechanisms.Indeed, NO increases cGMP levels via activation ofguanylyl cyclase, leading to subsequent activation of thecGMP-dependent protein kinases (Bredt and Snyder,1994). Due to its high redox potential, NO can alsodirectly modulate the activity of other enzymes via ni-trosation, nitration, and oxidation reactions (Kro¨ncke etal., 1997).

The observations that 8-bromo-cGMP did not mitigatethe toxic response evoked by tBu-OOH in CHP100 cellsand that the protective effects of the NO donors wereinsensitive to ODQ (Fig. 1B) rule out the possible in-volvement of cGMP-dependent pathways. Thus, theseresults are consistent with those previously obtained byGorbunov et al. (1997), indicating that the mechanism ofcytoprotection mediated by exogenous NO in erythroleu-kemia cells exposed to organic hydroperoxides does notdepend on cGMP formation and is causally linked tonitrosylation of heme and non-heme iron. It is possiblethat S-nitrosylation mediates cytoprotection under theconditions used in the present study, because the occur-rence of these reactions in response to exogenous NOwas described previously in various cellular proteins,including caspases (Dimmeler et al., 1997), NADH-ubiquinone Q reductase (Clementi et al., 1998), andglyceraldehyde-3-phosphate dehydrogenase (Molina yVedia et al., 1992).

The second important finding presented in this study isthat endogenous NO does not mitigate, but rather en-hances, the toxic response brought about by tBu-OOH inCHP100 cells. Endogenous generation of NO was ob-tained using the Ca21-mobilizing drug Cf, which, aspreviously shown in our laboratory (Guidarelli et al.,1998), promotes a threefold accumulation of nitrite thatis sensitive to the NOS inhibitorL-NAME.

The Cf-mediated enhancement of tBu-OOH toxicity(Fig. 2A) was found to be entirely dependent on thegeneration of endogenous NO, because the increasedcytotoxic response was prevented by the NOS inhibitorsL-NAME and L-NIO (Fig. 2B), as well as by the NOscavenger PTIO (Fig. 2B). It is unlikely that accumula-tion of nitrite was the cause of the increased lethalresponse, because addition of concentrations of nitrite fargreater than those produced by Cf exposure failed toaffect peroxide toxicity (data not shown). Along thesame lines, it is unlikely that the Cf-mediated increase inthe tBu-OOH-induced cytotoxic response was the con-sequence of NOS-dependent effects on hydroperoxideactivation to toxic intermediates, because the rate oftBu-OOH metabolism was not affected by Cf. The en-hancing effects of Cf were causally linked to the releaseof Ca21 from Ry-sensitive Ca21 stores, because theywere prevented by a high concentration of Ry (20mM;Fig. 2B). Importantly, as previously reported (Meissner,1994; Guidarelli et al., 1997b), 20 mM Ry abolished the

FIG. 4. The Cf-mediated release of Ca21 from Ry-sensitive Ca21

stores is NO-dependent. A: Fura-2-loaded cells were supple-mented with saline A (continuous trace) or Ry (20 mM, dashedline). After 5 min, the cells were treated with 10 mM Cf and afteran additional 5 min with tBu-OOH (3 mM ). The numbers on theleft indicate the [Ca21]i. Traces are representative of eight to 10consistent experiments. B: Fura-2-loaded cells were supple-mented with saline A (continuous trace), L-NAME (300 mM,dashed line), or ODQ (1 mM, dotted line). After 5 min, the cellswere treated with 10 mM Cf and after an additional 5 min withtBu-OOH (3 mM ). The numbers on the left indicate the [Ca21]i.Traces are representative of eight to 10 consistent experiments.

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Cf-mediated release of Ca21 from Ry-sensitive Ca21

stores (Fig. 4A).Thus, Cf increases the toxicity mediated by tBu-OOH

via a mechanism involving endogenous NO and releaseof Ca21 from Ry-sensitive Ca21 stores. These two eventsappear to operate in the same pathway, because suppres-sion of either one of these events was a condition nec-essary and sufficient to prevent the Cf-mediated en-hancement of tBu-OOH toxicity. It was therefore impor-tant to assess whether release of Ca21 from Ry-sensitiveCa21 stores is an upstream or downstream event toformation of endogenous NO. In principle, both of thesesequences of events are possible. Indeed, on the onehand, the requirement of Ca21 for activation of consti-tutive NOS is very well established (Moncada et al.,1991) but, on the other hand, NO is also known topromote Ca21 release from Ry-sensitive Ca21 stores(Clementi et al., 1996b; Willmott et al., 1996; Xu et al.,1998). The observation that the Cf-induced Ca21 releasewas dramatically blunted by the NOS inhibitorL-NAME(Fig. 4B) is consistent with the notion that NO plays acritical role in the process of Cf-mediated Ca21 releasefrom Ry-sensitive Ca21 stores in CHP100 cells. Thus, apositive feedback mechanism appears to be operative inCHP100 cells whereby the initial release of Ca21 fromRy-sensitive Ca21 stores, induced by Cf, activates theCa21-dependent NOS and the resulting NO triggers fur-ther Ca21 release from the same Ca21 store.

Taken together, the above results lead to the conclu-sion that endogenous NO is responsible for the increasedtoxicity mediated by Cf in cells exposed to tBu-OOH andthat the involvement of NO is at the level of the Cf-mediated release of Ca21 from Ry-sensitive Ca21 stores.The fact that ODQ neither affected the Cf-mediatedenhancement of tBu-OOH toxicity (Fig. 1B) nor modi-fied the rate and extent of release of Ca21 in response toCf (Fig. 4B) suggests that NO does not act by stimulatingthe guanylyl cyclase pathway.

Previous studies indicated that NO generated by ex-ogenously added NO donors promotes release of Ca21

from Ry-sensitive Ca21 stores in cultured cells via eithercGMP-dependent generation of cyclic ADP-ribose(Clementi et al., 1996b; Willmott et al., 1996) or via acGMP-independent mechanism involvingS-nitrosylationof the receptor (Xu et al., 1998). However, the onlymechanism that has been proven to date to be operativein the case of endogenous NO is the one working via thecGMP/cyclic ADP-ribose pathway (Clementi et al.,1996b). The results reported in the present study repre-sent the first demonstration that endogenous NO canpromote release of Ca21 from Ry-sensitive Ca21 storesalso via a cGMP-independent mechanism. The nature ofthe cGMP-independent modification that mediates open-ing of the Ry receptor in response to endogenous NO iselusive. As Ca21-dependent NOS promotes formation ofnanomolar levels of NO, it may be predicted that NO willreact preferentially with hemes. Indeed, these eventswere shown to occur at the level of both guanylyl cyclase(Brown and Cooper, 1994) and cytochromec oxidase

(Cleeter et al., 1994). On the other hand, it is also widelyaccepted that NOS is a highly compartmentalized en-zyme; in particular, Brenman et al. (1996) demonstratedthat NOS is bound to the plasma membrane in neuronalcells. It is therefore possible that, under the conditionsused in this study, formation of NO takes place in spe-cific microenvironments and yields localized concentra-tions of the messenger sufficient to promoteS-nitrosyla-tion reactions. It is important to note that previous workby Lander et al. (1995) clearly established that nanomo-lar concentrations of NO promoteS-nitrosylation of thesmall GTP-binding protein p21 ras.

Thus,S-nitrosylation of the Ry receptor is a potentialmechanism that would explain the effects of endogenousNO on the release of Ca21 from Ry-sensitive Ca21 storesmediated by Cf in CHP100 cells.

In the present study, we also demonstrated that treat-ment with a toxic concentration of tBu-OOH (3 mM)promotes a transient elevation in [Ca21]i (Fig. 3). Thecation was released from internal stores of neutral pHand different from the endoplasmic reticulum-located,SERCA-containing, IP3-, and Ry-sensitive Ca21 stores(data not shown and Fig. 3). The finding that suppressionof NOS activity byL-NAME was not accompanied byinhibition of the tBu-OOH-mediated Ca21 mobilizationwould suggest that the hydroperoxide promotes Ca21

release via a mechanism that is not affected by NO.Similarly, the lack of effect of NOS inhibitors and PTIOon tBu-OOH toxicity (Fig. 2B) rules out any involve-ment of NO in the deleterious effects mediated by thehydroperoxide.

In conclusion, the results presented in this study are, toour knowledge, the first demonstration that NO promotesopposite effects in cells exposed to the organic hydroper-oxide tBu-OOH, depending on whether this pleiotropicmessenger is generated intra- or extracellularly. The ef-fects of both exogenous and endogenous NO are medi-ated by cGMP-independent mechanisms. EndogenousNO promotes the release of Ca21 from Ry-sensitiveCa21 stores in response to Cf, and this effect appears tobe causally linked to the Cf-mediated enhancement oftBu-OOH cytotoxicity.

Acknowledgment: The financial support of Telethon—Italy(grant no. 1110, to O.C.) and that of the Consiglio Nazionaledelle Ricerche (Target Project on Biotechnology grant97.01144.PF49, to E.C.) are gratefully acknowledged.

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