Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine

ABB Archives of Biochemistry and Biophysics 430 (2004) 170–177 www.elsevier.com/locate/yabbi

Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine Silvia Lores-Arnaiz*, Gabriela DÕAmico, Analı´a Czerniczyniec, Juanita Bustamante, Alberto Boveris Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Received 24 March 2004, and in revised form 12 July 2004 Available online 14 August 2004

Abstract Mouse brain mitochondria have a nitric oxide synthase (mtNOS) of 147 kDa that reacts with anti-nNOS antibodies and that shows an enzymatic activity of 0.31–0.48 nmol NO/min mg protein. Addition of chlorpromazine to brain submitochondrial membranes inhibited mtNOS activity (IC50 = 2.0 ± 0.1 lM). Brain mitochondria isolated from chlorpromazine-treated mice (10 mg/ kg, i.p.) show a marked (48%) inhibition of mtNOS activity and a markedly increased state 3 respiration (40 and 29% with malate–glutamate and succinate as substrates, respectively). Respiration of mitochondria isolated from control mice was 16% decreased by arginine and 56% increased by NNA (Nx-nitro-L -arginine) indicating a regulatory activity of mtNOS and NO on mitochondrial respiration. Similarly, mitochondrial H2O2 production was 55% decreased by NNA. The effect of NNA on mitochondrial respiration and H2O2 production was significantly lower in chlorpromazine-added mitochondria and absent in mitochondria isolated from chlorpromazine-treated mice. Results indicate that chlorpromazine inhibits brain mtNOS activity in vitro and can exert the same action in vivo. Ó 2004 Elsevier Inc. All rights reserved. Keywords: mtNOS; Nitric oxide; Brain mitochondria; Chlorpromazine; Hydrogen peroxide

The production of NO by a specialized form of nitric oxide synthase (NOS)1 located in rat liver mitochondria was simultaneously reported by Ghafourifar and Richter [1] and by Giulivi et al. [2,3]. Further studies reported relevant regulatory properties of mitochondrial nitric oxide synthase (mtNOS) activity: endogenously generated NO has a functional activity in the regulation of the rates of respiration [4,5] and of H2O2 production [5,6], and a role as an early apoptotic signal [7,8]. The activity of mtNOS has been recognized in a series of organs: liver [1–4,6], brain [9–11], heart [12–14], kidney cortex [5], thymus [8,15], and skeletal muscle [16]. *

Corresponding author. Fax: +5411 4508 3646. E-mail address: slarnaiz@ffyb.uba.ar (S. Lores-Arnaiz). 1 Abbreviations used: NOS, nitric oxide synthase; mtNOS, mitochondrial nitric oxide synthase. 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.07.012

Recently, Guilivi and co-workers [17] sequenced liver mtNOS and identified it as nNOS splice variant a with posttranslational (myristoylation and phosphorylation) modifications and recognized the corresponding transcripts in brain, heart, muscle, kidney, lung, testis, and spleen. This important work established mtNOS as an integral protein of the inner mitochondrial membrane. Of note, mtNOS activity has been observed susceptible of in vivo endocrine regulation: it is down-regulated by thyroxine in liver [18] and by angiotensin II in heart and liver [14,19]. While there is growing evidence for mtNOS, the issue of the possibility of contamination of mitochondria with cytosolic NOS during isolation is still a matter of debate [20]. The enzymatic and functional activities of brain mtNOS are assessed in this study taking advantage of the inhibitory effect of chlorpromazine on mtNOS

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activity [21]. The effects of chlorpromazine on mtNOS activities were determined in vitro and in vivo. Chlorpromazine is a known inhibitor of NOS: it decreases the NO-mediated activation of soluble guanylate cyclase [22] and the nNOS activity of rat brain cytosol [23]. Concerning its pharmacological actions, chlorpromazine acts by blocking postsynaptic dopamine receptors, thus altering the turnover and release of dopamine [24]. It has been reported that dopamine agonists increase nitric oxide synthase activity in the paraventricular nucleus of the hypothalamus [25]. These findings raised the question whether in vivo effects of chlorpromazine may be mediated by NOS inhibition. The enzymatic and functional activities of mouse brain mtNOS were assessed through the determination of: (a) NO generation, (b) O2 consumption, and (c) H2O2 production in mitochondrial preparations.

Materials and methods Isolation of brain mitochondria Female Swiss mice (20–25 g) were killed by cervical dislocation and the brains were immediately excised. Experiment groups of 6 mice were used and brains from 2 mice were pooled for each experimental point. Brains were weighed and homogenized in a medium consisting of 0.23 M mannitol, 0.07 M sucrose, 10 mM Tris–HCl, and 1 mM EDTA, pH 7.4, and homogenized at a ratio of 1 g brain/8 ml homogenization medium. Homogenates were centrifuged at 700g for 10 min to discard nuclei and cell debris and the pellet was washed to enrich the supernatant that was centrifuged at 8000g for 10 min. The obtained pellet, washed and suspended in the same buffer, consisted of intact mitochondria able to carry out oxidative phosphorylation [26]. The supernatant of the 8000g centrifugation was called ‘‘cytosolic fraction.’’ The operations were carried out at 0–2 °C. Submitochondrial membranes were obtained from mitochondria by twice freezing, thawing, and homogenizing by passing the suspension through a 15/10 hypodermic needle [21]. Further mitochondrial purification for Western blot analysis was performed by Ficoll gradient [27]. Mitochondrial pellets were washed with isolation buffer and dialyzed against it at 4 °C for 4 h. Submitochondrial membranes and purified mitochondria were less than 2% contaminated with cytosolic components according to b-actin Western blot analysis. Proteins were determined by the Folin phenol reagent using bovine serum albumin as standard.

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1 lg/ml aprotinin), were separated by SDS–PAGE (7.5%), blotted onto a nitrocellulose membrane, and probed primarily with rabbit polyclonal antibodies (dilution 1:500) for nitric oxide synthase neuronal constitutive form (nNOS or NOS-1), reacting with the amino terminus (H-299) or with the carboxy terminus (R-20) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then, the nitrocellulose membrane was incubated with a secondary goat anti-rabbit antibody conjugated with horseradish peroxidase (dilution 1:5000) and revealed by chemiluminescence with ECL reagent [8,21]. Densitometric analysis of nNOS bands was performed by NIH Image 1.54 software. All experiments were performed in triplicate. Experimental design for in vivo chlorpromazine effect Chlorpromazine dissolved in 145 mM NaCl was i.p. injected to the animals at 10 mg/kg, in a single administration, and sacrificed 1 h after injection. The dose, as expressed (mg/kg), is at the upper limit of human doses [28], and expressed in terms of basal metabolic rate (0.17 mg/(kJ/h) in mice) is at the lower limit of human doses (0.24–2.4 mg/(kJ/h) in mice. Control animals were injected with 145 mM NaCl solution. Nitric oxide production Nitric oxide production was measured in brain submitochondrial membranes and in cytosolic fractions by following spectrophotometrically the oxidation of oxyhemoglobin to methemoglobin at 37 °C, in an air-saturated (0.21 mM O2) reaction medium containing 50 mM phosphate buffer (pH 5.8 for mitochondrial preparations and pH 7.4 for the cytosolic fraction), 1 mM CaCl2, 50 lM L -arginine, 100 lM NADPH, 10 lM dithiothreitol, 4 lM Cu–Zn SOD (to avoid interference by O2 ), 0.1 lM catalase (to avoid oxyhemoglobin oxidation by H2O2), 0.5–1.0 mg submitochondrial protein/ml, and 25 lM oxyhemoglobin (in heme group). Kinetics were followed at 577–591 nm (e = 11 mM 1 cm 1) in a Perkin–Elmer Model 356 double-beam double-wavelength spectrophotometer [21]. Controls adding 2 mM NNA (Nx-nitro-L -arginine) as inhibitor of mtNOS were performed in all cases to give specificity to the assay; addition of NNA inhibited by about 90% the rate of hemoglobin oxidation. The absorbance changes that were inhibitable by NNA were expressed as nmol NO/min mg protein [21]. For the in vitro assay of the direct chlorpromazine effect on mtNOS, submitochondrial membranes were incubated with 0.25–3 lM chlorpromazine during 5 min at 37 °C before the determination of NO production.

Western blots Mitochondrial respiration Submitochondrial membranes (150 lg), in the presence of protease inhibitors (1 lg/ml pepstatin, 1 lg/ml leupeptin, 0.4 mM phenylmethylsulfonyl fluoride, and

Mitochondrial respiratory rates were measured with an oxygen electrode in a reaction medium containing

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0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 4 mM MgCl2, 5 mM phosphate, and 0.2% bovine serum albumin at 37 °C. Malate and glutamate (6 mM each) or succinate (7 mM) were used as substrates to measure state 4 respiration; 1 mM ADP was added to measure state 3 respiration [29]. Respiratory controls, the ratio of oxygen uptake in state 3/state 4, were in the range of 3.2–3.6 for succinate and 4.3–4.7 for malate–glutamate. Arginine (0.3 mM) or NNA (2 mM) was added to brain mitochondria in order to assay the functional NO effects on mitochondrial respiration. Hydrogen peroxide production H2O2 generation was determined in intact brain mitochondria by the scopoletin–HRP method, following the decrease in fluorescence intensity at 365–450 nm (kexc– kem) at 37 °C [30]. The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 0.8 lM HRP, 1 lM scopoletin, 7 mM succinate, and 0.3 lM SOD. Mitochondria (0.1–0.3 mg protein/ ml) were either incubated for 2 min with 0.3 mM GSNO or supplemented with 0.3 mM arginine or 2 mM NNA. Calibration was made using H2O2 (0.05–0.35 lM) as standard to express the fluorescence changes as nmol H2O2/min mg protein. Arginine content Purified mitochondria were extracted with 9 volumes of cold methanol, centrifuged at 10,000g for 15 min, and evaporated to half volume. A TLC was performed in order to verify the purity of the sample before aminoacid determination. Aminoacids were quantified by derivatization with phenylisothiocyanate and separation of the phenylthiocarbamyl aminoacids by HPLC, using a aminoacid analyzer Model 420 of Applied Biosystems.

Results Mitochondrial nitric oxide production: in vitro and in vivo effects of chlorpromazine The NO production of brain submitochondrial membranes was 0.31–0.48 nmol NO/min mg protein, understood as the enzymatic activity of mtNOS. Production of NO by mitochondria isolated from different brain regions was as follows (in nmol NO/min mg protein): whole brain, 0.48 ± 0.05; cortex, 0.42 ± 0.06; and cerebellum, 1.0 ± 0.1. Supplementation of whole brain mitochondria with chlorpromazine inhibited mtNOS activity in a concentration-dependent manner with a half-maximal inhibition at 2.0 ± 0.1 lM (Fig. 1). The NO production of mouse brain cytosol was similarly inhibited by chlorpromazine, in this case with a halfmaximal effect at 3.6 ± 0.2 lM, thus showing that the inhibitory action of chlorpromazine is not selective for mtNOS and that mtNOS and cytosolic nNOS have different properties (Fig. 1). The optimal pH of mtNOS activity was 5.8, whereas the one of the NOS activity of the cytosolic fraction (nNOS) was 7.4 (Fig. 1, inset). A similar inhibitory effect of chlorpromazine was reproduced in vivo: the mitochondrial production of NO was decreased by 48% in brain mitochondrial membranes isolated from mice treated with chlorpromazine (10 mg/kg) (Fig. 2). To further assess the direct effect of chlorpromazine on mtNOS, a brain homogenate was incubated with 3 and 10 lM chlorpromazine at 37 °C for 5 min. Then, mitochondria were isolated and NO production was measured; 50 and 67% decreased NO production was observed after incubation with 3 and 10 lM chlorpromazine, respectively, as compared with mitochondria iso-

Drugs and chemicals Chlorpromazine was a gift from Rhone-Poulenc Rorer, Argentina; ADP, L -arginine, catalase, dithiothreitol, EDTA, glutamic acid, GSH, HRP, malic acid, mannitol, NADPH, Nx-nitro-L -arginine, hemoglobin, scopoletin, succinate, sucrose, superoxide dismutase, and Trizma base were purchased from Sigma Chemical (St. Louis, Missouri). Other reagents were of analytical grade. Statistics Results are expressed as mean values ± SEM. StudentÕs t test was used to analyze the significance of differences between paired groups.



Fig. 1. In vitro effect of chlorpromazine on mitochondrial ( ) and cytosolic (s) NOS activities of mouse brain. Inset: pH dependence of mitochondrial ( ) and cytosolic (s) NOS activities.



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Table 1 Respiratory parameters in brain mitochondria isolated from control and in vivo chlorpromazine-treated mice Substrate/condition

Malate–glutamate (a) Control (b) Chlorpromazinetreated Chlorpromazine effect (b a) Succinate (a) Control (b) Chlorpromazinetreated Chlorpromazine effect (b a) *

Fig. 2. In vivo effect of chlorpromazine (one dose, 10 mg/kg i.p., 1 h) on the mtNOS activity of mouse brain. Bars represent mean values ± SEM of four experiments with five mice each. (*p < 0.02).

lated from homogenates not supplemented with chlorpromazine. Western blot analysis of mtNOS Western blot analysis identified an important amount of a protein of 147 kDa reacting with anti-nNOS antibodies in brain mitochondrial membranes. The anti-nNOS antibodies were directed against the carboxy terminus and the amino terminus of the protein (Fig. 3). In vivo chlorpromazine treatment did not modify the amount of mitochondrial nNOS protein detected by Western blot as shown by densitometric analysis (Figs. 3A and B).

Oxygen consumption (ng-atom O/min mg protein)

Respiratory control

State 4

State 3

15 ± 2 21 ± 3

67 ± 4 94 ± 5*

4.5 ± 0.3 4.5 ± 0.3

27 ± 6 (40%)

—

76 ± 5 98 ± 4*

3.4 ± 0.2 3.4 ± 0.2

22 ± 6 (29%)

—

6 ± 3 (40%)

22 ± 3 29 ± 3 7 ± 4 (32%)

p < 0.005.

Malate–glutamate- and succinate-dependent oxygen consumption rates were measured in state 4 (resting or controlled respiration) and in state 3 (active respiration, the maximal physiological rate of O2 uptake, and ATP synthesis) and the respiratory control ratios (the most sensitive indicator of mitochondrial oxidative phosphorylation coupling) were calculated [31]. Both types of isolated mitochondria were well coupled and able to carry out oxidative phosphorylation (Table 1). In vivo treatment with chlorpromazine produced a marked increase in the oxygen uptake of isolated brain mitochondria, both in state 4 and in state 3: 40% with malate– glutamate and 29–32% with succinate as substrates

Mitochondrial respiration The respiratory parameters indicative of mitochondrial function were determined in brain mitochondria isolated from control and chlorpromazine-treated mice.

Fig. 3. Western blot analysis of the NOS present in brain mitochondrial membranes (mtNOS), reacting with: (A) an anti-nNOS antibody (carboxy terminus) in control (untreated) and chlorpromazine-treated (CPZ) mice; (B) an anti-nNOS antibody (amino terminus). Molecular markers at 100 and 150 kDa.

Fig. 4. Oxygen consumption rate as a function of O2 concentration in control and 3.3 lM chlorpromazine-supplemented mouse brain mitochondria added with malate–glutamate and ADP. Differential rates were normalized at equal rates at 25 lM O2 in order to better show Vi/ V (see text). The line was adjusted from the experimental points by non-linear regression to a hyperbolic curve.

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(Table 1). This effect agrees with the observed in vivo effect of chlorpromazine in decreasing mtNOS activity (Fig. 2) and reveals the functional regulatory activity of mtNOS on brain mitochondrial respiration, as it was reported for liver and kidney mitochondria [4,5]. The effect of chlorpromazine on O2 uptake was higher at lower O2 concentrations (Fig. 4). Values of [O2]0.5, the O2 concentration that allows a half-maximal rate of respiration, of 4.7 ± 0.6 and 1.2 ± 0.1 lM were obtained for control and chlorpromazine-treated mitochondria, in agreement with previous reports of [O2]0.5 values of 3 and 1 lM for liver and heart mitochondria in conditions of active and inactive mtNOS, respectively [4,31]. The functional role of brain mtNOS on the regulation of mitochondrial respiration was further characterized by using mitochondria with: (a) normal mtNOS activity and (b) with decreased mtNOS activity, this latter, both by in vitro added chlorpromazine and after in vivo chlorpromazine treatment. In both cases, either maximal or minimal mtNOS biochemical activities were approached by supplementation with either the substrate arginine or the competitive inhibitor NNA. The addition of arginine to control, in vitro chlorpromazine-supplemented and in vivo chlorpromazine-treated mitochondria in state 3 produced slight decreases in respiration: 16, 7, and 6% (Table 2). This slight effect of arginine indicates an important intramitochondrial Table 2 Effects of arginine and of NNA on the malate–glutamate supported state 3 respiration of mouse brain mitochondria, supplemented with chlorpromazine, and isolated from chlorpromazine-treated mice

(1) Mouse brain mitochondria (a) +0.3 mM arginine (b) +2 mM NNA mtNOS functional activity (b

a)

(2) Id. + 3.3 lM chlorpromazine (a) +0.3 mM arginine (b) +2 mM NNA mtNOS functional activity (b

Relative rate (%)a

81 ± 3 68 ± 2* 126 ± 6*

64 54 100

58 ± 5 95 ± 3 88 ± 4* 102 ± 2*

a)

(3) Brain mitochondria isolated from chlorpromazine-treated mice (a) +0.3 mM arginine (b) +2 mM NNA mtNOS functional activity (b

Respiratory rate (ng-atom O/min mg protein)

a)

14 ± 4**

46** 93 86 100 14**

108 ± 5

96

102 ± 6 113 ± 4

90 100

11 ± 6**

10**

Values represent mean value of four experiments, each obtained from a pool of two brains. a Percentage of the maximal rate of respiration, i.e., in the presence of NNA. * p < 0.01, between arginine and NNA supplementation (high and low mtNOS activity). ** p < 0.01, mtNOS functional activity in respect of normal brain mitochondria.

Table 3 Aminoacid content in mouse brain mitochondria Aminoacid

Content (nmol/mg protein)

Arg Asp Glu

19.0 ± 0.5 12.8 ± 0.9 20 ± 2

Mouse brain mitochondrial contents of Ser, Gly, His, Thr, Ala, Pro, Tyr, Val, Met, Cys, Ile, Leu, Phe, and Lys are below 1 nmol/mg protein.

arginine pool. This was confirmed by quantification of the mitochondrial content of L -arginine in the mitochondrial fraction resulting in an approximate value of 19.0 ± 0.5 nmol/mg of mitochondrial protein, which is comparable to glutamate and aspartate contents (Table 3). At variance, the addition of the NOS inhibitor NNA markedly increased the respiratory rate by 56% in control brain mitochondria, and only 7 and 5% in in vitro chlorpromazine-supplemented mitochondria and in mitochondria isolated from in vivo chlorpromazinetreated mice, respectively (Table 2). The functional activity of mtNOS, expressed as the difference in maximal state 3 respiratory rates (in the presence of NNA) and minimal state 3 respiratory rates (in the presence of arginine), was estimated as 58 ± 5 ng-atom O/min mg protein for control mouse brain mitochondria and was significantly decreased (75–80%) both in brain mitochondria incubated with chlorpromazine and in brain mitochondria obtained from chlorpromazine-treated animals (Table 2). Hydrogen peroxide production Mitochondrial H2O2 production is increased by NO, either added [32] or produced by mtNOS [5,6]. In vitro chlorpromazine-supplementation or in vivo chlorpromazine treatment only slightly modified the state 4 H2O2 production of brain mitochondria (Table 3), again as an indication of an important intramitochondrial arginine pool. Supplementation with arginine had also a slight effect in control and chlorpromazine groups, but NNA markedly (55%) decreased H2O2 production in the mitochondria from control mice. The effect of NNA on H2O2 production rate was lower (47%) in the case of chlorpromazine-supplemented mitochondria and absent in mitochondria obtained from chlorpromazine-treated animals (Table 4). The functional activity of mtNOS on the regulation of H2O2 production, estimated as the difference in H2O2 production rate between the conditions of maximal and minimal NO levels, was 0.26 ± 0.03 nmol/min mg protein and was 50 and 77% decreased in mouse brain mitochondria incubated with chlorpromazine and in mouse brain mitochondria obtained from chlorpromazine-treated animals, respectively.

S. Lores-Arnaiz et al. / Archives of Biochemistry and Biophysics 430 (2004) 170–177 Table 4 Effects of arginine and of NNA on the succinate supported state 4 hydrogen peroxide production of mouse brain mitochondria, supplemented with chlorpromazine, and isolated from chlorpromazinetreated mice H2O2 production (nmol/min mg protein)

Relative rate (%)a

(1) Mouse brain mitochondria (a) +0.3 mM arginine (b) +2 mM NNA (c) +0.3 mM GSNO mtNOS functional activity (a b)

0.40 ± 0.03 0.44 ± 0.02 0.18 ± 0.02* 0.75 ± 0.04 0.26 ± 0.03

53 59 24 100 35

(2) Id. + 3.3 lM chlorpromazine (a) +0.3 mM arginine (b) +2 mM NNA (c) +0.3 mM GSNO mtNOS functional activity (a b)

0.19 ± 0.02 0.23 ± 0.01 0.10 ± 0.01 0.71 ± 0.04 0.13 ± 0.01**

27 32 14 100 18

(3) Brain mitochondria isolated from chlorpromazine-treated mice (a) +0.3 mM arginine (b) +2 mM NNA (c) +0.3 mM GSNO mtNOS functional activity (a b)

0.29 ± 0.02 0.34 ± 0.02 0.28 ± 0.02 0.72 ± 0.03 0.06 ± 0.03**

40 47 39 100 8

Values represent the mean value of four experiments, each obtained from a pool of two brains. a Percentage of the maximal rate of H2O2 production, i.e., in the presence of GSNO. * p < 0.01, between arginine and NNA supplementation (high and low mtNOS activity). ** p < 0.01, mtNOS functional activity in respect of normal brain mitochondria.

Discussion This study further recognizes brain mitochondrial NO production [9–11,21] as the activity of a mitochondrial protein of 147 kDa, identified through the use of specific anti-nNOS antibodies. The Western blot analysis of Fig. 3 shows a brain mtNOS reacting with antinNOS antibodies directed both to the carboxy- and the amino ends of the protein; however, other studies reported reactivity with anti-nNOS carboxy end and not with anti-nNOS amino end [11], and reactivity with anti-eNOS antibodies [10,33]. The unclear situation is likely explained by protein homology and is not unique for brain mtNOS; in rat liver, a mtNOS of 130 kDa was reported to react with anti-iNOS antibodies [3,12,21] and with anti-eNOS antibodies [33]. The rates of NO production by brain mtNOS determined in this study (0.31–0.48 nmol/min mg protein) are in the range (0.5–1.6 nmol/min mg protein) given in previous reports [9,10], and about 20 times higher than other reported rates (17 pmol/min mg protein) [11]. Neurons show a bimodal distribution of NOS activities: a synaptosomal and cytosolic nNOS and a mtNOS (Fig. 1 and [11]). Riobo et al. [11] recently recognized a different and complementary sequential expression of mtNOS and nNOS in rat brain during development.

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Hepatocytes and thymocytes also exhibit a bimodal intracellular NOS distribution, with a mitochondrial mtNOS and a cytosolic eNOS at the endoplasmic reticulum [8,18]. Both, brain mtNOS and nNOS, require Ca2+ for their in vitro activity and are understood as physiologically activated by Ca2+ pulses for their physiological function. An interesting difference was observed in optimal pH, that was 5.8 for mtNOS and 7.4 for cytosolic nNOS. Under physiological conditions, mtNOS seems limited by the intramitochondrial pH of about 7.8. Considering that there is skepticism concerning a possible mitochondrial contamination with cytosolic NOS during isolation, we measured NO production after incubation of brain mitochondria with 0.05% trypsin for 3 min at room temperature and we found an increased NOS activity after that incubation, as compared with mitochondria incubated for the same period of time without trypsin (data not shown). This gives support to the idea of the presence of the enzyme inside the mitochondria and not as a result of contamination. Chlorpromazine, both by in vitro supplementation and by in vivo treatment, inhibited mtNOS activity and increased the respiration of isolated mitochondria. The inhibitory effect of chlorpromazine on brain mtNOS activity observed after in vivo treatment unveiled the regulatory effect of mtNOS on mitochondrial O2 uptake and indicated that isolated brain mitochondria contain the substrates and co-factors required for mtNOS activity. The instantaneous distribution of the given dose (10 mg/kg) would correspond to about 30 lM chlorpromazine in the whole mouse. The direct inhibitory effect was reproduced by addition of 3 and 10 lM chlorpromazine to a brain homogenate followed by mitochondrial isolation. As the chlorpromazine inhibition of mtNOS is observed after in vivo chlorpromazine treatment and mitochondrial isolation, and after homogenate incubation with chlorpromazine, it seems clear that chlorpromazine is tightly bound to the mitochondrial inner membrane. Moreover, it was observed that chlorpromazine treatment did not alter the amount of mtNOS detected by Western blot analysis indicating a direct effect on the protein. Arginine at high concentrations was not able to reverse chlorpromazine inhibition of mtNOS, indicating an effect of the drug at a site other than the substrate site. Chlorpromazine seems to inhibit mtNOS activity by binding to the calmodulin site; phenotiazines are known calmodulin antagonists [34]. Similar to chlorpromazine, the neuroleptic drug haloperidol inhibited brain mitochondrial NO production after 1 and 24 h of drug administration [9]. The hidden cytochrome oxidase inhibition by endogenous NO in isolated brain mitochondria (46%) observed after addition of L -NNA to control mouse brain mitochondria was decreased to 7 and 4% in chlorpromazine-supplemented mitochondria and in mitochondria isolated from chlorpromazine-treated animals

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(Table 2). Chlorpromazine effect in increasing mitochondrial respiration was higher at lower pO2 in agreement with the condition of O2-competitive inhibition of cytochrome oxidase by NO (Fig. 4). It is then clear that mtNOS activity is able to regulate the respiration and ATP synthesis of isolated mitochondria through the O2-competitive inhibition of cytochrome oxidase [32,35–37] that limits the coupled ATP synthesis [38]. The phenomenon is highly favored by the vicinity of the generation and target sites. Recently, the discoverers of the NO inhibition of cytochrome oxidase considered such phenomenon of relevant physiological importance [39,40]. However, it is not clear how much of this respiratory regulation occurs in myoglobin containing tissues and in blood (hemoglobin) perfused organs [41]. A brain mitochondrial steady state concentration of 20 nM NO has been calculated on the basis of a brain mtNOS KmO2 of 73 lM [42]; this NO level should inhibit cytochrome oxidase activity by about 31% under physiological conditions. The kinetic data of Fig. 4 and the application of equation Vi/V = 1/(1 + 100 [NO]/[O2]) [31] for the ratios of O2 uptake between the inhibited respiration by an active mtNOS (Vi) and an inactive (chlorpromazine-inhibited) mtNOS (V) give a [NO] steady state concentration of 18 nM in normal mouse brain mitochondria at the considered [O2], in excellent agreement with the referred level of 20 nM. Brain mitochondrial production of H2O2 was also regulated by mtNOS activity. The effect was clear after supplementation with the NOS inhibitor L -NNA. Arginine has a limited effect in brain mitochondria (control and chlorpromazine-added or -treated), in agreement with the results of oxygen consumption that indicate an important intramitochondrial arginine pool in brain mitochondria. Brain mitochondrial L -arginine content quantification yielded similar levels as glutamate and aspartate, clearly indicating that the enzyme is not limited by L -arginine in physiological conditions. There is a tissue specificity for the arginine effect; the mitochondrial production of O2 and H2O2 in liver mitochondria, which is a NO-dependent process, is markedly enhanced by L -arginine [5,6]. There is increasing evidence that O2 , H2O2, and NO steady state levels regulate physiological cell functions such as proliferation, differentiation, and apoptosis [43]. Clinical treatments with chlorpromazine would affect the signaling levels of mitochondrial O2 , H2O2, and NO, and such alterations would contribute to chlorpromazine pharmacological actions and toxicity.

Acknowledgments The authors thank Dr. Clara Pen˜a for advice in the measurement of aminoacids content. This research was

supported by grants from Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (PIP 02272), Agencia Nacional de Promocio´n Cientı´ficay Tecnolo´gica (PICT 01-8710), and Universidad de Buenos Aires (B075 and B411) (Argentina).

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