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Effects of clozapine, olanzapine and haloperidol on nitric oxide production by lipopolysaccharide-activated N9 cells
Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) 1523 – 1528 www.elsevier.com/locate/pnpbp
Short communication
Effects of clozapine, olanzapine and haloperidol on nitric oxide production by lipopolysaccharide-activated N9 cells Yue Hou a,b , Chun Fu Wu a,⁎, Jing Yu Yang a , Xiang He b , Xiu Li Bi a , Liang Yu a , Tao Guo b a
Department of Pharmacology, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China b General Hospital of Shenyang Military Region, 110016, Shenyang, PR China Received 6 January 2006; received in revised form 9 May 2006; accepted 9 May 2006 Available online 27 June 2006
Abstract Schizophrenia is a devastating illness of unknown etiology and the basis for its treatment rests in the symptomatic response to antipsychotics. It was found that some of the patients with schizophrenia elicited microglia activation. The present study used lipopolysaccharide (LPS)-activated mouse microglial cell line N9 as an in vitro model to mimic microglia activation seen in the patients with schizophrenia. The effects of clozapine, olanzapine and haloperidol on the release of nitric oxide (NO) by LPS-stimulated N9 cells were investigated. The results showed that olanzapine significantly inhibited NO release by LPS-stimulated N9 cells. Clozapine and haloperidol did not show significant effects on this model. The present study suggested that the inhibiting effect of olanzapine on the NO release by LPS-stimulated microglial cells might be a new mechanism through which olanzapine exhibits its therapeutic effect in the treatment of schizophrenia. © 2006 Elsevier Inc. All rights reserved. Keywords: Clozapine; Haloperidol; N9 cells; Nitric oxide (NO); Olanzapine
1. Introduction Schizophrenia is a severe illness that affects approximately 1% of the world's population. The etiology of schizophrenia is only partially understood and, consequently, the basis for its treatment rests in the symptomatic response to antipsychotics. Although classic antipsychotic drugs, such as haloperidol, produce a marked reduction in positive symptoms of schizophrenia, they do not improve the negative symptoms such as apathy, confusion, and social withdrawal, nor do they alter the progressive deterioration in the mental abilities of the patients. In recent years, several new drugs, such as clozapine and olanzapine, have been shown to improve both positive and negative symptoms of schizophrenia, and seem to prevent further worsening of psychotic symptoms (Buckley, 1997; Blin, 1999; Bhana et al., 2001). The atypical antipsychotic Abbreviations: CNS, central nervous system; DMSO, dimethyl sulfoxide; LPS, lipopolysaccharide; NO, nitric oxide; NOS, nitric oxide synthase. ⁎ Corresponding author. Tel./fax: +86 24 23843567. E-mail addresses: [email protected], [email protected] (C.F. Wu). 0278-5846/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2006.05.006
drugs are potent 5-HT2A and weak D2 antagonist, which distinguishes them from typical antipsychotic drugs (Altar et al., 1986; Meltzer et al., 1989; Meltzer, 1999; Rasmussen and Aghajanian, 1988). They also have many other pharmacologic properties that may contribute to their superior therapeutic actions in schizophrenia and that could be the basis for their usefulness in controlling psychotic symptoms in other disorders as well (Tran et al., 1997; Stoppe and Staedt, 1999; Wolfgang, 1999). Microglial cells are ubiquitously distributed in the central nervous system (CNS) and comprise up to 20% of the total glial cell population in the brain (Lawson et al., 1991). As a kind of cells of the macrophage lineage in the CNS, microglial cells are quiescent in the normal brain. However, these cells can be activated by cytokines produced by infiltrating immune effector cells after CNS injury or by LPS during bacterial infection (Gonzalez-Scarano and Baltuch, 1999; Stoll and Jander, 1999). Activation of microglial cells is associated with increased phagocytosis and release of NO, oxygen radicals, proteases as well as pro-inflammatory cytokines (Stoll and Jander, 1999; Lee et al., 2002). In recent years, microglial cells have been shown
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to be involved in many CNS illnesses. For example, prolonged and excessive stimulation of microglial cells initiates an inflammatory cascade in the CNS that contributes to the pathogenesis of Alzheimer's disease, Parkinson's disease (Stoll and Jander, 1999; Mcgeer et al., 1993), multiple sclerosis (Boyle and McGeer, 1990) and HIV-associated dementia (Kaul et al., 2001). Recently, Munn (2000) has proposed microglia dysfunction in schizophrenia as an integrated theory. The study of Bayer et al. (1999) showed that some of the patients with schizophrenia elicited microglia activation, suggesting microglia activation might be involved in the pathophysiological process of schizophrenia. Taken together, the present study used LPS-activated mouse microglial cell line N9 as an in vitro model to mimic microglia activation seen in the patients with schizophrenia. The effects of clozapine, olanzapine and haloperidol on the release of NO by LPS-stimulated N9 cells were investigated.
Cell viability was evaluated by the MTT reduction assay (Chang et al., 1998). In brief, cells were seeded onto 96-well microtiter plates and treated with various reagents for the indicated time period. After various treatments, medium was removed and the cells were incubated with MTT (0.25 mg/ml) for 3 h at 37 °C. The formazan crystals in the cells were solubilized with a solution containing 50% dimethylformamide and 20% sodium dodecyl sulfate (pH 4.7). The level of MTT formazan was determined by measuring its absorbance at the wavelength of 490 nm with a SPECTRA (shell) Reader (TECAN, Austria).
2. Materials and methods
2.4. Nitrite assays
2.1. Materials
Accumulation of nitrite (NO2− ) in the culture supernatants, an indicator of NO synthase activity, was measured by Griess reaction (Barger and Harmon, 1997). Fifty microliter culture supernatants were mixed with 50 μl Griess reagent (part I: 1% sulfanilamide; part II: 0.1% naphthylethylene diamide dihydrochloride and 2% phosphoric acid) at room temperature. Fifteen minutes later, the absorbance was determined at 540 nm using the SPECTRA (shell) Reader. Nitrite concentration was calculated with reference to a standard curve of sodium nitrite generated by known concentrations.
Clozapine and haloperidol were purchased from Sigma (St. Louis, MO, USA). Olanzapine was purified from the tablets (Eli Lilly and Co. Ltd.) by the Department of Pharmaceutical Engineering, Shenyang Pharmaceutical University (Shenyang, China). The drug concentrations were determined according to the results of our preliminary study. In our preliminary study, the highest concentrations tested for haloperidol, olanzapine and clozapine were all up to 100 μM. It was found that haloperidol and clozapine, at the concentrations of 30 and 100 μM, significantly reduced the viability of N9 cells. In contrast, olanzapine (up to 100 μM) did not affect the viability of N9 cells. In order to devoid the possible effects of reduced viability on NO release, the drug concentrations not affecting the viability of N9 cells were selected in the present study. Fetal bovine serum (FBS) was purchased from TBD Biotechnology Development (Tianjin, China). LPS (E5:055) was purchased from Sigma (St. Louis, MO, USA). Thiazolylblue (MTT) was from Sino-American Biotechnology (Beijing, China). Iscove's modified dulbecco's medium (IMDM) was from Gibco (Grand Island, USA). Clozapine, olanzapine and haloperidol were dissolved initially in dimethyl sulfoxide (DMSO) and were diluted with PBS for experiments. DMSO at the highest concentration possibly present in experimental conditions (0.1%) was not toxic to cells. 2.2. Cell culture The murine microglial cell line N9 was a kind gift from Dr. J. M. Wang (NCI, Frederick, USA). The cells were grown in IMDM supplemented with 5% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 × 10− 5 M 2-mercaptoethanol. Cells at density of 3 × 104 cells/well were plated onto 96-well microtiter plates for MTT and nitrite assay. Clozapine, olanzapine or haloperidol with or without LPS (1 μg/ml) was added to
the culture medium of N9 cells for 24 h for the experiments. The drug doses used were according to our preliminary study, not affecting the viability of N9 cells. 2.3. Cell viability
2.5. Data analysis Results were expressed as mean ± S.E.M. Statistical significance (P < 0.05) was assessed by one-way ANOVA followed by Dunnett's t-test (SPSS12.0 software, SPSS, USA). 3. Results 3.1. The effect of clozapine on N9 cell viability and LPSinduced NO release Treatment with clozapine (1–10 μM) alone or with 1 μg/ml of LPS for 24 h did not cause any change in MTT absorbance in N9 cells, indicating that clozapine did not affect the viability of N9 cells at the doses used (Fig. 1). Next, the release of NO by LPS-stimulated N9 cells was examined. In unstimulated N9 cells, only small amounts of NO2− (4.23 ± 2.18 μM) occurred in the medium. Pretreatment of unstimulated cells with clozapine (1–10 μM) for 24 h did not result in any change of NO2−. The stimulation of N9 cells with LPS resulted in a manifold increase in NO2− production (11.1 ± 5.1 μM). LPS significantly induced NO release in N9 cells, which was in consistence with the previous report (Bi et al., 2005). Simultaneous treatment of N9 cells with LPS and clozapine (1–10 μM) was not different from the result observed by LPS alone.
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4. Discussion This is the first report showing that olanzapine suppressed LPS-induced NO release, reflecting the activation of microglial cells, suggesting that it might be beneficial for the treatment of neurodegenerative diseases. In fact, olanzapine has been shown to be safe and effective in the treatment of schizophrenia (Beasley et al., 1996a,b, 1997; Tollefson et al., 1997; Bhana et al., 2001). Olanzapine showed neuroproliferative effect above 60 μM in the study of Dwyer et al. (2003). In fact, olanzapine also showed its protective effect at lower doses. For example, olanzapine stimulated phosphorylation of Akt at the dose of 10 μM and was mitogenic and neuroprotective at concentrations as low as 10– 40 μM (Lu et al., 2004). It has also been reported that olanzapine increased the gene expression of superoxide dismutase (SOD1) at the concentration of 10 μM, which was associated with its neuroprotective effect (Li et al., 1999). Therapeutic levels of olanzapine in serum are in the range of 0.1–0.3 μM (Olesen and Linnet, 1999; Robertson and McMullin, 2000). It is also known that antipsychotic drugs are accumulated in brain and fat tissue to levels that are 25–30-fold Fig. 1. (a) The effect of clozapine on N9 cell viability. N9 cells were treated with clozapine in the presence or absence of LPS (1 μg/ml) for 24 h. Cell viability was examined by MTT reduction assays and the results were expressed as percentage of surviving cells over control cells (no addition of clozapine). Data were represented as mean ± S.E.M. of three separate experiments. (b) The effect of clozapine on NO release by LPS-activated N9 cells. N9 cells were treated with clozapine alone for 24 h or clozapine and LPS (1 μg/ml) simultaneously for 24 h. The results were expressed as the percentage values taking LPS treatment group as 100%. Data were represented as mean ± S.E.M. of three separate experiments. ###P < 0.001 compared with the control group.
3.2. The effect of olanzapine on N9 cell viability and LPSinduced NO release Treatment with olanzapine (3–100 μM) alone or with 1 μg/ ml of LPS for 24 h did not cause any change in MTT absorbance in N9 cells, indicating that olanzapine did not affect the viability of N9 cells at the doses used (Fig. 2). Pretreatment of unstimulated cells with olanzapine (3– 100 μM) for 24 h did not result in any change of NO2−. However, simultaneous treatment of N9 cells with LPS and olanzapine (10–100 μM) markedly reduced NO production. 3.3. The effect of haloperidol on N9 cell viability and LPSinduced NO release Treatment with haloperidol (1–10 μM) alone or with 1 μg/ml of LPS for 24 h did not cause any change in MTT absorbance in N9 cells, indicating that haloperidol (1–10 μM) did not affect the viability of N9 cells at the doses used (Fig. 3). Pretreatment of unstimulated cells with haloperidol (1– 10 μM) for 24 h did not result in any change of NO2−. Simultaneous treatment of N9 cells with LPS and haloperidol (1–10 μM) was not different from the result observed by LPS alone.
Fig. 2. (a) The effect of olanzapine on N9 cell viability. N9 cells were treated with olanzapine in the presence or absence of LPS (1 μg/ml) for 24 h. Cell viability was examined by MTT reduction assays and the results were expressed as percentage of surviving cells over control cells (no addition of olanzapine). Data were represented as mean ± S.E.M. of three separate experiments. (b) The effect of olanzapine on NO release by LPS-activated N9 cells. N9 cells were treated with olanzapine alone for 24 h or olanzapine and LPS (1 μg/ml) simultaneously for 24 h. The results were expressed as the percentage values taking LPS treatment group as 100%. Data were represented as mean ± S.E.M. of three separate experiments. ###P < 0.001 compared with the control group. ⁎P < 0.05, ⁎⁎⁎P < 0.001 compared with the LPS group.
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Fig. 3. (a) The effect of haloperidol on N9 cell viability. N9 cells were treated with haloperidol in the presence or absence of LPS (1 μg/ml) for 24 h. Cell viability was examined by MTT reduction assays and the results were expressed as percentage of surviving cells over control cells (no addition of haloperidol). Data were represented as mean ± S.E.M. of three separate experiments. (b) The effect of haloperidol on NO release by LPS-activated N9 cells. N9 cells were treated with haloperidol alone for 24 h or haloperidol and LPS (1 μg/ml) simultaneously for 24 h. The results were expressed as the percentage values taking LPS treatment group as 100%. Data were represented as mean ± S.E.M. of three separate experiments. ###P < 0.001 compared with the control group.
higher than this (Aravagiri et al., 1999; Baldessarini et al., 1993; Kornhuber et al., 1999). Thus, olanzapine may reach 5–10 μM in the brain. Taking these into account, concentrations higher than 100 μM could not been reached in vivo. Thus, the effect of olanzapine at higher concentrations was not investigated. Unlike olanzapine, clozapine and haloperidol did not show significant effects on LPS-induced microglial cell activation. There are already existing data showing the differential effects between the two drugs and olanzapine. For example, clozapine and haloperidol were toxic for PC12 cells, while olanzapine stimulated cell proliferation under the same experimental condition (Dwyer et al., 2003). In a delayed radial maze task, olanzapine significantly reduced the number of errors made during the retention phase, while clozapine and haloperidol failed to affect the total number of retention phase errors (Wolff and Leander, 2003). The present study provides new data for further understanding the differential properties between olanzapine and the other two drugs in regulating some neurochemical events in the central nervous system. In the present study, the effects of haloperidol and clozapine were studied up to 10 μM concentrations. This is because haloperidol and clozapine, at the concentrations of 30 and 100 μM, significantly reduced the viability of N9 cells. To devoid the possible effects of reduced viability on NO release,
the drug concentrations not affecting the viability of N9 cells were selected in the present study. Nitric oxide (NO) is now accepted as an active messenger molecule and effector in the immune, cardiovascular and nervous systems. NO is formed endogenously by the conversion of L-arginine to L-citrulline by the enzyme nitric oxide synthase (NOS) (Mayer et al., 1989). NOS is known to exist in at least three isoforms (neuronal NOS, endothelial NOS and inducible NOS) broadly corresponding to that present in neuronal, endothelial and macrophage cells (Snyder and Bredt, 1991). NO has been studied in relation to the etiology of various neurological and psychiatric diseases, including schizophrenia. A postmortem histochemical study (Akbarian et al., 1993) found that nNOS levels were increased in deep layers of dorsolateral prefrontal cortex and decreased in hippocampus in brain tissue from schizophrenia vs control subjects. Another study (Karson et al., 1996) found elevated concentrations of nNOS in cerebellum from patients diagnosed with schizophrenia. Das et al. (1995, 1996) reported accumulation of an endogenous nNOS inhibitor in plasma of patients with schizophrenia and detected elevated Ca2+-induced NOS activity in blood platelets of drug naïve schizophrenia patients compared to others treated with antipsychotic drugs and to controls. The study of Yanik et al. (2003) showed that total nitrite level in the plasma was higher in patients with schizophrenia compared with controls. Postmortem study showed that nNOS expression in the prefrontal cortex was significantly higher in individuals with schizophrenia, suggesting the abnormalities of nNOS expression in the brain might contribute to the development of schizophrenia (Baba et al., 2004). Therefore, the inhibiting effect of olanzapine on the NO release by LPS-stimulated microglial cells suggested that this might be a new mechanism through which olanzapine exhibits its therapeutic effect in the treatment of schizophrenia. In fact, the effects of both typical and atypical antipsychotic drugs on NO have been investigated. Suzuki et al. (2002) found that haloperidol induced iNOS mRNA after acute, subacute and chronic treatment. Repeated treatment of rats with olanzapine did not change the density of nNOS enzyme in cortical, limbic, or extrapyramidal brain regions (Tarazi et al., 2002). Haloperidol did not reverse NOS inhibitor-induced deficit in social interaction (SI) before pronounced secondary effects on general behavior were seen at high doses, while clozapine and olanzapine were able to significantly reverse this deficit at doses which did not effect baseline SI values (Black et al., 2002). It was reported that rats treated with haloperidol for 28 days developed significant suppression of striatal NOS activity, while acute challenge with olanzapine significantly reversed it (Nel and Harvey, 2003). In the present study, olanzapine and haloperidol also showed differential effects on NO release by LPS-activated N9 cells. Although olanzapine closely resembles clozapine in terms of chemical structure and pharmacological profiles, they showed differential effects on the present model. In fact, in vitro radioligand studies in animal tissues and cloned human receptors have demonstrated that olanzapine has higher affinity for dopamine D2, serotonin 5-HT2A and muscarinic M1 receptors,
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whereas has lower affinity for serotonin 5-HT1A, α1-adrenergic and histamine H1 receptors compared with clozapine (Bhana et al., 2001). Such discrepancy between olanzapine and clozapine on the affinity for these neurotransmitter receptors might contribute to their differential effects on the release of NO by LPS-stimulated N9 cells. 5. Conclusions In conclusion, the present study demonstrated for the first time that olanzapine suppressed LPS-induced microglial cell activation. Clozapine and haloperidol showed no effect on this model. However, the mechanisms underlying the effect of olanzapine on the release of NO by LPS-stimulated microglial cells merits further investigation. References Akbarian S, Vinuela A, Kim JJ, Potkin SG, Bunney Jr WE, Jones EG. Distorted distribution of nicotinamide-adenine dinucleotide phosphate-diaphorase neurons in temporal lobe of schizophrenics implies anomalous cortical development. Arch Gen Psychiatry 1993;50:178–87. Altar CA, Wasley AM, Neale RF, Stone GA. Typical and atypical antipsychotic occupancy of D2 and S2 receptors: an autoradiographic analysis in rat brain. Brain Res Bull 1986;16:517–25. Aravagiri M, Teper Y, Marder SR. Pharmacokinetics and tissue distribution of olanzapine in rats. Biopharm Drug Dispos 1999;20:369–77. Baba H, Suzuki T, Arai H, Emson PC. Expression of nNOS and soluble guanylate cyclase in schizophrenic brain. NeuroReport 2004;15:677–80. Baldessarini RJ, Centorrino F, Flood JG, Volpicelli SA, Huston-Lyons D, Cohen BM. Tissue concentrations of clozapine and its metabolites in the rat. Neuropsychopharmacology 1993;9:117–24. Barger SW, Harmon AD. Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E. Nature 1997;388:878–81. Bayer TA, Buslei R, Havas L, Falkai P. Evidence for activation of microglia in patients with psychiatric illnesses. Neurosci Lett 1999;271:126–8. Beasley Jr CM, Sanger T, Satterlee W, Tollefson G, Tran P, Hamilton S. Olanzapine versus placebo: results of a double-blind, fixed-dose olanzapine trial. Psychopharmacology 1996a;124:159–67. Beasley Jr CM, Tollefson G, Tran P, Satterlee W, Sanger T, Hamilton S. Olanzapine versus placebo and haloperidol: acute phase results of the North American double-blind olanzapine trial. Neuropsychopharmacology 1996b; 14:111–23. Beasley Jr CM, Hamilton SH, Crawford AM, Dellva MA, Tollefson GD, Tran PV, et al. Olanzapine versus haloperidol: acute phase results of the international double-blind olanzapine trial. Eur Neuropsychopharmacol 1997;7:125–37. Bhana N, Foster RH, Olney R, Plosker GL. Olanzapine: an updated review of its use in the management of schizophrenia. Drugs 2001;61:111–61. Bi XL, Yang JY, Dong YX, Wang JM, Cui YH, Ikeshima T, et al. Resveratrol inhibits nitric oxide and TNF-alpha production by lipopolysaccharideactivated microglia. Int Immunopharmacol 2005;5:185–93. Black MD, Simmonds J, Senyah Y, Wettstein JG. Neonatal nitric oxide synthase inhibition: social interaction deficits in adulthood and reversal by antipsychotic drugs. Neuropharmacology 2002;42:414–20. Blin O. A comparative review of new antipsychotics. Can J Psychiatry 1999;44:235–44. Boyle EA, McGeer PL. Cellular immune response in multiple sclerosis plaques. Am J Pathol 1990;137:575–84. Buckley PF. New dimensions in the pharmacologic treatment of schizophrenia and related psychoses. J Clin Pharmacol 1997;37:363–78. Chang JY, Chavis JA, Liu LZ, Drew PD. Cholesterol oxides induce programmed cell death in microglial cells. Biochem Biophys Res Commun 1998;249: 817–21.
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Short communication
Effects of clozapine, olanzapine and haloperidol on nitric oxide production by lipopolysaccharide-activated N9 cells Yue Hou a,b , Chun Fu Wu a,⁎, Jing Yu Yang a , Xiang He b , Xiu Li Bi a , Liang Yu a , Tao Guo b a
Department of Pharmacology, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China b General Hospital of Shenyang Military Region, 110016, Shenyang, PR China Received 6 January 2006; received in revised form 9 May 2006; accepted 9 May 2006 Available online 27 June 2006
Abstract Schizophrenia is a devastating illness of unknown etiology and the basis for its treatment rests in the symptomatic response to antipsychotics. It was found that some of the patients with schizophrenia elicited microglia activation. The present study used lipopolysaccharide (LPS)-activated mouse microglial cell line N9 as an in vitro model to mimic microglia activation seen in the patients with schizophrenia. The effects of clozapine, olanzapine and haloperidol on the release of nitric oxide (NO) by LPS-stimulated N9 cells were investigated. The results showed that olanzapine significantly inhibited NO release by LPS-stimulated N9 cells. Clozapine and haloperidol did not show significant effects on this model. The present study suggested that the inhibiting effect of olanzapine on the NO release by LPS-stimulated microglial cells might be a new mechanism through which olanzapine exhibits its therapeutic effect in the treatment of schizophrenia. © 2006 Elsevier Inc. All rights reserved. Keywords: Clozapine; Haloperidol; N9 cells; Nitric oxide (NO); Olanzapine
1. Introduction Schizophrenia is a severe illness that affects approximately 1% of the world's population. The etiology of schizophrenia is only partially understood and, consequently, the basis for its treatment rests in the symptomatic response to antipsychotics. Although classic antipsychotic drugs, such as haloperidol, produce a marked reduction in positive symptoms of schizophrenia, they do not improve the negative symptoms such as apathy, confusion, and social withdrawal, nor do they alter the progressive deterioration in the mental abilities of the patients. In recent years, several new drugs, such as clozapine and olanzapine, have been shown to improve both positive and negative symptoms of schizophrenia, and seem to prevent further worsening of psychotic symptoms (Buckley, 1997; Blin, 1999; Bhana et al., 2001). The atypical antipsychotic Abbreviations: CNS, central nervous system; DMSO, dimethyl sulfoxide; LPS, lipopolysaccharide; NO, nitric oxide; NOS, nitric oxide synthase. ⁎ Corresponding author. Tel./fax: +86 24 23843567. E-mail addresses: [email protected], [email protected] (C.F. Wu). 0278-5846/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2006.05.006
drugs are potent 5-HT2A and weak D2 antagonist, which distinguishes them from typical antipsychotic drugs (Altar et al., 1986; Meltzer et al., 1989; Meltzer, 1999; Rasmussen and Aghajanian, 1988). They also have many other pharmacologic properties that may contribute to their superior therapeutic actions in schizophrenia and that could be the basis for their usefulness in controlling psychotic symptoms in other disorders as well (Tran et al., 1997; Stoppe and Staedt, 1999; Wolfgang, 1999). Microglial cells are ubiquitously distributed in the central nervous system (CNS) and comprise up to 20% of the total glial cell population in the brain (Lawson et al., 1991). As a kind of cells of the macrophage lineage in the CNS, microglial cells are quiescent in the normal brain. However, these cells can be activated by cytokines produced by infiltrating immune effector cells after CNS injury or by LPS during bacterial infection (Gonzalez-Scarano and Baltuch, 1999; Stoll and Jander, 1999). Activation of microglial cells is associated with increased phagocytosis and release of NO, oxygen radicals, proteases as well as pro-inflammatory cytokines (Stoll and Jander, 1999; Lee et al., 2002). In recent years, microglial cells have been shown
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to be involved in many CNS illnesses. For example, prolonged and excessive stimulation of microglial cells initiates an inflammatory cascade in the CNS that contributes to the pathogenesis of Alzheimer's disease, Parkinson's disease (Stoll and Jander, 1999; Mcgeer et al., 1993), multiple sclerosis (Boyle and McGeer, 1990) and HIV-associated dementia (Kaul et al., 2001). Recently, Munn (2000) has proposed microglia dysfunction in schizophrenia as an integrated theory. The study of Bayer et al. (1999) showed that some of the patients with schizophrenia elicited microglia activation, suggesting microglia activation might be involved in the pathophysiological process of schizophrenia. Taken together, the present study used LPS-activated mouse microglial cell line N9 as an in vitro model to mimic microglia activation seen in the patients with schizophrenia. The effects of clozapine, olanzapine and haloperidol on the release of NO by LPS-stimulated N9 cells were investigated.
Cell viability was evaluated by the MTT reduction assay (Chang et al., 1998). In brief, cells were seeded onto 96-well microtiter plates and treated with various reagents for the indicated time period. After various treatments, medium was removed and the cells were incubated with MTT (0.25 mg/ml) for 3 h at 37 °C. The formazan crystals in the cells were solubilized with a solution containing 50% dimethylformamide and 20% sodium dodecyl sulfate (pH 4.7). The level of MTT formazan was determined by measuring its absorbance at the wavelength of 490 nm with a SPECTRA (shell) Reader (TECAN, Austria).
2. Materials and methods
2.4. Nitrite assays
2.1. Materials
Accumulation of nitrite (NO2− ) in the culture supernatants, an indicator of NO synthase activity, was measured by Griess reaction (Barger and Harmon, 1997). Fifty microliter culture supernatants were mixed with 50 μl Griess reagent (part I: 1% sulfanilamide; part II: 0.1% naphthylethylene diamide dihydrochloride and 2% phosphoric acid) at room temperature. Fifteen minutes later, the absorbance was determined at 540 nm using the SPECTRA (shell) Reader. Nitrite concentration was calculated with reference to a standard curve of sodium nitrite generated by known concentrations.
Clozapine and haloperidol were purchased from Sigma (St. Louis, MO, USA). Olanzapine was purified from the tablets (Eli Lilly and Co. Ltd.) by the Department of Pharmaceutical Engineering, Shenyang Pharmaceutical University (Shenyang, China). The drug concentrations were determined according to the results of our preliminary study. In our preliminary study, the highest concentrations tested for haloperidol, olanzapine and clozapine were all up to 100 μM. It was found that haloperidol and clozapine, at the concentrations of 30 and 100 μM, significantly reduced the viability of N9 cells. In contrast, olanzapine (up to 100 μM) did not affect the viability of N9 cells. In order to devoid the possible effects of reduced viability on NO release, the drug concentrations not affecting the viability of N9 cells were selected in the present study. Fetal bovine serum (FBS) was purchased from TBD Biotechnology Development (Tianjin, China). LPS (E5:055) was purchased from Sigma (St. Louis, MO, USA). Thiazolylblue (MTT) was from Sino-American Biotechnology (Beijing, China). Iscove's modified dulbecco's medium (IMDM) was from Gibco (Grand Island, USA). Clozapine, olanzapine and haloperidol were dissolved initially in dimethyl sulfoxide (DMSO) and were diluted with PBS for experiments. DMSO at the highest concentration possibly present in experimental conditions (0.1%) was not toxic to cells. 2.2. Cell culture The murine microglial cell line N9 was a kind gift from Dr. J. M. Wang (NCI, Frederick, USA). The cells were grown in IMDM supplemented with 5% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 × 10− 5 M 2-mercaptoethanol. Cells at density of 3 × 104 cells/well were plated onto 96-well microtiter plates for MTT and nitrite assay. Clozapine, olanzapine or haloperidol with or without LPS (1 μg/ml) was added to
the culture medium of N9 cells for 24 h for the experiments. The drug doses used were according to our preliminary study, not affecting the viability of N9 cells. 2.3. Cell viability
2.5. Data analysis Results were expressed as mean ± S.E.M. Statistical significance (P < 0.05) was assessed by one-way ANOVA followed by Dunnett's t-test (SPSS12.0 software, SPSS, USA). 3. Results 3.1. The effect of clozapine on N9 cell viability and LPSinduced NO release Treatment with clozapine (1–10 μM) alone or with 1 μg/ml of LPS for 24 h did not cause any change in MTT absorbance in N9 cells, indicating that clozapine did not affect the viability of N9 cells at the doses used (Fig. 1). Next, the release of NO by LPS-stimulated N9 cells was examined. In unstimulated N9 cells, only small amounts of NO2− (4.23 ± 2.18 μM) occurred in the medium. Pretreatment of unstimulated cells with clozapine (1–10 μM) for 24 h did not result in any change of NO2−. The stimulation of N9 cells with LPS resulted in a manifold increase in NO2− production (11.1 ± 5.1 μM). LPS significantly induced NO release in N9 cells, which was in consistence with the previous report (Bi et al., 2005). Simultaneous treatment of N9 cells with LPS and clozapine (1–10 μM) was not different from the result observed by LPS alone.
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4. Discussion This is the first report showing that olanzapine suppressed LPS-induced NO release, reflecting the activation of microglial cells, suggesting that it might be beneficial for the treatment of neurodegenerative diseases. In fact, olanzapine has been shown to be safe and effective in the treatment of schizophrenia (Beasley et al., 1996a,b, 1997; Tollefson et al., 1997; Bhana et al., 2001). Olanzapine showed neuroproliferative effect above 60 μM in the study of Dwyer et al. (2003). In fact, olanzapine also showed its protective effect at lower doses. For example, olanzapine stimulated phosphorylation of Akt at the dose of 10 μM and was mitogenic and neuroprotective at concentrations as low as 10– 40 μM (Lu et al., 2004). It has also been reported that olanzapine increased the gene expression of superoxide dismutase (SOD1) at the concentration of 10 μM, which was associated with its neuroprotective effect (Li et al., 1999). Therapeutic levels of olanzapine in serum are in the range of 0.1–0.3 μM (Olesen and Linnet, 1999; Robertson and McMullin, 2000). It is also known that antipsychotic drugs are accumulated in brain and fat tissue to levels that are 25–30-fold Fig. 1. (a) The effect of clozapine on N9 cell viability. N9 cells were treated with clozapine in the presence or absence of LPS (1 μg/ml) for 24 h. Cell viability was examined by MTT reduction assays and the results were expressed as percentage of surviving cells over control cells (no addition of clozapine). Data were represented as mean ± S.E.M. of three separate experiments. (b) The effect of clozapine on NO release by LPS-activated N9 cells. N9 cells were treated with clozapine alone for 24 h or clozapine and LPS (1 μg/ml) simultaneously for 24 h. The results were expressed as the percentage values taking LPS treatment group as 100%. Data were represented as mean ± S.E.M. of three separate experiments. ###P < 0.001 compared with the control group.
3.2. The effect of olanzapine on N9 cell viability and LPSinduced NO release Treatment with olanzapine (3–100 μM) alone or with 1 μg/ ml of LPS for 24 h did not cause any change in MTT absorbance in N9 cells, indicating that olanzapine did not affect the viability of N9 cells at the doses used (Fig. 2). Pretreatment of unstimulated cells with olanzapine (3– 100 μM) for 24 h did not result in any change of NO2−. However, simultaneous treatment of N9 cells with LPS and olanzapine (10–100 μM) markedly reduced NO production. 3.3. The effect of haloperidol on N9 cell viability and LPSinduced NO release Treatment with haloperidol (1–10 μM) alone or with 1 μg/ml of LPS for 24 h did not cause any change in MTT absorbance in N9 cells, indicating that haloperidol (1–10 μM) did not affect the viability of N9 cells at the doses used (Fig. 3). Pretreatment of unstimulated cells with haloperidol (1– 10 μM) for 24 h did not result in any change of NO2−. Simultaneous treatment of N9 cells with LPS and haloperidol (1–10 μM) was not different from the result observed by LPS alone.
Fig. 2. (a) The effect of olanzapine on N9 cell viability. N9 cells were treated with olanzapine in the presence or absence of LPS (1 μg/ml) for 24 h. Cell viability was examined by MTT reduction assays and the results were expressed as percentage of surviving cells over control cells (no addition of olanzapine). Data were represented as mean ± S.E.M. of three separate experiments. (b) The effect of olanzapine on NO release by LPS-activated N9 cells. N9 cells were treated with olanzapine alone for 24 h or olanzapine and LPS (1 μg/ml) simultaneously for 24 h. The results were expressed as the percentage values taking LPS treatment group as 100%. Data were represented as mean ± S.E.M. of three separate experiments. ###P < 0.001 compared with the control group. ⁎P < 0.05, ⁎⁎⁎P < 0.001 compared with the LPS group.
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Fig. 3. (a) The effect of haloperidol on N9 cell viability. N9 cells were treated with haloperidol in the presence or absence of LPS (1 μg/ml) for 24 h. Cell viability was examined by MTT reduction assays and the results were expressed as percentage of surviving cells over control cells (no addition of haloperidol). Data were represented as mean ± S.E.M. of three separate experiments. (b) The effect of haloperidol on NO release by LPS-activated N9 cells. N9 cells were treated with haloperidol alone for 24 h or haloperidol and LPS (1 μg/ml) simultaneously for 24 h. The results were expressed as the percentage values taking LPS treatment group as 100%. Data were represented as mean ± S.E.M. of three separate experiments. ###P < 0.001 compared with the control group.
higher than this (Aravagiri et al., 1999; Baldessarini et al., 1993; Kornhuber et al., 1999). Thus, olanzapine may reach 5–10 μM in the brain. Taking these into account, concentrations higher than 100 μM could not been reached in vivo. Thus, the effect of olanzapine at higher concentrations was not investigated. Unlike olanzapine, clozapine and haloperidol did not show significant effects on LPS-induced microglial cell activation. There are already existing data showing the differential effects between the two drugs and olanzapine. For example, clozapine and haloperidol were toxic for PC12 cells, while olanzapine stimulated cell proliferation under the same experimental condition (Dwyer et al., 2003). In a delayed radial maze task, olanzapine significantly reduced the number of errors made during the retention phase, while clozapine and haloperidol failed to affect the total number of retention phase errors (Wolff and Leander, 2003). The present study provides new data for further understanding the differential properties between olanzapine and the other two drugs in regulating some neurochemical events in the central nervous system. In the present study, the effects of haloperidol and clozapine were studied up to 10 μM concentrations. This is because haloperidol and clozapine, at the concentrations of 30 and 100 μM, significantly reduced the viability of N9 cells. To devoid the possible effects of reduced viability on NO release,
the drug concentrations not affecting the viability of N9 cells were selected in the present study. Nitric oxide (NO) is now accepted as an active messenger molecule and effector in the immune, cardiovascular and nervous systems. NO is formed endogenously by the conversion of L-arginine to L-citrulline by the enzyme nitric oxide synthase (NOS) (Mayer et al., 1989). NOS is known to exist in at least three isoforms (neuronal NOS, endothelial NOS and inducible NOS) broadly corresponding to that present in neuronal, endothelial and macrophage cells (Snyder and Bredt, 1991). NO has been studied in relation to the etiology of various neurological and psychiatric diseases, including schizophrenia. A postmortem histochemical study (Akbarian et al., 1993) found that nNOS levels were increased in deep layers of dorsolateral prefrontal cortex and decreased in hippocampus in brain tissue from schizophrenia vs control subjects. Another study (Karson et al., 1996) found elevated concentrations of nNOS in cerebellum from patients diagnosed with schizophrenia. Das et al. (1995, 1996) reported accumulation of an endogenous nNOS inhibitor in plasma of patients with schizophrenia and detected elevated Ca2+-induced NOS activity in blood platelets of drug naïve schizophrenia patients compared to others treated with antipsychotic drugs and to controls. The study of Yanik et al. (2003) showed that total nitrite level in the plasma was higher in patients with schizophrenia compared with controls. Postmortem study showed that nNOS expression in the prefrontal cortex was significantly higher in individuals with schizophrenia, suggesting the abnormalities of nNOS expression in the brain might contribute to the development of schizophrenia (Baba et al., 2004). Therefore, the inhibiting effect of olanzapine on the NO release by LPS-stimulated microglial cells suggested that this might be a new mechanism through which olanzapine exhibits its therapeutic effect in the treatment of schizophrenia. In fact, the effects of both typical and atypical antipsychotic drugs on NO have been investigated. Suzuki et al. (2002) found that haloperidol induced iNOS mRNA after acute, subacute and chronic treatment. Repeated treatment of rats with olanzapine did not change the density of nNOS enzyme in cortical, limbic, or extrapyramidal brain regions (Tarazi et al., 2002). Haloperidol did not reverse NOS inhibitor-induced deficit in social interaction (SI) before pronounced secondary effects on general behavior were seen at high doses, while clozapine and olanzapine were able to significantly reverse this deficit at doses which did not effect baseline SI values (Black et al., 2002). It was reported that rats treated with haloperidol for 28 days developed significant suppression of striatal NOS activity, while acute challenge with olanzapine significantly reversed it (Nel and Harvey, 2003). In the present study, olanzapine and haloperidol also showed differential effects on NO release by LPS-activated N9 cells. Although olanzapine closely resembles clozapine in terms of chemical structure and pharmacological profiles, they showed differential effects on the present model. In fact, in vitro radioligand studies in animal tissues and cloned human receptors have demonstrated that olanzapine has higher affinity for dopamine D2, serotonin 5-HT2A and muscarinic M1 receptors,
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