Hydroxyl radical scavengers enhance nitric oxide-evoked acetylcholine release from mouse cerebral cortical neurons

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 34 (1995) 347-350

Short communication

Hydroxyl radical scavengers enhance nitric oxide-evoked acetylcholine release from mouse cerebral cortical neurons Seitaro Ohkuma, Masashi Katsura, Da-Zhi Chen, Jin-Long Guo, Kinya Kuriyama * Departmentof Pharmacology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji,Kamikyo-Ku,Kyoto 602, Japan Accepted 16 August 1995

Abstract The effect of hydroxyl radical scavengers on acetylcholine (ACh) release evoked by nitric oxide (NO) generators and N-methyl-oaspartate (NMDA) was investigated. Dimethylthiourea enhanced dose-dependently NO generators-evoked ACh release. Similarly, uric acid and mannitol significantly facilitated ACh release evoked by NO generators. The NMDA-induced ACh release was also significantly facilitated by hydroxyl radical scavengers. These scavengers themselves showed no effects on ACh release. These results suggest that hydroxyl radicals may modify the mechanism for NO-evoked ACh release.

Keywords: Nitric oxide; Peroxynitrite; Acetylcholine release; Hydroxyl radical; Hydroxyl radical scavenger; Cerebral cortical neurons

Nitric oxide (NO) is an unstable radical and has a high potential to react with superoxide [2]. By this reaction, peroxynitrite is assumed to be formed with the rate constant of 6.7 + 0.9 × 109 M - 1. s - 1 [5] and such formation of peroxynitrite has been reported to occur under physiological conditions [6]. Peroxynitrite is also unstable and is rapidly degraded to hydroxyl radical and .NO 2 at pH 7.4 at 37°C [1]. One of the functional roles of NO in the central nervous system is to evoke neurotransmitter release. NO has been reported to induce the release of acetylcholine (ACh) from the forebrain [15] and hippocampus slices [7]. Similar release of ACh induced by NO, which was generated from NO donors such as sodium nitroprusside (SNP) and Snitroso-N-acetylpenicillamine (SNAP) and produced by N-methyl-D-aspartate (NMDA) receptor stimulation, was noted in primary cultured cerebral cortical neurons [10,11]. In addition, we have reported that NO-induced ACh release is partially mediated by peroxynitrite [10,11]. In the present study, therefore, we have examined the effect of removal of hydroxyl radical on NO-induced ACh release with use of mouse cerebral cortical neurons in primary culture to clarify the role of hydroxyl radical on neurotransmitter release mediated by NO.

* Corresponding author. Fax: (8l) (75) 241-0824. 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

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Cerebral cortical neurons were prepared according to the method described previously [12]. The neopallium dissected from 15-day-old fetus of mouse was treated with trypsin, centrifuged and finally suspended in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal calf serum (Hazelton Research Products Inc., Nutick, USA). The cells were then added into a poly-L-lysinecoated culture dish and cultured at 37°C for 3 days under humidified 95% air-5% CO 2. After the exposure of the cells to 10 /xM cytosine arabinoside in DMEM with 10% horse serum (Hazelton Research Products Inc.) for 24 h, the neurons were continued in culture with DMEM supplemented with 10% horse serum under the same conditions described above, and the culture medium was changed to a fresh one every 4 days. The neurons cultured for 13 days were used in the following experiments. The measurement of ACh release from the neurons was carried out as previously described [10,13]. In brief, the culture medium was discarded by aspiration followed by washing the neurons three times with ice-cold Krebs-Ringer bicarbonate buffer (KRB; pH 7.4:137 mM NaC1, 4.8 mM KC1, 1.2 mM K H 2 P O 4 , 2.7 mM CaC12.2H20, 1.2 mM MgSO4.6H20, 10 mM glucose and 25 mM NaHCO3), and the neurons were preincubated with KRB at 37°C for 10 min. Thereafter, the neurons were incubated with warm (37°C) KRB at an interval of 5 min for a total of 25 min. The incubation buffer was collected after each interval and used to measure ACh released into the incubation buffer

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by HPLC [9,10]. 100 /zM SNP (Sigma Chemicals, St. Louis, USA), 5 /zM SNAP (Research Biochemical Inc. Ltd., Nutick, USA), and NMDA (Research Biochemical Inc. Ltd.) were added to the incubation buffer at the beginning of the fourth interval of the incubation. Uric acid, dimethylthiourea (DMTU; Sigma Chemicals) and mannitol were added to the incubation buffer immediately before the addition of NO generators or NMDA. All the incubation buffers were supplemented with 100 /zM eserine to prevent the enzymatic degradation of ACh released into the incubation buffer. Mg2+-free KRB was employed, when examining the effect of NMDA, to minimize the inhibitory action of Mg 2+ on NMDA receptor function. The release of ACh determined during the third interval of the incubation was defined as the basal release and the stimulated release of ACh was expressed as a percentage of the basal release. Protein in the neurons was determined by the method of Lowry et al. [8] using bovine serum albumin as standard. Each value of ACh release was expressed as the mean _+ SEM and statistical significance was analyzed as described in each legend of the figures and the table following the application of the one-way ANOVA. Fig. 1A shows the effects of one of the hydroxyl radical scavengers, DMTU, used in this study on the SNP (100 /xM)-induced ACh release from the cerebral cortical neurons. DMTU enhanced ACh release evoked by 100 /zM SNP in a dose-dependent manner. The enhancement of the SNP-induced ACh release by DMTU attained a plateau at 10 ~ M of DMTU. Uric acid (100 /zM) and mannitol (1 mM) also significantly increased the SNP-evoked ACh release (Fig. 1B). The SNAP (5 /~M)-evoked release of ACh from the neurons was also dose-dependently increased by DMTU (Fig, 2A). Uric acid (100 /xM) and mannitol (1 mM) showed a significant elevation of the SNAP-induced ACh release (Fig. 2B). In addition, the order of magnitude of these hydroxyl radical scavengers to enhance the release of ACh induced by SNAP tended to be similar to that by SNP. As shown in Table 1, these hydroxyl radical scavengers themselves did not affect the release of ACh determined in the absence of SNP and SNAP. NMDA, an agonist specific to NMDA receptors, induced ACh release from the neurons. This NMDA-evoked ACh release was significantly facilitated by two different hydroxyl radical scavengers, DMTU and mannitol, as shown in Fig. 3. The present study demonstrated that the removal of hydroxyl radical by hydroxyl radical scavengers such as DMTU, uric acid and mannitol enhanced the ACh release, which was induced by NO liberated from NO generators and produced by NMDA, from the cerebral cortical neurons, whereas each hydroxyl radical scavenger alone did not affect the release of ACh. Previous reports have revealed that neurotransmitter release induced by NMDA is mediated by NO production through NMDA receptor acti-

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Fig. 1. Effects of (A) dimethylthiourea (DMTU), (B) uric acid and mannitol on sodium nitroprusside (SNP: 100 /zM)-induced acetylcholine (ACh) release from cerebral cortical neurons in primary culture. The neurons were incubated with Krebs-Ringer bicarbonate buffer (pH 7.4) containing 100 # M eserine at 37°C at an interval of 5 min for a total of 25 rain. SNP was added into the incubation buffer at the initiation of the fourth interval of the incubation. Hydroxyl radical scavengers were added immediately before the addition of SNP. The incubation buffer obtained during each interval was used to measure ACh by HPLC. The release of ACh determined during the third interval of the incubation was defined as the basal release and the stimulated release of ACh by the agents used here was expressed as percent of the basal release. Each value represents the mean_+SEM obtained from four separate experiments. The basal release of ACh was 5.63 + 0.77 n m o l / m g protein/fraction. (A) * P < 0.05, * * P < 0.01, compared with the release of ACh determined in the presence of SNP alone (Dunnett's test). (B) * * P < 0.01, compared with the release of ACh determined in the presence of SNP alone (Bonferroni's test).

vation subsequent to the activation of Ca2+-dependent NO synthase [9,10]. Although the mechanisms of such enhancement of the NO-induced ACh release by hydroxyl radical scavengers are not clear at present, several possibilities are considered. In previous reports, we have indicated that peroxynitrite formed by the reaction of NO with superoxide participates in neurotransmitter release mediated by NO [10,13]. Peroxynitrite is reported to be formed under physiological conditions [6], suggesting that, even in the brain, the formation of peroxynitrite may occur under the situation that NO and superoxide are present. Peroxynitrite is rapidly

S. Ohkuma et al. /Molecular Brain Research 34 (1995) 347-350

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Fig. 2. Effects of (A) dimethylthiourea (DMTU), (B) uric acid and mannitol on S-nitroso-N-acetylpenicillamine (SNAP; 5 /xM)-induced acetylcholine (ACh) release from cerebral cortical neurons in primary culture. The measurement of ACh release was carried out as described in the legend of Fig. 1. Each value represents the mean_+ SEM obtained from four separate experiments. The basal release of ACh was 5.77 + 0.61 n m o l / m g protein/fraction. (A) * P < 0.05, * * P < 0.01, compared with the release of ACh determined in the presence of SNAP alone (Dunnett's test). (B) * * P < 0.01, compared with the release of ACh determined in the presence of SNP alone (Bonferroni's test).

decomposed to hydroxyl radical and nitrogen dioxide [1]. Based on these data, it is assumed that hydroxyl radical produced from peroxynitrite may in turn suppress the release of ACh evoked by NO a n d / o r peroxynitrite, that is, the removal of hydroxyl radical counteracts the inhibitory action of hydroxyl radical on release system as an Table 1 Effects of uric acid, dimethylthiourea (DMTU), and mannitol on the basal release of ACh from cerebral cortical neurons in primary culture ACh release (% of the basal release) DMTU (100 /zM) Uric acid (100 /zM) Mannitol (1000/xM)

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neuronal membrane function. In fact, several investigators have reported that hydroxyl radical mediates dysfunction of biological membranes [4,14]. Another possible mechanism is that hydroxyl radical, which is assumed to be produced from peroxynitrite, may decrease Ca 2+ influx into the neurons. Lonart and his co-workers reported that NO-evoked release of neurotransmitter required extracellular Ca z + [7]. In addition, NO has the ability to induce Ca 2÷ influx into sympathetic neurons [3]. These data are assumed to suggest that the enhancement of the NO-induced ACh release by hydroxyl radical scavengers may be mediated by an increase in Ca 2÷ influx into the cerebral cortical neurons subsequent to the removal of hydroxyl radical, although it is not clear that hydroxyl radical has a potency to inhibit Ca 2+ influx into neurons. However, the exact mechanisms of the stimulatory action of hydroxyl radical scavengers on NO-evoked ACh release remain to be elucidated at present.

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[5] Hule, R.E. and Padmaja, S., The reaction of NO with superoxide, Free Radical Res. Commun., 18 (1994) 195-199. [6] Ischiropoulos, H., Zhu, L. and Beckman, J.S., Peroxynitrite formation from macrophage-derived nitric oxide, Arch. Biochem. Biophys., 298 (1992) 446-452. [7] Lonart, G., Wang, J. and Johnson, K.M., Nitric oxide induces neurotransmitter release from hippocampal slices, Eur. J. Pharmacol., 220 (1992) 271-272. [8] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R3., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [9] Montague, P.R., Gancayco, C.D., Winn, M.J., Marchase, R.B. and Friedlander, M.J., Role of NO production in NMDA receptor-mediated neurotransmitter release in cerebral cortex, Science, 263 (1994) 973-977. [10] Ohkuma, S., Katsura, M., Guo, J.-L., Hasegawa, T. and Kuriyama, K., Involvement of peroxynitrite in N-methyl-o-aspartate- and

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