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Oxidative metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde, induces dopamine release from PC12 cells by a Ca2+-independent mechanism
Brain Research 931 (2002) 96–99 www.elsevier.com / locate / bres
Short communication
Oxidative metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde, induces dopamine release from PC12 cells by a Ca 21 -independent mechanism Tsuneichi Hashimoto, Chihiro Yabe-Nishimura* Department of Pharmacology, Kyoto Prefecutural University of Medicine, Kawaramachi-Hirokoji, Kamikyoku, Kyoto 602 -8566, Japan Accepted 20 December 2001
Abstract 3,4-Dihydroxyphenylacetaldehyde (DOPALD), an oxidative metabolite of dopamine (DA), induced dose-dependent DA release from pheochromocytoma (PC12) cells without affecting leakage of lactate dehydrogenase from the cells. DOPALD-induced DA release was independent of extracellular Ca 21 concentration and was not blocked by nifedipine, an L-type Ca 21 channel antagonist. These results indicated a novel intrinsic role of DOPALD in dopaminergic nerve terminals that may take part in the activation of dopamine neurons. 2002 Elsevier Science B.V. All rights reserved. Keywords: 3,4-Dihydroxyphenylacetaldehyde; Dopamine release; Parkinson’s disease; PC12 cell
3,4-Dihydoxyphenylacetaldehyde (DOPALD) is generated from dopamine (DA) by type B monoamine oxidase (MAO-B) as the initial metabolite of DA. DOPALD is subsequently oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH) [19], or reduced to 3,4-dihydroxyphenylethanol by members of the aldo–keto reductase family, aldose reductase (AKR1B4) and aldehyde reductase (AKR1A3) [18]. Similarly to dopaminergic neurotoxins such as 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), DOPALD is thought to be one of the substances that provoke idiopathic Parkinson’s disease [10]. In fact, DOPALD was detected in the substantia nigra of parkinsonian patients but not in control subjects [9]. Augmented formation of DOPALD accompanied by accelerated cell death was demonstrated in PC12 cells cultured under metabolic stress [8]. Direct toxic effects of 100 mM DOPALD on these cells were observed after 24 h, leading to increased leakage of lactate dehydrogenase (LDH) into the medium. Neurotoxicity of DOPALD was also documented in other dopaminergic neurons such as neostriatal synaptosomes and cultured dissociated mesencephalon [10] as well as norepinephrine *Corresponding author. Tel.: 181-75-251-5333; fax: 181-75-2515348. E-mail address: [email protected] (C. Yabe-Nishimura).
metabolite, 3,4-dihydroxyphenylglycolaldehyde, in PC12 cells [2]. In these studies, however, the viability of the cells was examined after 24 h of incubation with DOPALD, and short-term effects of DOPALD on cellular responses have not been investigated. In our previous study, we observed that a single inhalation of acetaldehyde facilitated the metabolic turnover of catecholamines in the mouse brain [6]. On the other hand, it has been reported that 4-hydroxynonenal (HNE), an aldehyde product of membrane lipid peroxidation, inhibits dopamine transporter [11,12] and induces neuronal apoptosis in PC12 cells [7,17]. As DOPALD belongs to the aldehyde group similarly to acetaldehyde and HNE, we set out to examine the direct effects of DOPALD on DA release using PC12 cells. The novel DA-releasing action of DOPALD demonstrated in the present study suggested a possible role of this oxidative metabolite of DA in the development of the degenerative processes leading to parkinsonism. DOPALD was synthesized from epinephrine by the method of Robins [15]. After purification by column chromatography (Sep-Pak C 18 , Waters, Milford, USA), the final product was identified by liquid chromatography– mass spectrometry (LC–MS) equipped with a DB-1 column interfaced with a Perkin Elmer Q-Mass. PC12 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated horse serum, 5%
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02233-3
T. Hashimoto, C. Yabe-Nishimura / Brain Research 931 (2002) 96 – 99
fetal calf serum, 100 units / ml penicillin and 0.05 mg / ml streptomycin. Cells plated on 0.2% gelatin-coated dishes were washed twice with Hanks’ balanced salt solution. After 1-min adaptation in medium containing 135 mM NaCl, 3 mM KCl, 1.7 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 1 mM ascorbic acid (pH7.4), incubations were started by the addition of DOPALD at 37 8C. High potassium (56 mM) stimulation was applied by subtracting an equimolar concentration of NaCl. In Ca 21 -free medium, CaCl 2 was replaced with 1.7 mM MgCl 2 . After 5 min incubation, medium was transferred into a tube containing 10% trichloroacetic acid for subsequent DA analysis [6]. The release of DA was expressed as the percentage of DA detected in the medium versus total DA, released plus DA remaining in the cells. LDH activity was measured as previously described [5]. LDH leakage was calculated as the percentage of LDH activity leaked into the medium versus total LDH activity. Statistical analysis was carried out by Student’s t-test or ANOVA followed by Dunnett’s multiple comparison. As shown in Fig. 1A, a more than twofold increase in DA release was observed by stimulation with 56 mM potassium. When treated with 10 mM DOPALD, DA release was augmented to a level similar to that induced by high potassium stimulation. To elucidate the specificity of the DOPALD-induced DA release from PC12 cells, the effects of another endogenous aldehyde, HNE, were tested on DA release. At concentrations up to 100 mM, however, HNE did not affect DA release (Fig. 1B). Thus, the effect of DOPALD on DA release was specific, and not common to other reactive aldehydes. As DOPALD is one of the toxic aldehydes generated during catecholamine metabolism, damage to PC12 cells was next evaluated (Fig. 2). The leakage of LDH was unchanged at concentrations of DOPALD up to 10 mM, although 100 mM DOPALD significantly induced LDH leakage. These results indicated that the cytotoxicity of DOPALD was negligible at 10 mM at which significant DA release was demonstrated. In the absence of extracellular Ca 21 , 56 mM KClinduced DA release was markedly suppressed, while 10 mM DOPALD-induced DA release was not affected (Table 1). In accordance with these findings, the L-type Ca 21 channel blocker nifedipine significantly reduced high potassium-evoked DA release, while DA release elicited by DOPALD was not affected. Distinct from potassiumevoked DA release, DOPALD-induced DA release was therefore independent of extracellular Ca 21 . Short-term exposure to DOPALD evoked DA release from PC12 cells to a level similar to that induced by high potassium. This effect of DOPALD on DA release was not associated with its cytotoxicity against the cells, since no increase in LDH leakage was observed at DOPALD concentrations up to 10 mM. Mattermal et al. previously reported DOPALD-induced decreases in DA and 3,4dihydroxyphenylacetic acid (DOPAC) contents in striatal synaptosomes and differentiated PC12 cells [10]. Lamen-
97
Fig. 1. DOPALD induced DA release from PC12 cells (A), while 4hydroxy-2-nonenal (HNE) had no effect on DA release (B). PC12 cells were incubated with the indicated concentrations of DOPALD, KCl or HNE for 5 min. Each value represents the mean6S.E.M. obtained from three to six separate experiments. Data are expressed as percent of control. Basal DA release of control was 14.764.2% (A), and 15.566.0% (B) of total DA contents. *, P,0.05, compared with control value.
sdorf et al. recently reported negligible cytotoxic effects of 10 mM DOPALD on differentiated PC12 cells, in that little increase in LDH leakage was observed following exposure of the cells to DOPALD for 24 h [8]. Our findings and these previous observations indicated a novel function of DOPALD on DA release at concentrations lower than those that show apparent cytotoxicity. This DA-releasing action of DOPALD appeared to be specific, and was not common to other aldehydes such as HNE, an endogenous aldehyde generated during lipid peroxidation. HNE is known to induce dose-dependent inhibition of dopamine uptake in rat striatal synaptosomes, potentially leading to decreased turnover of dopamine [11,12]. Furthermore, HNE-induced apoptosis was reported in PC12 cells [7,17]. Similar cytotoxicity of
98
T. Hashimoto, C. Yabe-Nishimura / Brain Research 931 (2002) 96 – 99
Table 1 DOPALD-induced DA release from PC 12 cells was Ca 21 -independent DA release (% of control)
Control KCl (56 mM) DOPALD (10 mM)
Ca 21 (1)
Ca 21 (2)
Nifedipine (10 nM)
100618.7 285646.2** 306.5645.9**
N.D. 96.2618.5[[ 385.7677.8
83.768.0 187.7625.3[ 305.4697.9
PC12 cells were incubated with each drug for 5 min in the presence or absence of Ca 21 . Each value represents the mean6S.E.M. obtained from four to ten separate experiments. N.D., not determined. **, P,0.01, compared with control; [, P,0.05, [[, P,0.01, compared with Ca 21 (1).
DOPALD was reported in DA-containing terminals of neostriatal synaptosomes, in PC12 cells, and mesencephalic cell cultures [10]. While direct neurotoxic effects of DOPALD have been implicated in the development of degenerative diseases such as parkinsonism, the present study provided the first evidence for the possible involvement of DOPALD-induced DA release in the pathogenesis of neuronal degeneration. DOPALD-induced DA release from PC 12 cells was independent of extracellular Ca 21 and was not inhibited by nifedipine, a voltage-sensitive calcium channel antagonist. Perturbation of DA transport into synaptic vesicles is among the mechanisms that could lead to Ca 21 -independent DA release. When direct effects of DOPALD on DA transport were tested, up to 10 mM of DOPALD did not affect [ 3 H]DA uptake into PC12 cells (data not shown). Ca 21 -independent neurotransmitter release from PC12 cells was previously demonstrated in cells stimulated by diacylglycerol, an activator of protein kinase C [14]. Recently, the excitatory neurotoxin, pardaxin was also reported to induce DA release from PC 12 cells in a Ca 21 -independent manner, and the involvement of the
lipoxygenase pathway was suggested [1]. There is as yet no direct evidence that DOPALD-induced DA release is mediated by protein kinase C or arachidonic acid cascade. However, these possibilities remain under consideration. MPTP, after conversion by MAO-B into the toxic metabolite 1-methyl-4-phenylpyridine (MPP 1 ), elicits cell death in dopaminergic neurons. Recent studies in PC12 cells indicate that MPP 1 interferes with complex I of the electron transport chains and impairs energy metabolism, leading to cell damage [4,16]. On the other hand, MPP 1 is a potent DA-releasing agent, and nonenzymatic autoxidation of DA leads to formation of hydroxy radicals. In fact, determination of the in vivo generation of free radicals by microdialysis indicated the involvement of hydroxy radicals derived from DA in MPTP-induced neurotoxicity [3,13]. Excess DA release induced by DOPALD and ensuing augmentation of oxidative stress may therefore be one of the mechanisms underlying neuronal degeneration leading to parkinsonism.
Acknowledgements This work was supported by a Grant-in-aid-for Scientific Research 11680760 from the Ministry of Education, Science, and Culture, Japan.
References
Fig. 2. LDH leakage from PC12 cells was unchanged by DOPALD treatment at concentrations up to 10 mM. PC12 cells were incubated with DOPALD or KCl for 5 min. Each column represents the mean6S.E.M. obtained from four separate experiments. **, P,0.01, compared with control value.
[1] S. Abu-Raya, E.B. Shilderman, P.I. Lelkes, V. Tremboler, E. Shohami, Y. Gutman, P. Lazarovici, Characterization of pardaxininduced dopamine release from pheochromocytoma cells: role of calcium and eicosanoids, J. Pharmacol. Exp.Ther. 288 (1999) 399– 406. [2] W.J. Burke, C.A. Schmitt, K.N. Gillespie, S.W. Li, Norepinephrine transmitter metabolite is a selective cell death messenger in differentiated rat pheochromocytoma cells, Brain Res. 722 (1996) 232–235. [3] C.C. Chiueh, R.-M. Wu, K.P. Mohanakunar, L.M. Sternberger, G. Krushna, T. Obata, D.L. Murphy, In vivo generation of hydroxyradicals and MPTP-induced dopaminergic toxicity in the basal ganglia, Ann. NY Acad. Sci. 738 (1994) 25–36. [4] C. Fonck, M. Baudry, Toxic effects of MPP(1) and MPTP in PC12 cells independent of reactive oxygen species formation, Brain Res. 905 (2001) 199–206. [5] R.J. Gay, R.B. McComb, G.N. Bowers, Optimum reaction con-
T. Hashimoto, C. Yabe-Nishimura / Brain Research 931 (2002) 96 – 99
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ditions for human lactate dehydrogenase isozymes as they affect total lactate dehydrogenase activity, Clin. Chem. 14 (1968) 740– 753. T. Hashimoto, T. Ueha, T. Kuriyama, M. Katsura, K. Kuriyama, Acetaldehyde-induced alterations in metabolism of monoamines in mouse brain, Alcohol Alcoholism 24 (1982) 91–99. I. Kruman, A.J. Bruce-Keller, D. Bredsen, G. Waeg, M.P. Mattson, Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis, J. Neurosci. 17 (1997) 5089–5100. I. Lamensdorf, G. Eisenhofer, J.H. White, Y. Hayakawa, K. Kirk, I.J. Kopin, Metabolic stress in PC12 cells induced the formation of the endogenous dopaminergic neurotoxin, 3,4-dihydroxyphenylacetaldehyde, J. Neurosci. Res. 60 (2000) 552–558. M.B. Mattermal, H.D. Chung, R. Strong, F.-F. Hsu, Confirmation of a dopamine metabolite in parkinsonian brain tissue by a gas chromatography–mass spectrometry, J. Chromatogr. 614 (1993) 205–212. M.B. Mattermal, J.H. Haring, H.D. Chung, G. Raghu, R. Strong, An endogenous dopamine neurotoxin: implication for Parkinson’s disease, Neurodegeneration 4 (1995) 271–281. P.F. Mopel, L. Barrier, B. Fauconneau, A. Piriou, F. Huguet, Origin of 4-hydroxynonenal incubation-induced inhibition of dopamine transporter and Na 1 / K 1 adenosine triphosphate in rat striatal synaptosomes, Neurosci. Lett. 277 (1999) 91–94. P. Mopel, C. Tallineau, R. Pontcharraud, A. Piriou, F. Huguet, Effects of 4-hydroxynonenal, a lipid peroxidation product, on
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dopamine transport and Na 1 / K 1 ATPase in rat synaptosomes, Neurochem. Int. 33 (1998) 531–540. T. Obata, C.C. Chiueh, In vivo trapping of hydroxy free radicals in the striatum utilizing intracranial microdialysis perfusion of salicylate: effect of MPTP, MPDP 1 and MPP 1 , J. Neural. Transm. Gen. Sect. 89 (1992) 139–145. T. Pozzan, G. Gatti, N. Dozio, L.M. Vicentini, J. Meldolesi, Ca 21 dependent and -independent release of neurotransmitter from PC12 cells: a role for protein kinase C activation?, J. Cell Biol. 99 (1984) 628–638. J.H. Robins, Preparation and properties of p-hydroxyphenylacetaldehyde and 3-methoxy-4-hydroxyphenylacetaldehyde, Arch. Biochem. Biophys. 114 (1966) 576–584. J. Seyfried, F. Soldner, W.S. Kunz, J.B. Schulz, T. Klockgether, K.A. Kovar, U. Wullner, Effect of 1-methyl-4-phenylpyridinium on glutathione in rat pheochromocytoma PC12 cells, Neurochem. Int. 36 (2000) 489–497. B.J. Song, Y. Soh, M. Bae, J. Wan, K. Jeong, Apoptosis of PC12 cells by 4-hydroxy-2-nonenal is mediated through selective activation of c-Jun N-terminal protein kinase pathway, Chem. Biol. Interact. 132 (2001) 943–954. A.J. Turner, K. Tipton, The characterization of two reduced nicotinamide-adenine dinucleotide phosphate-linked aldehyde reductases from pig brain, Biochem. J. 130 (1972) 765–772. H. Weiner, X. Wang, Aldehyde dehydrogenase and acetaldehyde metabolism, Alcohol Alcoholism Suppl. 2 (1994) 141–145.
Short communication
Oxidative metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde, induces dopamine release from PC12 cells by a Ca 21 -independent mechanism Tsuneichi Hashimoto, Chihiro Yabe-Nishimura* Department of Pharmacology, Kyoto Prefecutural University of Medicine, Kawaramachi-Hirokoji, Kamikyoku, Kyoto 602 -8566, Japan Accepted 20 December 2001
Abstract 3,4-Dihydroxyphenylacetaldehyde (DOPALD), an oxidative metabolite of dopamine (DA), induced dose-dependent DA release from pheochromocytoma (PC12) cells without affecting leakage of lactate dehydrogenase from the cells. DOPALD-induced DA release was independent of extracellular Ca 21 concentration and was not blocked by nifedipine, an L-type Ca 21 channel antagonist. These results indicated a novel intrinsic role of DOPALD in dopaminergic nerve terminals that may take part in the activation of dopamine neurons. 2002 Elsevier Science B.V. All rights reserved. Keywords: 3,4-Dihydroxyphenylacetaldehyde; Dopamine release; Parkinson’s disease; PC12 cell
3,4-Dihydoxyphenylacetaldehyde (DOPALD) is generated from dopamine (DA) by type B monoamine oxidase (MAO-B) as the initial metabolite of DA. DOPALD is subsequently oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH) [19], or reduced to 3,4-dihydroxyphenylethanol by members of the aldo–keto reductase family, aldose reductase (AKR1B4) and aldehyde reductase (AKR1A3) [18]. Similarly to dopaminergic neurotoxins such as 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), DOPALD is thought to be one of the substances that provoke idiopathic Parkinson’s disease [10]. In fact, DOPALD was detected in the substantia nigra of parkinsonian patients but not in control subjects [9]. Augmented formation of DOPALD accompanied by accelerated cell death was demonstrated in PC12 cells cultured under metabolic stress [8]. Direct toxic effects of 100 mM DOPALD on these cells were observed after 24 h, leading to increased leakage of lactate dehydrogenase (LDH) into the medium. Neurotoxicity of DOPALD was also documented in other dopaminergic neurons such as neostriatal synaptosomes and cultured dissociated mesencephalon [10] as well as norepinephrine *Corresponding author. Tel.: 181-75-251-5333; fax: 181-75-2515348. E-mail address: [email protected] (C. Yabe-Nishimura).
metabolite, 3,4-dihydroxyphenylglycolaldehyde, in PC12 cells [2]. In these studies, however, the viability of the cells was examined after 24 h of incubation with DOPALD, and short-term effects of DOPALD on cellular responses have not been investigated. In our previous study, we observed that a single inhalation of acetaldehyde facilitated the metabolic turnover of catecholamines in the mouse brain [6]. On the other hand, it has been reported that 4-hydroxynonenal (HNE), an aldehyde product of membrane lipid peroxidation, inhibits dopamine transporter [11,12] and induces neuronal apoptosis in PC12 cells [7,17]. As DOPALD belongs to the aldehyde group similarly to acetaldehyde and HNE, we set out to examine the direct effects of DOPALD on DA release using PC12 cells. The novel DA-releasing action of DOPALD demonstrated in the present study suggested a possible role of this oxidative metabolite of DA in the development of the degenerative processes leading to parkinsonism. DOPALD was synthesized from epinephrine by the method of Robins [15]. After purification by column chromatography (Sep-Pak C 18 , Waters, Milford, USA), the final product was identified by liquid chromatography– mass spectrometry (LC–MS) equipped with a DB-1 column interfaced with a Perkin Elmer Q-Mass. PC12 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated horse serum, 5%
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02233-3
T. Hashimoto, C. Yabe-Nishimura / Brain Research 931 (2002) 96 – 99
fetal calf serum, 100 units / ml penicillin and 0.05 mg / ml streptomycin. Cells plated on 0.2% gelatin-coated dishes were washed twice with Hanks’ balanced salt solution. After 1-min adaptation in medium containing 135 mM NaCl, 3 mM KCl, 1.7 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 1 mM ascorbic acid (pH7.4), incubations were started by the addition of DOPALD at 37 8C. High potassium (56 mM) stimulation was applied by subtracting an equimolar concentration of NaCl. In Ca 21 -free medium, CaCl 2 was replaced with 1.7 mM MgCl 2 . After 5 min incubation, medium was transferred into a tube containing 10% trichloroacetic acid for subsequent DA analysis [6]. The release of DA was expressed as the percentage of DA detected in the medium versus total DA, released plus DA remaining in the cells. LDH activity was measured as previously described [5]. LDH leakage was calculated as the percentage of LDH activity leaked into the medium versus total LDH activity. Statistical analysis was carried out by Student’s t-test or ANOVA followed by Dunnett’s multiple comparison. As shown in Fig. 1A, a more than twofold increase in DA release was observed by stimulation with 56 mM potassium. When treated with 10 mM DOPALD, DA release was augmented to a level similar to that induced by high potassium stimulation. To elucidate the specificity of the DOPALD-induced DA release from PC12 cells, the effects of another endogenous aldehyde, HNE, were tested on DA release. At concentrations up to 100 mM, however, HNE did not affect DA release (Fig. 1B). Thus, the effect of DOPALD on DA release was specific, and not common to other reactive aldehydes. As DOPALD is one of the toxic aldehydes generated during catecholamine metabolism, damage to PC12 cells was next evaluated (Fig. 2). The leakage of LDH was unchanged at concentrations of DOPALD up to 10 mM, although 100 mM DOPALD significantly induced LDH leakage. These results indicated that the cytotoxicity of DOPALD was negligible at 10 mM at which significant DA release was demonstrated. In the absence of extracellular Ca 21 , 56 mM KClinduced DA release was markedly suppressed, while 10 mM DOPALD-induced DA release was not affected (Table 1). In accordance with these findings, the L-type Ca 21 channel blocker nifedipine significantly reduced high potassium-evoked DA release, while DA release elicited by DOPALD was not affected. Distinct from potassiumevoked DA release, DOPALD-induced DA release was therefore independent of extracellular Ca 21 . Short-term exposure to DOPALD evoked DA release from PC12 cells to a level similar to that induced by high potassium. This effect of DOPALD on DA release was not associated with its cytotoxicity against the cells, since no increase in LDH leakage was observed at DOPALD concentrations up to 10 mM. Mattermal et al. previously reported DOPALD-induced decreases in DA and 3,4dihydroxyphenylacetic acid (DOPAC) contents in striatal synaptosomes and differentiated PC12 cells [10]. Lamen-
97
Fig. 1. DOPALD induced DA release from PC12 cells (A), while 4hydroxy-2-nonenal (HNE) had no effect on DA release (B). PC12 cells were incubated with the indicated concentrations of DOPALD, KCl or HNE for 5 min. Each value represents the mean6S.E.M. obtained from three to six separate experiments. Data are expressed as percent of control. Basal DA release of control was 14.764.2% (A), and 15.566.0% (B) of total DA contents. *, P,0.05, compared with control value.
sdorf et al. recently reported negligible cytotoxic effects of 10 mM DOPALD on differentiated PC12 cells, in that little increase in LDH leakage was observed following exposure of the cells to DOPALD for 24 h [8]. Our findings and these previous observations indicated a novel function of DOPALD on DA release at concentrations lower than those that show apparent cytotoxicity. This DA-releasing action of DOPALD appeared to be specific, and was not common to other aldehydes such as HNE, an endogenous aldehyde generated during lipid peroxidation. HNE is known to induce dose-dependent inhibition of dopamine uptake in rat striatal synaptosomes, potentially leading to decreased turnover of dopamine [11,12]. Furthermore, HNE-induced apoptosis was reported in PC12 cells [7,17]. Similar cytotoxicity of
98
T. Hashimoto, C. Yabe-Nishimura / Brain Research 931 (2002) 96 – 99
Table 1 DOPALD-induced DA release from PC 12 cells was Ca 21 -independent DA release (% of control)
Control KCl (56 mM) DOPALD (10 mM)
Ca 21 (1)
Ca 21 (2)
Nifedipine (10 nM)
100618.7 285646.2** 306.5645.9**
N.D. 96.2618.5[[ 385.7677.8
83.768.0 187.7625.3[ 305.4697.9
PC12 cells were incubated with each drug for 5 min in the presence or absence of Ca 21 . Each value represents the mean6S.E.M. obtained from four to ten separate experiments. N.D., not determined. **, P,0.01, compared with control; [, P,0.05, [[, P,0.01, compared with Ca 21 (1).
DOPALD was reported in DA-containing terminals of neostriatal synaptosomes, in PC12 cells, and mesencephalic cell cultures [10]. While direct neurotoxic effects of DOPALD have been implicated in the development of degenerative diseases such as parkinsonism, the present study provided the first evidence for the possible involvement of DOPALD-induced DA release in the pathogenesis of neuronal degeneration. DOPALD-induced DA release from PC 12 cells was independent of extracellular Ca 21 and was not inhibited by nifedipine, a voltage-sensitive calcium channel antagonist. Perturbation of DA transport into synaptic vesicles is among the mechanisms that could lead to Ca 21 -independent DA release. When direct effects of DOPALD on DA transport were tested, up to 10 mM of DOPALD did not affect [ 3 H]DA uptake into PC12 cells (data not shown). Ca 21 -independent neurotransmitter release from PC12 cells was previously demonstrated in cells stimulated by diacylglycerol, an activator of protein kinase C [14]. Recently, the excitatory neurotoxin, pardaxin was also reported to induce DA release from PC 12 cells in a Ca 21 -independent manner, and the involvement of the
lipoxygenase pathway was suggested [1]. There is as yet no direct evidence that DOPALD-induced DA release is mediated by protein kinase C or arachidonic acid cascade. However, these possibilities remain under consideration. MPTP, after conversion by MAO-B into the toxic metabolite 1-methyl-4-phenylpyridine (MPP 1 ), elicits cell death in dopaminergic neurons. Recent studies in PC12 cells indicate that MPP 1 interferes with complex I of the electron transport chains and impairs energy metabolism, leading to cell damage [4,16]. On the other hand, MPP 1 is a potent DA-releasing agent, and nonenzymatic autoxidation of DA leads to formation of hydroxy radicals. In fact, determination of the in vivo generation of free radicals by microdialysis indicated the involvement of hydroxy radicals derived from DA in MPTP-induced neurotoxicity [3,13]. Excess DA release induced by DOPALD and ensuing augmentation of oxidative stress may therefore be one of the mechanisms underlying neuronal degeneration leading to parkinsonism.
Acknowledgements This work was supported by a Grant-in-aid-for Scientific Research 11680760 from the Ministry of Education, Science, and Culture, Japan.
References
Fig. 2. LDH leakage from PC12 cells was unchanged by DOPALD treatment at concentrations up to 10 mM. PC12 cells were incubated with DOPALD or KCl for 5 min. Each column represents the mean6S.E.M. obtained from four separate experiments. **, P,0.01, compared with control value.
[1] S. Abu-Raya, E.B. Shilderman, P.I. Lelkes, V. Tremboler, E. Shohami, Y. Gutman, P. Lazarovici, Characterization of pardaxininduced dopamine release from pheochromocytoma cells: role of calcium and eicosanoids, J. Pharmacol. Exp.Ther. 288 (1999) 399– 406. [2] W.J. Burke, C.A. Schmitt, K.N. Gillespie, S.W. Li, Norepinephrine transmitter metabolite is a selective cell death messenger in differentiated rat pheochromocytoma cells, Brain Res. 722 (1996) 232–235. [3] C.C. Chiueh, R.-M. Wu, K.P. Mohanakunar, L.M. Sternberger, G. Krushna, T. Obata, D.L. Murphy, In vivo generation of hydroxyradicals and MPTP-induced dopaminergic toxicity in the basal ganglia, Ann. NY Acad. Sci. 738 (1994) 25–36. [4] C. Fonck, M. Baudry, Toxic effects of MPP(1) and MPTP in PC12 cells independent of reactive oxygen species formation, Brain Res. 905 (2001) 199–206. [5] R.J. Gay, R.B. McComb, G.N. Bowers, Optimum reaction con-
T. Hashimoto, C. Yabe-Nishimura / Brain Research 931 (2002) 96 – 99
[6]
[7]
[8]
[9]
[10]
[11]
[12]
ditions for human lactate dehydrogenase isozymes as they affect total lactate dehydrogenase activity, Clin. Chem. 14 (1968) 740– 753. T. Hashimoto, T. Ueha, T. Kuriyama, M. Katsura, K. Kuriyama, Acetaldehyde-induced alterations in metabolism of monoamines in mouse brain, Alcohol Alcoholism 24 (1982) 91–99. I. Kruman, A.J. Bruce-Keller, D. Bredsen, G. Waeg, M.P. Mattson, Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis, J. Neurosci. 17 (1997) 5089–5100. I. Lamensdorf, G. Eisenhofer, J.H. White, Y. Hayakawa, K. Kirk, I.J. Kopin, Metabolic stress in PC12 cells induced the formation of the endogenous dopaminergic neurotoxin, 3,4-dihydroxyphenylacetaldehyde, J. Neurosci. Res. 60 (2000) 552–558. M.B. Mattermal, H.D. Chung, R. Strong, F.-F. Hsu, Confirmation of a dopamine metabolite in parkinsonian brain tissue by a gas chromatography–mass spectrometry, J. Chromatogr. 614 (1993) 205–212. M.B. Mattermal, J.H. Haring, H.D. Chung, G. Raghu, R. Strong, An endogenous dopamine neurotoxin: implication for Parkinson’s disease, Neurodegeneration 4 (1995) 271–281. P.F. Mopel, L. Barrier, B. Fauconneau, A. Piriou, F. Huguet, Origin of 4-hydroxynonenal incubation-induced inhibition of dopamine transporter and Na 1 / K 1 adenosine triphosphate in rat striatal synaptosomes, Neurosci. Lett. 277 (1999) 91–94. P. Mopel, C. Tallineau, R. Pontcharraud, A. Piriou, F. Huguet, Effects of 4-hydroxynonenal, a lipid peroxidation product, on
[13]
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[16]
[17]
[18]
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