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Lysophosphatidylcholine Decreases Locomotor Activities and Dopamine Turnover Rate in Rats
NeuroToxicology 26 (2005) 27–38
Lysophosphatidylcholine Decreases Locomotor Activities and Dopamine Turnover Rate in Rats Eun-Sook Y. Lee*, Karam F.A. Soliman, Clivel G. Charlton1 College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA Received 23 August 2003; accepted 21 July 2004 Available online 11 September 2004
Abstract Lysophosphatidylcholine (lyso-PTC), a secondary product of S-adenosylmethionine (SAM)-dependent phosphatidylethanolamine (PTE) methylation, is a potent cytotoxin and might be involved in the pathogenesis of Parkinson’s disease (PD). Our previous studies showed that the injection of SAM into the brain caused PD-like changes in rodents. Moreover, 1-methyl-4-phenylpyridinium (MPP+), a Parkinsonism-inducing agent, increased lyso-PTC formation via the stimulation of PTE methylation pathway. These results indicate a possible role of lyso-PTC in the PD-like changes seen following the injection of SAM or MPP+. In the present study, lyso-PTC was injected into the lateral ventricle of rats and locomotor activities and the biogenic amine levels were measured to evaluate the effects of lyso-PTC on the dopaminergic system. Quinacrine, a phospholipase A2 (PLA2) inhibitor, was employed to determine its protective effect on SAM-induced PDlike changes by the inhibition of lyso-PTC formation. The results showed that 1 h after the injection, 0.4 and 0.8 mmol of lyso-PTC increased striatal dopamine (DA) by 20 and 24%, decreased 3,4-dihydroxyphenylacetic acid (DOPAC) by 37 and 45% and decreased homovanilic acid (HVA) by 24 and 13%, respectively. Consequently, dopamine turnover rate, (DOPAC + HVA)/DA, was significantly reduced by 44 and 48% in the rat striatum. Meanwhile, the administration of 0.4 or 0.8 mmol of lyso-PTC decreased movement time by 52 and 63%, total distance by 44 and 48% and the number of movements by 43 and 64%, respectively. Quinacrine attenuated SAM-induced hypokinesia without affecting SAM metabolism prior to its action on rat brain. The results obtained indicate that the hypokinesia observed following the administration of lyso-PTC might be related to the decline in DA turnover in the striatum in response to lyso-PTC exposure. The present study suggests that inhibitory effects of lyso-PTC on dopaminergic neurotransmission is one of the contributing factors in SAM and MPP+-induced PD-like changes.
# 2004 Elsevier Inc. All rights reserved. Keywords: Lysophosphatidylcholine; Methylation; S-adenosylmethionine; Parkinson’s disease; Dopamine; Locomotor activity
INTRODUCTION The symptoms associated with Parkinson’s disease (PD) include rigidity, resting tremors and hypokinesia, caused by the degeneration of dopaminergic neurons in the nigrostriatal pathway (Selby, 1975). The major biochemical change is dopamine (DA) depletion * Corresponding author. Tel.: +1 850 599 8445; fax: +1 850 412 7261. E-mail address: [email protected] (E.Y. Lee). 1 Current address: Department of Pharmacology, Meharry Medical College, Nashville, TN, USA.
(Hornykiewicz, 1966), but serotonin (5-HT) and norepinephrine (NE) are also depleted in advanced PD (Scatton et al., 1983; Tohgi et al., 1993). Although the etiology of PD is still unknown, two endogenous classes of compounds, b-carbolines and isoquinoline derivatives which have been suggested as causative factors for PD, cause toxic effects via methylation (Collins et al., 1992; McNaught et al., 1996; Gearhart et al., 1997; Matsubara et al., 1998). It is of interest to note that high N-methylation activity was found in PD patients (Williams et al., 1993; Naoi et al., 1994; Aoyama et al., 2000, 2001) and S-adenosylmethionine (SAM), an endogenous methyl donor, can induce
0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2004.07.009
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PD-like symptoms (Crowell et al., 1993), suggesting a possible relationship between hypermethylation and PD. Excess methylation has been proposed as a possible causing factor of PD (Charlton and Mack, 1994; Lee et al., 2004a) and phospholipid methylation is believed to play an important role in the excessive methylationinduced Parkinsonism. Phosphatidylcholine (PTC), a major product of SAM-dependent phospholipid methylation, is synthesized from phosphatidylethanolamine (PTE) by phosphatidylethanolamine Nmethyl transferase (2.1.1.17, PENMT) and readily hydrolyzes to lysophosphatidylcholine (lyso-PTC) by phospholipase A2 (3.1.1.4, PLA2) (Fig. 1). LysoPTC is a normal component of tissues, representing 5– 20% of total phospholipids and 0.2–0.25 mM concentrations in mammalian plasma (Thies et al., 1992). Although the cellular mechanisms of lyso-PTCmediated action are unclear, it has been recognized as an important cell signaling agent (Shier et al., 1976; Yuan et al., 1996; Ikeuchi et al., 1997). Moreover, lysoPTC was identified as a ligand for the immunoregulatory receptor G2A (Kabarowski et al., 2001; Xu, 2002). As a result of various actions of lyso-PTC in different cellular processes, the elevated level of lysoPTC has been suggested to be involved in various pathological conditions such as atherosclerosis, inflammation (Quinn et al., 1988), gastric ulcerogen-
esis (Maksem et al., 1984), and ischemia (Magishi et al., 1996). In the nervous system, lyso-PTC is involved in exocytosis (Poole et al., 1970) and in reversibly arresting exocytosis at a stage between triggering and membrane merger (Vogel et al., 1993). PLA2, the responsible enzyme for the formation of lyso-PTC, is associated with the regulation of the degranulation process leading to a release of neurotransmitters in the exocytosis process (Matsuzawa et al., 1996) and the fusion process of synaptic vesicles with the presynaptic membranes (Nishio et al., 1996). Previously we have shown that lyso-PTC inhibited dopamine D1 and D2 receptor binding activities (Lee et al., 2004b), indicating that lyso-PTC might be involved in modulating neural transmission. Classical PLA2 inhibitors such as mepacrine and p-bromophenacyl bromide are reported to inhibit neurotransmitter release from neuroendocrine cells (Moskowitz et al., 1982; Bradford et al., 1983). Lyso-PTC also exerts detergent-like effects (Slomiany et al., 1981; Maksem et al., 1984; Nishizuka, 1995) and a potent cytolytic property, which may damage neuronal cells. Quinacrine has frequently been used as an inhibitor of PLA2 for its attenuation of stimulated release of arachidonic acid in different cell lines (Valenzuela et al., 1992). It has been widely studied on signal transduction, blood clotting, ischemia, hypertension, and
Fig. 1. Schematic diagram of the formation of phosphatidylcholine (PTC) and lysophosphatidylcholine (lyso-PTC) in PENMT pathway. SAM is a methyl donor and a substrate for PENMT; MPP+ increases PTE methylation by stimulating PENMT. Both SAM and MPP+ increase PTC production which, in turn, serves as a substrate for PLA2 to increase the formation of lyso-PTC. Quinacrine is an inhibitor of PLA2 resulting in the reduction of lyso-PTC formation. PTE: phosphatidylethanolamine; SAM: S-adenosylmethionine; SAH, S-adenosylhomocysteine; PENMT, phosphatidylethanolamine N-methyltransferases; MPP+, 1methyl-4-phenyl-pyridinium; PTC, phosphatidylcholine; PLA2, phospholipase A2; lyso-PTC, lysophosphatidylcholine; AA, arachidonic acid.
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inflammation. Quinacrine decreases experimentallyinduced myocardial necrosis in rats and dogs (Chiariello et al., 1987; Chiariello et al., 1990) and reduces infarct size in rats after transient middle cerebral artery occlusion (Estevez and Phillis, 1997). It has also shown to inhibit the Torpedo nicotinic acetylcholine receptor (Johnson and Ayres, 1996; Arias, 1997). Quinacrine prevented the desensitization of b-adrenergic receptors associated with increment of PTC degradation to lysoPTC and arachidonic acid (AA) (Torda et al., 1981) and inhibited phospholipid methylation in human blood monocytes (Hurst et al., 1986). Moreover, quinacrine has been shown to have a protective effect against MPTP and 6-OHDA neurotoxicities in animal models (Tariq et al., 2001). It is well established that methylation is increased during aging and PD is an age-related disorder (Sellinger et al., 1988). The administration of 1-methyl-4-phenyltetrahydropyridine (MPTP) causes Parkinsonism (Langston et al., 1983) via its metabolite 1-methyl-4-phenylpyridinium (MPP+) which in turn increases phospholipid methylation by enhancing PENMT activity (Lee and Charlton, 2001). SAM, the biological methyl donor, also increases the formation of lyso-PTC through the PTE methylation pathway (Fig. 1). Since there are similarities between the effects of SAM and those of MPTP on impairing motor functions and damaging dopaminergic neurons (Crowell et al., 1993), their abilities to increase lyso-PTC suggest that lyso-PTC may play an important role in the actions of MPP+ and SAM. Coinciding with the major role of the striatum in PD, we found that PENMT activity is highest in the striatum and that the striatum manifested the highest rate in the formation of lyso-PTC following the methylation of PTE by SAM (Lee and Charlton, 2001). Therefore, the present study was designed to determine the effects of lyso-PTC administration on locomotor activities as well as biogenic amine levels in the rat and to determine the role of lyso-PTC in SAMinduced PD-like changes by the inhibition of lyso-PTC formation using quinacrine.
MATERIALS AND METHODS Materials Lysophosphatidylcholine (lyso-PTC), MPP+ I, dopamine HCl (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT), SAM, quinacrine and 5-hydroxyindoleacetic acid (5HIAA), were obtained from Sigma Chemical Company
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(St. Louis, MO, USA). [3H-methyl]SAM (82.4 Ci/mmol) was purchased from New England Nuclear (Boston, MA, USA). Other chemicals and agents were purchased from Fisher Scientific Company (Pittsburgh, PA, USA). Animals Male Sprague–Dawley rats weighing 250–350 g were purchased from Harlan (Indianapolis, IN, USA) and acclimatized for at least one week in a room with 12 h-light and 12 h-dark cycles, 22 1 8C. Water and food were supplied ad libitum. PENMT (Phosphatidylethanolamine N-methyltransferases) Assay PENMT assay described previously (Hirata et al., 1978) was used with some modifications. Rats were sacrificed by decapitation, followed by homogenizing the rat brain or liver tissues in five volume of 50 mM Tris–HCl buffer at pH 7.5. Aliquots of rat liver tissue homogenate (0.2–0.5 mg protein) were incubated with various concentrations of SAM or MPP+ in the medium containing 50 mM Tris–HCl buffer, 15 mM Mg2+ and 20 mM EDTA at pH 7.5 in a total volume of 100 ml. The reaction was initiated with the addition of [3Hmethyl]SAM as a tracer and continued for 1 h at 37 8C. The reaction was terminated by the addition of 3 ml of chloroform:methanol:HCl (100:50:1, v/v/v). Lipids were extracted for 10 min and washed twice with 2 ml of 0.1 M KCl in 50% methanol, followed by thin layer chromatography (TLC) analysis. Thin Layer Chromatography (TLC) Analysis The formation of lyso-PTC was identified by applying aliquots of the organic phase to a silica gel TLC plate (LK5D Silica Gel). The mobile phase was chloroform:methanol:water (65:25:4, v/v/v). Standard lysoPTC dissolved in chloroform: methanol (2:1) was co-chromatographed. The chromatograms were then visualized by exposing the plate to iodine vapor. Spots that matched the standards were scraped off the plates and solublized in scintillation cocktail and the radioactivity was measured in a liquid scintillation counter (Beckman, LS 6500). Cannulation and Drug Treatments in Rats Rats were cannulated prior to the injection of the different compounds (lyso-PTC, SAM or quinacrine) into the lateral ventricles of rat brains as previously
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described (Charlton and Mack, 1994). Male Sprague– Dawley rats weighing 250–350 g were anesthetized with chloral hydrate (400 mg/kg, i.p.). A 22 gauge stainless cannula was positioned at 1.5 mm lateral and 0.6 mm caudal with reference to the bregma from which it extended to the lateral ventricles. The rats were allowed to recover for 3 days before the experiments. Injections were made through polyethylene tube (PE 20) which was attached to a 10 ml Hamilton syringe. For lyso-PTC study, lyso-PTC was dissolved in phosphate-buffered saline (PBS) at pH 7.4 and PBS solution was used as a control. The rats were injected with 5 ml of PBS or lyso-PTC (0.4 or 0.8 mmol) by i.c.v. route single time for acute effects or once a day for 3 days for subacute effects. For SAM and quinacrine study, SAM or quinacrine was dissolved in PBS and the injection amount of each compound was 0.8 mmole in 2.5 ml. Four groups (PBS and PBS, PBS and quinacrine, PBS and SAM, or quinacrine and SAM) were prepared and each rat was administered with two injections at 5 min interval between the injections. Measurement of Locomotor Activities After the last injections, rats were placed in the locomotor activity monitor and the measurement was started 3 min after placed in the monitor. The changes in motor activity of the animals were measured in an Activity Monitor (Degiscan Instruments Inc., Columbus, OH, USA). The measurements were performed in a quiet isolated place with a dim light. Movement time (MT), total distance (TD) and the number of movements (NM) were determined. The locomotor activities were determined for 60 min post-injection. HPLC Analysis The measurement for the levels of DA, DOPAC, HVA, 5-HT, and 5-HIAA were performed by high performance liquid chromatography (HPLC) combined with electrochemical detection (EC), and minor modification of a previously described procedure (Lowry et al., 1996). Briefly, after the dissection of the rat brains into the striatum, hippocampus and cortex, brain tissues were homogenized in three volumes (w/v) of ice-cold 0.4 M perchloric acid and centrifuged at 9000 g for 20 min at 4 8C. Supernatant was filtered (0.45 mm) and 30 ml of the filtered supernatant was injected into the HPLC system. A stainless steel hypersil ODS C-18 reverse-phase column (5 mm, 250 mm 4.6 mm, Whatman EQC) was connected to
the HPLC-EC system. A dual electrochemical detector (Coulochem II, ESA) was set with 350 mV at the guard cell and E1 = 50 mV, E2 = +700 mV at the analytical cell. The mobile phase consisted of 0.1 M sodium acetate, 60 mM citric acid, 0.6 mM octanesulfonic acid sodium salt, 0.5 mM disodium EDTA, in 15% methanol in water, pH 3.5 and pumped at a rate of 1.5 ml/min. For the measurement of SAM and SAH, the rat striatal regions were prepared for HPLC analysis by homogenizing tissues in threevolumes of 0.4 M perchloric acid followed by centrifugation of samples at 9000 g for 20 min at 4 8C and filtration of the supernatant of tissue samples (0.45 mm). The HPLC–UV system consisted of Shimazu HPLC with a hypersil ODS C-18 reverse-phase column (5 mm, 250 mm 4.6 mm, Whatman EQC) with a UVof 260 nm and a flow rate of 1.5 ml/min. The mobile phase was 75 mM NaH2PO4H2O, 4.7 mM 1-octanesulfonic acid sodium salt, 10 mM EDTA and 10% acetonitrile adjusting the final pH to 3.0 with phosphoric acid. Statistical Analysis The mean and standard error of the mean (S.E.M.) were determined for each set of data. Data analysis was performed using one-way analysis of variance (ANOVA) followed by post-hoc Newman–Keuls test to evaluate differences between the different groups. A probability of 0.05 or less was considered a significant difference.
RESULTS A Parallel Effect of SAM and MPP+ on the Formation of Lyso-PTC PTE is methylated by PENMT using SAM as a methyl donor to produce PTC which serves as a substrate for PLA2. Subsequently, lyso-PTC formation is increased when this methylation pathway is accelerated. MPP+ increased phospholipid methylation by stimulating PENMT activity. SAM and MPP+ revealed a parallel action on phospholipid methylation pathway. As shown in Table 1, MPP+ increased the formation of lyso-PTC significantly in a concentration-dependent manner in rat liver tissue (F4,10 = 15.71; p = 0.0003). Effect of Lyso-PTC on Locomotor Activities in Rats Since both SAM and MPP+ increased the formation of lyso-PTC significantly, the effects of lyso-PTC on
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Table 1 Effect of SAM and MPP+ on the formation of lyso-PTC in rat liver tissue A
B
SAM (mM)
Lyso-PTC (pmol/mg protein/h)
15 32 63 125 250
0.18 0.89 1.73 4.52 11.2
0.01 0.01 0.25 0.41* 1.78***
MPP+ (mM)
Lyso-PTC (pmol/mg protein/h)
0 0.3 1 3 10
2.17 2.60 4.41 5.40 7.63
0.36 0.38 0.59 0.76* 0.63***
Rat liver tissue homogenate was incubated with various concentrations of SAM with 0.28 mCi [3H-methyl]SAM as a tracer (A) or incubated with various concentrations of MPP+ in the presence of 100 mM SAM containing 0.55 mCi [3H-methyl]SAM as a tracer (B) for 1 h at 37 8C. The lipids were extracted and separated by thin layer chromatography. The values shown are the means S.E.M. for triplicate samples (N = 3). One-way ANOVA followed by post-hoc Newman–Keuls test was used for statistical analysis. (*) Indicates significant increases when compared to the control (*p < 0.05, ***p < 0.001).
animal behavior were studied to determine the role of lyso-PTC in SAM or MPP+-induced PD-like symptoms. The injection of lyso-PTC into the lateral ventricle of rat brain caused severe hypokinesia as indicated by the decrease MT, TD and NM (Fig. 3). Following the single injection of 0.4 and 0.8 mmol of lyso-PTC, MT was significantly decreased by 52 and 63% compared to the control group (Fig. 2A, F2,12 = 3.915; p = 0.0491). The repeated injections of lysoPTC showed further significant decreases in locomotor activities following 3 days of treatment (Fig. 3A–C). The administration of 0.4 and 0.8 mmol of lyso-PTC decreased MT by 48 and 79% (F2,12 = 3.617; p = 0.0326), TD by 51 and 89% (F2,12 = 39.22; p < 0.0001), and NM by 32 and 66% (F2,12 = 3.920; p = 0.0490). Effects of Lyso-PTC on Dopaminergic Nervous System In this experiment, the effect of lyso-PTC on the levels of DA and its metabolites was investigated to determine whether lyso-PTC alters dopaminergic neurotransmission. As shown in Table 2, the content of DA in the striatum was increased 1 h after a single dose of 0.4 and 0.8 mmol of lyso-PTC by 20 and 24%, respectively (F2,12 = 4.894; p = 0.0279). However, these doses of lyso-PTC significantly decreased the levels of DOPAC by 37 and 45%, respectively (F2,12 = 14.04; p = 0.0007). HVA level was also decreased by 24 and 13%, respectively. Consequently, the turnover rate of dopamine which was represented as (DOPAC + HVA)/ DA was reduced by 44 and 48% by the single doses of lyso-PTC (F2,12 = 18.50; p = 0.0002). Following three daily injections of 0.4 or 0.8 mmol of lyso-PTC, there was a slight decrease in the content of DA, whereas DOPAC level was decreased by 37 and 30% (F2,12 = 17.84; p = 0.0003). The consequent decrements in the ratio of (DOPAC + HVA)/DA were 22 and 26%,
respectively (F2,12 = 5.167; p = 0.0241). The results reveal that lyso-PTC significantly decreases the turnover rate of DA neurotransmission in the rat striatal region. In contrast to the striatum, the effects of lysoPTC on the content of DA, DOPAC and HVA in the cortex or hippocampus were not as significant as those on the striatum by single as well as repeated doses of lyso-PTC although some of the levels of HVA and DA showed statistically significant differences (Table 3). Effects of Lyso-PTC on Serotonergic System In this experiment the administration of a single dose of 0.4 and 0.8 mmol of lyso-PTC resulted in significant increases in the content of 5-HT in the striatum by 27 and 74%, respectively (Table 4, F2,12 = 17.62; p = 0.0003). 5-HIAA level was decreased by 32% at 0.8 mmol of lyso-PTC dose. Consequently, the 5-HIAA/5-HT value was reduced from 0.55 to 0.39 (29%) for the 0.4 mmol dose and 0.21 (62%) for the 0.8 mmol dose, indicating that acute administration of lyso-PTC reduced the turnover of 5-HT (F2,12 = 39.45; p < 0.0001). The repeated administration of lyso-PTC did not cause significant changes on the levels of 5-HT and 5-HIAA. In the cortex and hippocampus serotonergic activity was significantly decreased by the administration of lyso-PTC (Table 5), as shown by the reduction of both 5-HT and 5-HIAA. Single doses of lyso-PTC showed more significant reduction of serotonergic activity than repeated doses. Effect of Quinacrine on SAM-Induced PD-Like Symptoms in Rats: A Possible Role of Lyso-PTC in SAM-Induced Hypokinesia Quinacrine is a PLA2 inhibitor and decreases the formation of lyso-PTC. Quinacrine was used in the present study to determine the role of lyso-PTC in SAM-induced PD-like symptoms and whether quina-
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Fig. 2. Effects of single injection of lyso-PTC on locomotor activities in rats. After the rats were treated (i.c.v.) with 5 ml of PBS or lyso-PTC (0.4 or 0.8 mmol), rats were placed in the locomotor activity monitor after the injection and the measurement was started 3 min later. Lyso-PTC was dissolved in PBS. Movement times, total distances, and the numbers of movement were measured for 60 min post injection. Data are expressed as means S.E.M. (N = 5) and represent two experiments. One-way ANOVA followed by post-hoc Newman–Keuls test was used for statistical analysis to compare different groups. (*) Indicates significantly different from the control (*p < 0.05).
Fig. 3. Effects of multiple injections of lyso-PTC on the locomotor activities in rats. Rats were injected (i.c.v.) with 5 ml of PBS or lysoPTC (0.4 or 0.8 mmol) once a day for 3 days. Movement times, total distances, and the numbers of movement were measured for 60 min post injection. Data are expressed as means S.E.M. (N = 5) and represent two experiments. One-way ANOVA followed by post-hoc Newman–Keuls test was used for statistical analysis to compare different groups. (*) Indicates significantly different from the control (*p < 0.05, **p < 0.01, *** p < 0.001).
crine attenuates the effects of SAM on PD-like symptoms. It was observed that SAM caused significant hypokinesia, by decreasing MT by 42%, TD by 34% (Fig. 4, third bar). The group of SAM co-administered with quinacrine showed a significant attenuation
effects on SAM-induced hypokinesia (F3,26 = 3.506, p = 0.0315 for MT; F3,26 = 3.371, p = 0.0358 for TD; F3,26 = 3.050, p = 0.0489 for NM; Fig. 4, fourth bar). The attenuation effects of the SAM/quinacrine group compared to the SAM group for MT, TD and NM were
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Table 2 Effect of lyso-PTC on levels of DA, DOPAC and HVA and (DOPAC + HVA)/DA in the rat striatum (ng/100 mg tissue) Lyso-PTC
DOPAC
HVA
(DOPAC + HVA)/DA
Acute (mmol, i.c.v., 1 h) 0 763.4 63.4 0.4 955.5 26.6* 0.8 1,000 43.9*
DA
97.0 8.8 61.2 5.2# 53.8 3.1#
75.9 5.0 58.0 2.3# 66.1 6.7
0.23 0.02 0.13 0.01 0.12 0.01
Subacute (mmol, i.c.v., 3 days) 0 720.6 26.9 0.4 667.0 26.6 0.8 661.6 5.4
104.5 9.0 66.2 3.1# 72.8 5.4#
62.3 4.0 54.1 2.1 58.7 3.3
0.23 0.02 0.18 0.01 0.17 0.01
Rats were treated (i.c.v.) with 5 ml of PBS or two different doses (0.4 or 0.8 mmol) of lyso-PTC dissolved in 5 ml PBS for 1 h or once a day for 3 days and sacrificed 1 h after the single injection or 24 h after the last injection. Rat striatal regions were dissected out and analyzed by HPLC. Data are expressed as means S.E.M. (N = 5). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis to compare different groups. (*) Indicates significantly increased from the control (p < 0.05). (#) Indicates significantly decreased from the control (p < 0.05).
Table 3 Effect of lyso-PTC on the content of DA, DOPAC, HVA in the rat cortex and hippocampus Lyso-PTC
Cortex (ng/100 mg tissue) DA
Hippocampus (ng/100 mg tissue)
DOPAC
HVA
DA
DOPAC
HVA
Acute (mmol, i.c.v., 1 h) 0 34.7 4.8 0.4 25.1 2.9 0.8 26.4 8.3
19.0 1.2 10.8 8.3 15.4 1.5
12.4 1.2 11.0 1.1 9.2 1.7*
3.4 0.4 3.9 0.6 2.2 0.2*
6.8 0.6 5.5 1.0 6.6 0.7
4.9 0.3 4.1 0.6 3.2 0.2*
Subacute (mmol, i.c.v., 3 days) 0 36.3 2.6 0.4 38.7 1.9 0.8 34.5 4.3
20.5 2.5 22.3 2.0 21.8 2.5
11.7 1.1 14.6 1.0 12.5 2.5
5.2 0.3 5.5 0.4 5.2 0.5
8.7 1.3 7.2 0.8 6.1 0.7
5.1 0.8 3.6 0.3 3.7 0.5
After i.c.v. injection with 5 ml of PBS or lyso-PTC (0.4 or 0.8 mmol), rats were sacrificed 1 h after the single injection or 24 h after the last injection of three daily consecutive treatments. Rat cortex and hippocampus regions were dissected out and DA, DOPAC and HVA were analyzed by HPLC. Data are expressed as means S.E.M. (N = 5). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis to compare different groups. (*) Indicates significantly different from the control (p < 0.05).
Table 4 Effect of lyso-PTC on content of 5-HT and 5-HIAA in the rat striatum (ng/100 mg tissue) Lyso-PTC
5-HT
Table 5 Effect of lyso-PTC on the content of 5-HT and 5-HIAA in the rat brain cortex and hippocampus Lyso-PTC Cortex (ng/100 mg tissue) Hippocampus (ng/100 mg tissue) 5HT
5HIAA
5HT
5HIAA
5-HIAA
5-HIAA/5-HT
Acute (mmol, i.c.v.,1 h) 0 61.4 2.6 0.4 78.0 2.8* 0.8 106.9 8.7*
33.7 1.0 30.7 2.1 22.9 1.9
0.55 0.03 0.39 0.03* 0.21 0.02*
Acute (mmol, i.c.v., 1 h) 0 47.9 2.4 12.5 0.5 30.7 1.8 20.1 1.2 0.4 39.1 5.7 9.5 1.1* 26.9 1.7 13.0 0.3* * 0.8 35.9 2.5 10.0 1.2 23.9 1.4* 11.2 1.8*
Subacute (mmol, i.c.v., 3 days) 0 52.8 4.3 0.4 49.1 3.2 0.8 39.9 3.5
35.4 4.2 36.0 7.7 42.4 10.4
0.67 0.11 0.73 0.16 1.06 0.36
Subacute (mmol, i.c.v., 3 0 46.8 4.3 0.4 43.7 2.6 0.8 36.7 s 2.4
Cannulated rats were treated (i.c.v.) with 5 ml PBS or lyso-PTC (0.4 or 0.8 mmol) for 1 h or once a day for three days and sacrificed 1 h after the single injection and 24 h after the last injection for three daily injections. Rat striatal regions were dissected out and 5-HT and 5-HIAA were analyzed by HPLC. Data are expressed as means S.E.M. (N = 5). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis to compare different groups. (*) Indicates that the group is significantly different from the PBS control group (p < 0.05).
days) 13.0 2.2 14.3 1.8 9.6 0.6
32.4 3.2 33.4 2.5 33.7 3.2
21.8 4.6 25.2 5.1 37.9 8.1
Rats were treated (i.c.v.) with 5 ml PBS or lyso-PTC (0.4 or 0.8 mmol) and sacrificed 1 h after the single injection or 24 h after the last injection for three daily treatment groups. Cortex and hippocampus regions were dissected out and 5-HT and 5-HIAA were analyzed by HPLC. Data are expressed as means S.E.M. (N = 5). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis to compare different groups. (*) Indicates significantly different from the control (p < 0.05).
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PBS nor quinacrine treated groups showed tremors. The SAM treated group showed severe tremors 5– 7 min after the injection. The group of SAM co-administered with quinacrine showed weak tremors about 10 min after the injection, indicating that quinacrine decreased the intensity of the SAM-induced tremors. One hour after the injections for each group, rats were sacrificed and brain striatal regions were dissected. The striata were homogenized and prepared for the HPLC analysis. Since SAM is a universal methyl donor and could be utilized in the metabolism of exogenous compounds, SAM and S-adenosylhomocysteine (SAH) levels were measured to eliminate the possibility that quinacrine might influence on SAM metabolism and reduce the availability of SAM prior to inducing PD-like changes. As shown in Fig. 5, SAM administrations significantly elevated the levels of SAM and SAH (F3,26 = 18.39, p < 0.0001 for SAM; F3,26 = 11.63, p < 0.0001 for SAH). However,
Fig. 4. Effect of quinacrine on SAM-induced hypokinesia in rats. SAM or quinacrine was dissolved in PBS and the injection amount of each compound was 0.8 mmole in 2.5 ml. Four groups (PBS and PBS, PBS and quinacrine, PBS and SAM, or quinacrine and SAM) were prepared and administered (i.c.v.) with two preparations at 5 min interval between injections as indicated in Materials and Methods. Movement time, total distances and the number of movements were measured for 60 min post injection in different groups of rats. Data are expressed as means S.E.M. (N = 6 for control group, N = 8 for the treated groups). Two tailed Student’s t-test was used for statistical analysis to compare corresponding groups. (#) Indicates significantly decreased from the PBS control group (#p < 0.05). (*) Indicates significantly increased from the SAM treated group (*p < 0.05, ** p < 0.01).
significant at p < 0.05. The quinacrine treated group did not show any significant changes on the locomotor activity compared to the control group. Whole body tremors induced in each group were also observed (data not shown) since SAM induces severe whole body tremors and resulting in abnormal posture to control the body poses in rats (Crowell et al., 1993). Neither the
Fig. 5. SAM and SAH levels in rat brain striatum 1 h after the injections (i.c.v.) of quinacrine, SAM, or combination of quinacrine and SAM. Rats were sacrificed 1 h after the treatment and rat brain striatal regions were dissected, homogenized, and prepared for HPLC analysis to measure SAM and SAH levels as described in the Method section. Data are expressed as means S.E.M. (N = 6 for the control group, N = 8 for the treated groups). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis of group comparison. (*) Indicates significantly increased from the SAM treated group (**p < 0.01, ***p < 0.001).
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quinacrine did not affect the level of SAM or SAH (p > 0.05 for SAM or SAH), indicating that quinacrine does not interact with SAM prior to its action.
DISCUSSION The present study demonstrates that lyso-PTC administration (i.c.v.) into the rat brain caused hypokinesia, which was accompanied with a decrease in DA turnover and an apparent reduction in dopaminergic neurotransmission. The (DOPAC + HVA)/DA ratio is frequently used as an index of dopaminergic activity (Lavielle et al., 1979). Reductions in dopaminergic neuronal activity and hypokinesia are parallel physiological functions that also represent the major symptoms of PD. It suggests, therefore, that lyso-PTC, a secondary product that was increased by excessive methylation, might play an important role in SAMinduced PD-like changes in rodents. Interestingly, the effects of lyso-PTC on the dopaminergic system occur most predominantly in the striatum among three regions tested in the present study. This result may be relevant to the fact that impairment of striatum plays a key role in Parkinsonism, and also supported by the previous result that the striatum has the highest activity of phosphatidylethanolamine PENMT (Lee and Charlton, 2001). The acute administration of lyso-PTC decreased the turnover of 5-HT in the striatum, whereas successive doses showed a slight increase of 5-HT turnover, suggesting that the serotonergic nervous system is responding in a similar manner to the dopaminergic system and could also be affected by lyso-PTC administration. The effect of lyso-PTC on dopaminergic neurotransmission show a very similar pattern to the effects of MPP+ in which the acute administration of MPP+ induced the accumulation of DA and decreased its metabolites and caused Parkinsonism in long term treatments (Burns et al., 1983; Chiueh et al., 1984; Desole et al., 1993; Miele et al., 1995). The result that MPP+ increased the formation of lyso-PTC indicates that lyso-PTC might play a role in MPTP neurotoxicity. The acute administration of lyso-PTC appears to cause the accumulation of DA, and the reduction of the levels of DA metabolites, DOPAC and HVA, in the striatum. This initial occurrence by the single dose of lyso-PTC suggests that lyso-PTC may interfere with dopaminergic neurotransmission process such as DA release, binding to postsynaptic receptors, uptake or metabolism, resulting in retarding DA turnover and inducing
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bradykinesia in animals. All these results indicate that lyso-PTC which is increased from S-adenosylmethionine (SAM)-mediated excessive phospholipid methylation or MPP+ might play a role, at least in part, in their actions on the dopaminergic system. It has been reported that lyso-PTC decreased DA D2 binding activity in caudate microsomal membranes (Oliveira et al., 1984). In their study, the effects of phospholipase A2 (PLA2) and lyso-PTC as a product of PLA2 on dopamine receptor binding activity were tested to determine which substance, PLA2 or lysoPTC, is actually responsible for the inhibition of dopamine receptor binding activity. It was found that the phospholipid hydrolysis products (lyso-PTC) rather than PLA2 are directly involved in the alteration of dopamine receptor binding activity, implicating the potential role of lyso-PTC. The results from our laboratory showed that lyso-PTC decreased DA D1 and D2 binding activities in rat striatal membranes (Lee et al., 2004b) and increased DA release and inhibited DA uptake in PC12 cells, implicating that lyso-PTC indeed impedes several steps of DA neurotransmission in the striatum. Several published studies that lyso-PTC is involved in exocytosis (Vogel et al., 1993) and vesicle fusion during the process of neurotransmitter release (Martin and Ruysschaert, 1995) strongly supports the role of lyso-PTC on the neurotransmission of catecholaminergic neurons. The fact that the DA content and the formation of lyso-PTC are highest in the striatum compared to other regions in the brain may explain the susceptibility of dopaminergic neurons and the selective damage of the nigrostriatal pathways. The reduction of dopaminergic neurotransmission induced by lyso-PTC in the rat striatum might be closely related to the effect of lyso-PTC on the impairment of locomotor activities in rats. Lyso-PTC is considered as the most potent substance among the secondary products of phospholipid methylation to alter dopaminergic action and basal ganglia activity and, therefore, it was attempted to block SAM-induced PD-like changes by the inhibition of lyso-PTC formation using quinacrine. The data obtained in the present study showed that quinacrine alleviated SAM-induced impairment of locomotor activity as indicated by compensating the decreased values of movement parameters such as movement time, total distances and the number of movement. The results that SAM and SAH levels were elevated in the striatum after the injections of SAM indicate that the inhibitory effects of quinacrine on SAM actions were not by decreasing SAM levels directly. It has been reported that quinacrine protected MPTP or 6-hydroxy dopamine (6-OHDA)-
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induced Parkinsonism in rodents (Tariq et al., 2001), suggesting the possible role of lyso-PTC in Parkinsonism. Quinacrine is a lipophilic compound and crosses the blood–brain barrier (Dubin et al., 1982; O’Regan et al., 1995). This lipophilicity of quinacrine might be an important characteristic to be used systemically as a therapeutic agent. PD is an age-related disorder and methylation reactions increase in aging (Sellinger et al., 1988) in which lyso-PTC is elevated as a consequence of excessive methylation of PTE to PTC. This may lead to the slow failure of striatal dopaminergic neurotransmission and result in hypokinesia. Moreover, the potent detergentlike cytotoxic effect of lyso-PTC could damage the dopaminergic nerve endings and impair dopaminergic functions. In conclusion, lyso-PTC, which is one of the products resulting from excessive methylation and MPP+, caused hypokinesia and biochemical changes that resemble PD. These results provide additional evidence that hypermethylation and lyso-PTC may be involved in the pathogenesis of PD and the MPTP neurotoxicity. The current findings may suggest the therapeutic potential of quinacrine as a PLA2 inhibitor for the treatment of PD. ACKNOWLEDGMENTS The authors would like to thank Ms. X. Liu for her excellent laboratory assistance. This work was supported by grants received from the National Institutes of Health (RO1 28432, RR 03020, and GM 08111). REFERENCES Aoyama K, Matsubara K, Okada K, Fukushima S, Shimizu K, Yamaguchi S, Uezono T, Satomi M, Hayase N, Ohta S, Shiono H, Kobayashi S. N-methylation ability for azaheterocyclic amines is higher in Parkinson’s disease: nicotinamide loading test. J Neural Transm 2000;107:985–95. Aoyama K, Matsubara K, Kondo M, Murakawa Y, Suno M, Yamashita K, Yamaguchi S, Kobayashi S. Nicotinamide-N-methyltransferase is higher in the lumbar cerebrospinal fluid of patients with Parkinson’s disease. Neurosci Lett 2001;298:78–80. Arias HR. The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor. Biochim Biophys Acta 1997;1347:9–22. Burns RS, Chiueh CC, Markey SP, Ebert MH, Jacobowitz DM, Kopin IJ. A primate model of Parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Nat Acad Sci USA 1983;80:4546–50.
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Naoi M, Maruyama W, Niwa T, Nagatsu T. Novel toxins and Parkinson’s disease: N-methylation and oxidation as metabolic bioactivation of neurotoxin. J Neural Transm 1994;Suppl. 41:197–205. Nishio H, Takeuchi T, Hata F, Yagasaki O. Ca2+-independent fusion of synaptic vesicles with phospholipase A2-treated presynaptic membranes in vitro. Biochem J 1996;318:981–7. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 1995;9:484–96. Oliveira CR, Duarte EP, Carvalho AP. Effect of phospholipase digestion and lysophosphatidylcholine on dopamine receptor binding. J Neurochem 1984;43:455–65. O’Regan MH, Smith-Barbour M, Perkins LM, Phillis JW. Arachidonic acid and lysophosphatidylcholine modulate excitatory transmitter amino acid release from the rat cerebral cortex. Neurosci Lett 1995;193:85–8. Poole AR, Howell JI, Lucy JA. Lysolecithin and cell fusion. Nature 1970;227:810–4. Quinn MT, Parthasarathy S, Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis. Proc Natl Acad Sci USA 1988;85:2805–9. Scatton B, Javoy-Agid F, Rouquier L, Dubois B, Agid Y. Reduction of cortical dopamine, noradrenalin, serotonin and their metabolites in Parkinson’s disease. Brain Res 1983;275: 321–8. Selby G. Parkinson’s disease. In: Vinken PJ, editor. Handbook of clinical neurology. Disease of the basal ganglia. 1975;173– 211. Sellinger OZ, Kramer CM, Conger A, Duboff GS. The carboxylmethylation of cerebral membrane-bound proteins increases with age. Mech Ageing Dev 1988;43:161–73. Shier WT, Baldwin JH, Nilsen-Hamilton M, Hamilton RT, Thanassi NM. Regulation of guanylate and adenylate cyclase activities by lysolecithin. Proc Natl Acad Sci USA 1976;73: 1586–90. Slomiany BL, Jerzy-Glass GB, Kojima K, Banas-Gruszka Z, Slomiany A. Effect of lysolecithin on the constituents of gastric mucus. In: Chantler N, Elder JB, Elstein M., editors. International symposium on mucus in health and disease; 1981. p. 163– 74. Tariq M, Khan HA, Al Moutaery K, Al Deeb S. Protective effect of quinacrine on striatal dopamine levels in 6-OHDA and MPTP models of Parkinsonism in rodents. Brain Res 2001;54:77–82. Thies F, Delachambre MC, Bentejac M, Lagarde M, Lecerf J. Unsaturated fatty acids esterified in 2-acyl-l-lysophosphatidylcholine bound to albumin are more efficiently taken up by the young rat brain than the un-esterified form. J Neurochem 1992;59:1110–6. Tohgi H, Abe T, Takahashi S, Takahashi J, Hamato H. Concentrations of serotonin and its related substances in the cerebrospinal fluid of Parkinsonian patients and their relations to the severity of symptoms. Neurosci Lett 1993;150:71–4. Torda T, Yamaguchi I, Hirata F, Kopin IJ, Axelrod J. Mepacrine treatment prevents immobilization-induced desensitization of beta-adrenergic receptors in rat hypothalamus and brain stem. Brain Res 1981;205:441–4. Valenzuela CF, Kerr JA, Johnson DA. Quinacrine binds to the lipidprotein interface of the Torpedo acetylcholine receptor: a fluorescence study. J Biol Chem 1992;267:8238–44.
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Lysophosphatidylcholine Decreases Locomotor Activities and Dopamine Turnover Rate in Rats Eun-Sook Y. Lee*, Karam F.A. Soliman, Clivel G. Charlton1 College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA Received 23 August 2003; accepted 21 July 2004 Available online 11 September 2004
Abstract Lysophosphatidylcholine (lyso-PTC), a secondary product of S-adenosylmethionine (SAM)-dependent phosphatidylethanolamine (PTE) methylation, is a potent cytotoxin and might be involved in the pathogenesis of Parkinson’s disease (PD). Our previous studies showed that the injection of SAM into the brain caused PD-like changes in rodents. Moreover, 1-methyl-4-phenylpyridinium (MPP+), a Parkinsonism-inducing agent, increased lyso-PTC formation via the stimulation of PTE methylation pathway. These results indicate a possible role of lyso-PTC in the PD-like changes seen following the injection of SAM or MPP+. In the present study, lyso-PTC was injected into the lateral ventricle of rats and locomotor activities and the biogenic amine levels were measured to evaluate the effects of lyso-PTC on the dopaminergic system. Quinacrine, a phospholipase A2 (PLA2) inhibitor, was employed to determine its protective effect on SAM-induced PDlike changes by the inhibition of lyso-PTC formation. The results showed that 1 h after the injection, 0.4 and 0.8 mmol of lyso-PTC increased striatal dopamine (DA) by 20 and 24%, decreased 3,4-dihydroxyphenylacetic acid (DOPAC) by 37 and 45% and decreased homovanilic acid (HVA) by 24 and 13%, respectively. Consequently, dopamine turnover rate, (DOPAC + HVA)/DA, was significantly reduced by 44 and 48% in the rat striatum. Meanwhile, the administration of 0.4 or 0.8 mmol of lyso-PTC decreased movement time by 52 and 63%, total distance by 44 and 48% and the number of movements by 43 and 64%, respectively. Quinacrine attenuated SAM-induced hypokinesia without affecting SAM metabolism prior to its action on rat brain. The results obtained indicate that the hypokinesia observed following the administration of lyso-PTC might be related to the decline in DA turnover in the striatum in response to lyso-PTC exposure. The present study suggests that inhibitory effects of lyso-PTC on dopaminergic neurotransmission is one of the contributing factors in SAM and MPP+-induced PD-like changes.
# 2004 Elsevier Inc. All rights reserved. Keywords: Lysophosphatidylcholine; Methylation; S-adenosylmethionine; Parkinson’s disease; Dopamine; Locomotor activity
INTRODUCTION The symptoms associated with Parkinson’s disease (PD) include rigidity, resting tremors and hypokinesia, caused by the degeneration of dopaminergic neurons in the nigrostriatal pathway (Selby, 1975). The major biochemical change is dopamine (DA) depletion * Corresponding author. Tel.: +1 850 599 8445; fax: +1 850 412 7261. E-mail address: [email protected] (E.Y. Lee). 1 Current address: Department of Pharmacology, Meharry Medical College, Nashville, TN, USA.
(Hornykiewicz, 1966), but serotonin (5-HT) and norepinephrine (NE) are also depleted in advanced PD (Scatton et al., 1983; Tohgi et al., 1993). Although the etiology of PD is still unknown, two endogenous classes of compounds, b-carbolines and isoquinoline derivatives which have been suggested as causative factors for PD, cause toxic effects via methylation (Collins et al., 1992; McNaught et al., 1996; Gearhart et al., 1997; Matsubara et al., 1998). It is of interest to note that high N-methylation activity was found in PD patients (Williams et al., 1993; Naoi et al., 1994; Aoyama et al., 2000, 2001) and S-adenosylmethionine (SAM), an endogenous methyl donor, can induce
0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2004.07.009
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PD-like symptoms (Crowell et al., 1993), suggesting a possible relationship between hypermethylation and PD. Excess methylation has been proposed as a possible causing factor of PD (Charlton and Mack, 1994; Lee et al., 2004a) and phospholipid methylation is believed to play an important role in the excessive methylationinduced Parkinsonism. Phosphatidylcholine (PTC), a major product of SAM-dependent phospholipid methylation, is synthesized from phosphatidylethanolamine (PTE) by phosphatidylethanolamine Nmethyl transferase (2.1.1.17, PENMT) and readily hydrolyzes to lysophosphatidylcholine (lyso-PTC) by phospholipase A2 (3.1.1.4, PLA2) (Fig. 1). LysoPTC is a normal component of tissues, representing 5– 20% of total phospholipids and 0.2–0.25 mM concentrations in mammalian plasma (Thies et al., 1992). Although the cellular mechanisms of lyso-PTCmediated action are unclear, it has been recognized as an important cell signaling agent (Shier et al., 1976; Yuan et al., 1996; Ikeuchi et al., 1997). Moreover, lysoPTC was identified as a ligand for the immunoregulatory receptor G2A (Kabarowski et al., 2001; Xu, 2002). As a result of various actions of lyso-PTC in different cellular processes, the elevated level of lysoPTC has been suggested to be involved in various pathological conditions such as atherosclerosis, inflammation (Quinn et al., 1988), gastric ulcerogen-
esis (Maksem et al., 1984), and ischemia (Magishi et al., 1996). In the nervous system, lyso-PTC is involved in exocytosis (Poole et al., 1970) and in reversibly arresting exocytosis at a stage between triggering and membrane merger (Vogel et al., 1993). PLA2, the responsible enzyme for the formation of lyso-PTC, is associated with the regulation of the degranulation process leading to a release of neurotransmitters in the exocytosis process (Matsuzawa et al., 1996) and the fusion process of synaptic vesicles with the presynaptic membranes (Nishio et al., 1996). Previously we have shown that lyso-PTC inhibited dopamine D1 and D2 receptor binding activities (Lee et al., 2004b), indicating that lyso-PTC might be involved in modulating neural transmission. Classical PLA2 inhibitors such as mepacrine and p-bromophenacyl bromide are reported to inhibit neurotransmitter release from neuroendocrine cells (Moskowitz et al., 1982; Bradford et al., 1983). Lyso-PTC also exerts detergent-like effects (Slomiany et al., 1981; Maksem et al., 1984; Nishizuka, 1995) and a potent cytolytic property, which may damage neuronal cells. Quinacrine has frequently been used as an inhibitor of PLA2 for its attenuation of stimulated release of arachidonic acid in different cell lines (Valenzuela et al., 1992). It has been widely studied on signal transduction, blood clotting, ischemia, hypertension, and
Fig. 1. Schematic diagram of the formation of phosphatidylcholine (PTC) and lysophosphatidylcholine (lyso-PTC) in PENMT pathway. SAM is a methyl donor and a substrate for PENMT; MPP+ increases PTE methylation by stimulating PENMT. Both SAM and MPP+ increase PTC production which, in turn, serves as a substrate for PLA2 to increase the formation of lyso-PTC. Quinacrine is an inhibitor of PLA2 resulting in the reduction of lyso-PTC formation. PTE: phosphatidylethanolamine; SAM: S-adenosylmethionine; SAH, S-adenosylhomocysteine; PENMT, phosphatidylethanolamine N-methyltransferases; MPP+, 1methyl-4-phenyl-pyridinium; PTC, phosphatidylcholine; PLA2, phospholipase A2; lyso-PTC, lysophosphatidylcholine; AA, arachidonic acid.
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inflammation. Quinacrine decreases experimentallyinduced myocardial necrosis in rats and dogs (Chiariello et al., 1987; Chiariello et al., 1990) and reduces infarct size in rats after transient middle cerebral artery occlusion (Estevez and Phillis, 1997). It has also shown to inhibit the Torpedo nicotinic acetylcholine receptor (Johnson and Ayres, 1996; Arias, 1997). Quinacrine prevented the desensitization of b-adrenergic receptors associated with increment of PTC degradation to lysoPTC and arachidonic acid (AA) (Torda et al., 1981) and inhibited phospholipid methylation in human blood monocytes (Hurst et al., 1986). Moreover, quinacrine has been shown to have a protective effect against MPTP and 6-OHDA neurotoxicities in animal models (Tariq et al., 2001). It is well established that methylation is increased during aging and PD is an age-related disorder (Sellinger et al., 1988). The administration of 1-methyl-4-phenyltetrahydropyridine (MPTP) causes Parkinsonism (Langston et al., 1983) via its metabolite 1-methyl-4-phenylpyridinium (MPP+) which in turn increases phospholipid methylation by enhancing PENMT activity (Lee and Charlton, 2001). SAM, the biological methyl donor, also increases the formation of lyso-PTC through the PTE methylation pathway (Fig. 1). Since there are similarities between the effects of SAM and those of MPTP on impairing motor functions and damaging dopaminergic neurons (Crowell et al., 1993), their abilities to increase lyso-PTC suggest that lyso-PTC may play an important role in the actions of MPP+ and SAM. Coinciding with the major role of the striatum in PD, we found that PENMT activity is highest in the striatum and that the striatum manifested the highest rate in the formation of lyso-PTC following the methylation of PTE by SAM (Lee and Charlton, 2001). Therefore, the present study was designed to determine the effects of lyso-PTC administration on locomotor activities as well as biogenic amine levels in the rat and to determine the role of lyso-PTC in SAMinduced PD-like changes by the inhibition of lyso-PTC formation using quinacrine.
MATERIALS AND METHODS Materials Lysophosphatidylcholine (lyso-PTC), MPP+ I, dopamine HCl (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT), SAM, quinacrine and 5-hydroxyindoleacetic acid (5HIAA), were obtained from Sigma Chemical Company
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(St. Louis, MO, USA). [3H-methyl]SAM (82.4 Ci/mmol) was purchased from New England Nuclear (Boston, MA, USA). Other chemicals and agents were purchased from Fisher Scientific Company (Pittsburgh, PA, USA). Animals Male Sprague–Dawley rats weighing 250–350 g were purchased from Harlan (Indianapolis, IN, USA) and acclimatized for at least one week in a room with 12 h-light and 12 h-dark cycles, 22 1 8C. Water and food were supplied ad libitum. PENMT (Phosphatidylethanolamine N-methyltransferases) Assay PENMT assay described previously (Hirata et al., 1978) was used with some modifications. Rats were sacrificed by decapitation, followed by homogenizing the rat brain or liver tissues in five volume of 50 mM Tris–HCl buffer at pH 7.5. Aliquots of rat liver tissue homogenate (0.2–0.5 mg protein) were incubated with various concentrations of SAM or MPP+ in the medium containing 50 mM Tris–HCl buffer, 15 mM Mg2+ and 20 mM EDTA at pH 7.5 in a total volume of 100 ml. The reaction was initiated with the addition of [3Hmethyl]SAM as a tracer and continued for 1 h at 37 8C. The reaction was terminated by the addition of 3 ml of chloroform:methanol:HCl (100:50:1, v/v/v). Lipids were extracted for 10 min and washed twice with 2 ml of 0.1 M KCl in 50% methanol, followed by thin layer chromatography (TLC) analysis. Thin Layer Chromatography (TLC) Analysis The formation of lyso-PTC was identified by applying aliquots of the organic phase to a silica gel TLC plate (LK5D Silica Gel). The mobile phase was chloroform:methanol:water (65:25:4, v/v/v). Standard lysoPTC dissolved in chloroform: methanol (2:1) was co-chromatographed. The chromatograms were then visualized by exposing the plate to iodine vapor. Spots that matched the standards were scraped off the plates and solublized in scintillation cocktail and the radioactivity was measured in a liquid scintillation counter (Beckman, LS 6500). Cannulation and Drug Treatments in Rats Rats were cannulated prior to the injection of the different compounds (lyso-PTC, SAM or quinacrine) into the lateral ventricles of rat brains as previously
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described (Charlton and Mack, 1994). Male Sprague– Dawley rats weighing 250–350 g were anesthetized with chloral hydrate (400 mg/kg, i.p.). A 22 gauge stainless cannula was positioned at 1.5 mm lateral and 0.6 mm caudal with reference to the bregma from which it extended to the lateral ventricles. The rats were allowed to recover for 3 days before the experiments. Injections were made through polyethylene tube (PE 20) which was attached to a 10 ml Hamilton syringe. For lyso-PTC study, lyso-PTC was dissolved in phosphate-buffered saline (PBS) at pH 7.4 and PBS solution was used as a control. The rats were injected with 5 ml of PBS or lyso-PTC (0.4 or 0.8 mmol) by i.c.v. route single time for acute effects or once a day for 3 days for subacute effects. For SAM and quinacrine study, SAM or quinacrine was dissolved in PBS and the injection amount of each compound was 0.8 mmole in 2.5 ml. Four groups (PBS and PBS, PBS and quinacrine, PBS and SAM, or quinacrine and SAM) were prepared and each rat was administered with two injections at 5 min interval between the injections. Measurement of Locomotor Activities After the last injections, rats were placed in the locomotor activity monitor and the measurement was started 3 min after placed in the monitor. The changes in motor activity of the animals were measured in an Activity Monitor (Degiscan Instruments Inc., Columbus, OH, USA). The measurements were performed in a quiet isolated place with a dim light. Movement time (MT), total distance (TD) and the number of movements (NM) were determined. The locomotor activities were determined for 60 min post-injection. HPLC Analysis The measurement for the levels of DA, DOPAC, HVA, 5-HT, and 5-HIAA were performed by high performance liquid chromatography (HPLC) combined with electrochemical detection (EC), and minor modification of a previously described procedure (Lowry et al., 1996). Briefly, after the dissection of the rat brains into the striatum, hippocampus and cortex, brain tissues were homogenized in three volumes (w/v) of ice-cold 0.4 M perchloric acid and centrifuged at 9000 g for 20 min at 4 8C. Supernatant was filtered (0.45 mm) and 30 ml of the filtered supernatant was injected into the HPLC system. A stainless steel hypersil ODS C-18 reverse-phase column (5 mm, 250 mm 4.6 mm, Whatman EQC) was connected to
the HPLC-EC system. A dual electrochemical detector (Coulochem II, ESA) was set with 350 mV at the guard cell and E1 = 50 mV, E2 = +700 mV at the analytical cell. The mobile phase consisted of 0.1 M sodium acetate, 60 mM citric acid, 0.6 mM octanesulfonic acid sodium salt, 0.5 mM disodium EDTA, in 15% methanol in water, pH 3.5 and pumped at a rate of 1.5 ml/min. For the measurement of SAM and SAH, the rat striatal regions were prepared for HPLC analysis by homogenizing tissues in threevolumes of 0.4 M perchloric acid followed by centrifugation of samples at 9000 g for 20 min at 4 8C and filtration of the supernatant of tissue samples (0.45 mm). The HPLC–UV system consisted of Shimazu HPLC with a hypersil ODS C-18 reverse-phase column (5 mm, 250 mm 4.6 mm, Whatman EQC) with a UVof 260 nm and a flow rate of 1.5 ml/min. The mobile phase was 75 mM NaH2PO4H2O, 4.7 mM 1-octanesulfonic acid sodium salt, 10 mM EDTA and 10% acetonitrile adjusting the final pH to 3.0 with phosphoric acid. Statistical Analysis The mean and standard error of the mean (S.E.M.) were determined for each set of data. Data analysis was performed using one-way analysis of variance (ANOVA) followed by post-hoc Newman–Keuls test to evaluate differences between the different groups. A probability of 0.05 or less was considered a significant difference.
RESULTS A Parallel Effect of SAM and MPP+ on the Formation of Lyso-PTC PTE is methylated by PENMT using SAM as a methyl donor to produce PTC which serves as a substrate for PLA2. Subsequently, lyso-PTC formation is increased when this methylation pathway is accelerated. MPP+ increased phospholipid methylation by stimulating PENMT activity. SAM and MPP+ revealed a parallel action on phospholipid methylation pathway. As shown in Table 1, MPP+ increased the formation of lyso-PTC significantly in a concentration-dependent manner in rat liver tissue (F4,10 = 15.71; p = 0.0003). Effect of Lyso-PTC on Locomotor Activities in Rats Since both SAM and MPP+ increased the formation of lyso-PTC significantly, the effects of lyso-PTC on
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Table 1 Effect of SAM and MPP+ on the formation of lyso-PTC in rat liver tissue A
B
SAM (mM)
Lyso-PTC (pmol/mg protein/h)
15 32 63 125 250
0.18 0.89 1.73 4.52 11.2
0.01 0.01 0.25 0.41* 1.78***
MPP+ (mM)
Lyso-PTC (pmol/mg protein/h)
0 0.3 1 3 10
2.17 2.60 4.41 5.40 7.63
0.36 0.38 0.59 0.76* 0.63***
Rat liver tissue homogenate was incubated with various concentrations of SAM with 0.28 mCi [3H-methyl]SAM as a tracer (A) or incubated with various concentrations of MPP+ in the presence of 100 mM SAM containing 0.55 mCi [3H-methyl]SAM as a tracer (B) for 1 h at 37 8C. The lipids were extracted and separated by thin layer chromatography. The values shown are the means S.E.M. for triplicate samples (N = 3). One-way ANOVA followed by post-hoc Newman–Keuls test was used for statistical analysis. (*) Indicates significant increases when compared to the control (*p < 0.05, ***p < 0.001).
animal behavior were studied to determine the role of lyso-PTC in SAM or MPP+-induced PD-like symptoms. The injection of lyso-PTC into the lateral ventricle of rat brain caused severe hypokinesia as indicated by the decrease MT, TD and NM (Fig. 3). Following the single injection of 0.4 and 0.8 mmol of lyso-PTC, MT was significantly decreased by 52 and 63% compared to the control group (Fig. 2A, F2,12 = 3.915; p = 0.0491). The repeated injections of lysoPTC showed further significant decreases in locomotor activities following 3 days of treatment (Fig. 3A–C). The administration of 0.4 and 0.8 mmol of lyso-PTC decreased MT by 48 and 79% (F2,12 = 3.617; p = 0.0326), TD by 51 and 89% (F2,12 = 39.22; p < 0.0001), and NM by 32 and 66% (F2,12 = 3.920; p = 0.0490). Effects of Lyso-PTC on Dopaminergic Nervous System In this experiment, the effect of lyso-PTC on the levels of DA and its metabolites was investigated to determine whether lyso-PTC alters dopaminergic neurotransmission. As shown in Table 2, the content of DA in the striatum was increased 1 h after a single dose of 0.4 and 0.8 mmol of lyso-PTC by 20 and 24%, respectively (F2,12 = 4.894; p = 0.0279). However, these doses of lyso-PTC significantly decreased the levels of DOPAC by 37 and 45%, respectively (F2,12 = 14.04; p = 0.0007). HVA level was also decreased by 24 and 13%, respectively. Consequently, the turnover rate of dopamine which was represented as (DOPAC + HVA)/ DA was reduced by 44 and 48% by the single doses of lyso-PTC (F2,12 = 18.50; p = 0.0002). Following three daily injections of 0.4 or 0.8 mmol of lyso-PTC, there was a slight decrease in the content of DA, whereas DOPAC level was decreased by 37 and 30% (F2,12 = 17.84; p = 0.0003). The consequent decrements in the ratio of (DOPAC + HVA)/DA were 22 and 26%,
respectively (F2,12 = 5.167; p = 0.0241). The results reveal that lyso-PTC significantly decreases the turnover rate of DA neurotransmission in the rat striatal region. In contrast to the striatum, the effects of lysoPTC on the content of DA, DOPAC and HVA in the cortex or hippocampus were not as significant as those on the striatum by single as well as repeated doses of lyso-PTC although some of the levels of HVA and DA showed statistically significant differences (Table 3). Effects of Lyso-PTC on Serotonergic System In this experiment the administration of a single dose of 0.4 and 0.8 mmol of lyso-PTC resulted in significant increases in the content of 5-HT in the striatum by 27 and 74%, respectively (Table 4, F2,12 = 17.62; p = 0.0003). 5-HIAA level was decreased by 32% at 0.8 mmol of lyso-PTC dose. Consequently, the 5-HIAA/5-HT value was reduced from 0.55 to 0.39 (29%) for the 0.4 mmol dose and 0.21 (62%) for the 0.8 mmol dose, indicating that acute administration of lyso-PTC reduced the turnover of 5-HT (F2,12 = 39.45; p < 0.0001). The repeated administration of lyso-PTC did not cause significant changes on the levels of 5-HT and 5-HIAA. In the cortex and hippocampus serotonergic activity was significantly decreased by the administration of lyso-PTC (Table 5), as shown by the reduction of both 5-HT and 5-HIAA. Single doses of lyso-PTC showed more significant reduction of serotonergic activity than repeated doses. Effect of Quinacrine on SAM-Induced PD-Like Symptoms in Rats: A Possible Role of Lyso-PTC in SAM-Induced Hypokinesia Quinacrine is a PLA2 inhibitor and decreases the formation of lyso-PTC. Quinacrine was used in the present study to determine the role of lyso-PTC in SAM-induced PD-like symptoms and whether quina-
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Fig. 2. Effects of single injection of lyso-PTC on locomotor activities in rats. After the rats were treated (i.c.v.) with 5 ml of PBS or lyso-PTC (0.4 or 0.8 mmol), rats were placed in the locomotor activity monitor after the injection and the measurement was started 3 min later. Lyso-PTC was dissolved in PBS. Movement times, total distances, and the numbers of movement were measured for 60 min post injection. Data are expressed as means S.E.M. (N = 5) and represent two experiments. One-way ANOVA followed by post-hoc Newman–Keuls test was used for statistical analysis to compare different groups. (*) Indicates significantly different from the control (*p < 0.05).
Fig. 3. Effects of multiple injections of lyso-PTC on the locomotor activities in rats. Rats were injected (i.c.v.) with 5 ml of PBS or lysoPTC (0.4 or 0.8 mmol) once a day for 3 days. Movement times, total distances, and the numbers of movement were measured for 60 min post injection. Data are expressed as means S.E.M. (N = 5) and represent two experiments. One-way ANOVA followed by post-hoc Newman–Keuls test was used for statistical analysis to compare different groups. (*) Indicates significantly different from the control (*p < 0.05, **p < 0.01, *** p < 0.001).
crine attenuates the effects of SAM on PD-like symptoms. It was observed that SAM caused significant hypokinesia, by decreasing MT by 42%, TD by 34% (Fig. 4, third bar). The group of SAM co-administered with quinacrine showed a significant attenuation
effects on SAM-induced hypokinesia (F3,26 = 3.506, p = 0.0315 for MT; F3,26 = 3.371, p = 0.0358 for TD; F3,26 = 3.050, p = 0.0489 for NM; Fig. 4, fourth bar). The attenuation effects of the SAM/quinacrine group compared to the SAM group for MT, TD and NM were
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Table 2 Effect of lyso-PTC on levels of DA, DOPAC and HVA and (DOPAC + HVA)/DA in the rat striatum (ng/100 mg tissue) Lyso-PTC
DOPAC
HVA
(DOPAC + HVA)/DA
Acute (mmol, i.c.v., 1 h) 0 763.4 63.4 0.4 955.5 26.6* 0.8 1,000 43.9*
DA
97.0 8.8 61.2 5.2# 53.8 3.1#
75.9 5.0 58.0 2.3# 66.1 6.7
0.23 0.02 0.13 0.01 0.12 0.01
Subacute (mmol, i.c.v., 3 days) 0 720.6 26.9 0.4 667.0 26.6 0.8 661.6 5.4
104.5 9.0 66.2 3.1# 72.8 5.4#
62.3 4.0 54.1 2.1 58.7 3.3
0.23 0.02 0.18 0.01 0.17 0.01
Rats were treated (i.c.v.) with 5 ml of PBS or two different doses (0.4 or 0.8 mmol) of lyso-PTC dissolved in 5 ml PBS for 1 h or once a day for 3 days and sacrificed 1 h after the single injection or 24 h after the last injection. Rat striatal regions were dissected out and analyzed by HPLC. Data are expressed as means S.E.M. (N = 5). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis to compare different groups. (*) Indicates significantly increased from the control (p < 0.05). (#) Indicates significantly decreased from the control (p < 0.05).
Table 3 Effect of lyso-PTC on the content of DA, DOPAC, HVA in the rat cortex and hippocampus Lyso-PTC
Cortex (ng/100 mg tissue) DA
Hippocampus (ng/100 mg tissue)
DOPAC
HVA
DA
DOPAC
HVA
Acute (mmol, i.c.v., 1 h) 0 34.7 4.8 0.4 25.1 2.9 0.8 26.4 8.3
19.0 1.2 10.8 8.3 15.4 1.5
12.4 1.2 11.0 1.1 9.2 1.7*
3.4 0.4 3.9 0.6 2.2 0.2*
6.8 0.6 5.5 1.0 6.6 0.7
4.9 0.3 4.1 0.6 3.2 0.2*
Subacute (mmol, i.c.v., 3 days) 0 36.3 2.6 0.4 38.7 1.9 0.8 34.5 4.3
20.5 2.5 22.3 2.0 21.8 2.5
11.7 1.1 14.6 1.0 12.5 2.5
5.2 0.3 5.5 0.4 5.2 0.5
8.7 1.3 7.2 0.8 6.1 0.7
5.1 0.8 3.6 0.3 3.7 0.5
After i.c.v. injection with 5 ml of PBS or lyso-PTC (0.4 or 0.8 mmol), rats were sacrificed 1 h after the single injection or 24 h after the last injection of three daily consecutive treatments. Rat cortex and hippocampus regions were dissected out and DA, DOPAC and HVA were analyzed by HPLC. Data are expressed as means S.E.M. (N = 5). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis to compare different groups. (*) Indicates significantly different from the control (p < 0.05).
Table 4 Effect of lyso-PTC on content of 5-HT and 5-HIAA in the rat striatum (ng/100 mg tissue) Lyso-PTC
5-HT
Table 5 Effect of lyso-PTC on the content of 5-HT and 5-HIAA in the rat brain cortex and hippocampus Lyso-PTC Cortex (ng/100 mg tissue) Hippocampus (ng/100 mg tissue) 5HT
5HIAA
5HT
5HIAA
5-HIAA
5-HIAA/5-HT
Acute (mmol, i.c.v.,1 h) 0 61.4 2.6 0.4 78.0 2.8* 0.8 106.9 8.7*
33.7 1.0 30.7 2.1 22.9 1.9
0.55 0.03 0.39 0.03* 0.21 0.02*
Acute (mmol, i.c.v., 1 h) 0 47.9 2.4 12.5 0.5 30.7 1.8 20.1 1.2 0.4 39.1 5.7 9.5 1.1* 26.9 1.7 13.0 0.3* * 0.8 35.9 2.5 10.0 1.2 23.9 1.4* 11.2 1.8*
Subacute (mmol, i.c.v., 3 days) 0 52.8 4.3 0.4 49.1 3.2 0.8 39.9 3.5
35.4 4.2 36.0 7.7 42.4 10.4
0.67 0.11 0.73 0.16 1.06 0.36
Subacute (mmol, i.c.v., 3 0 46.8 4.3 0.4 43.7 2.6 0.8 36.7 s 2.4
Cannulated rats were treated (i.c.v.) with 5 ml PBS or lyso-PTC (0.4 or 0.8 mmol) for 1 h or once a day for three days and sacrificed 1 h after the single injection and 24 h after the last injection for three daily injections. Rat striatal regions were dissected out and 5-HT and 5-HIAA were analyzed by HPLC. Data are expressed as means S.E.M. (N = 5). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis to compare different groups. (*) Indicates that the group is significantly different from the PBS control group (p < 0.05).
days) 13.0 2.2 14.3 1.8 9.6 0.6
32.4 3.2 33.4 2.5 33.7 3.2
21.8 4.6 25.2 5.1 37.9 8.1
Rats were treated (i.c.v.) with 5 ml PBS or lyso-PTC (0.4 or 0.8 mmol) and sacrificed 1 h after the single injection or 24 h after the last injection for three daily treatment groups. Cortex and hippocampus regions were dissected out and 5-HT and 5-HIAA were analyzed by HPLC. Data are expressed as means S.E.M. (N = 5). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis to compare different groups. (*) Indicates significantly different from the control (p < 0.05).
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PBS nor quinacrine treated groups showed tremors. The SAM treated group showed severe tremors 5– 7 min after the injection. The group of SAM co-administered with quinacrine showed weak tremors about 10 min after the injection, indicating that quinacrine decreased the intensity of the SAM-induced tremors. One hour after the injections for each group, rats were sacrificed and brain striatal regions were dissected. The striata were homogenized and prepared for the HPLC analysis. Since SAM is a universal methyl donor and could be utilized in the metabolism of exogenous compounds, SAM and S-adenosylhomocysteine (SAH) levels were measured to eliminate the possibility that quinacrine might influence on SAM metabolism and reduce the availability of SAM prior to inducing PD-like changes. As shown in Fig. 5, SAM administrations significantly elevated the levels of SAM and SAH (F3,26 = 18.39, p < 0.0001 for SAM; F3,26 = 11.63, p < 0.0001 for SAH). However,
Fig. 4. Effect of quinacrine on SAM-induced hypokinesia in rats. SAM or quinacrine was dissolved in PBS and the injection amount of each compound was 0.8 mmole in 2.5 ml. Four groups (PBS and PBS, PBS and quinacrine, PBS and SAM, or quinacrine and SAM) were prepared and administered (i.c.v.) with two preparations at 5 min interval between injections as indicated in Materials and Methods. Movement time, total distances and the number of movements were measured for 60 min post injection in different groups of rats. Data are expressed as means S.E.M. (N = 6 for control group, N = 8 for the treated groups). Two tailed Student’s t-test was used for statistical analysis to compare corresponding groups. (#) Indicates significantly decreased from the PBS control group (#p < 0.05). (*) Indicates significantly increased from the SAM treated group (*p < 0.05, ** p < 0.01).
significant at p < 0.05. The quinacrine treated group did not show any significant changes on the locomotor activity compared to the control group. Whole body tremors induced in each group were also observed (data not shown) since SAM induces severe whole body tremors and resulting in abnormal posture to control the body poses in rats (Crowell et al., 1993). Neither the
Fig. 5. SAM and SAH levels in rat brain striatum 1 h after the injections (i.c.v.) of quinacrine, SAM, or combination of quinacrine and SAM. Rats were sacrificed 1 h after the treatment and rat brain striatal regions were dissected, homogenized, and prepared for HPLC analysis to measure SAM and SAH levels as described in the Method section. Data are expressed as means S.E.M. (N = 6 for the control group, N = 8 for the treated groups). One-way ANOVA followed by post-hoc Newman–Keuls test were used for statistical analysis of group comparison. (*) Indicates significantly increased from the SAM treated group (**p < 0.01, ***p < 0.001).
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quinacrine did not affect the level of SAM or SAH (p > 0.05 for SAM or SAH), indicating that quinacrine does not interact with SAM prior to its action.
DISCUSSION The present study demonstrates that lyso-PTC administration (i.c.v.) into the rat brain caused hypokinesia, which was accompanied with a decrease in DA turnover and an apparent reduction in dopaminergic neurotransmission. The (DOPAC + HVA)/DA ratio is frequently used as an index of dopaminergic activity (Lavielle et al., 1979). Reductions in dopaminergic neuronal activity and hypokinesia are parallel physiological functions that also represent the major symptoms of PD. It suggests, therefore, that lyso-PTC, a secondary product that was increased by excessive methylation, might play an important role in SAMinduced PD-like changes in rodents. Interestingly, the effects of lyso-PTC on the dopaminergic system occur most predominantly in the striatum among three regions tested in the present study. This result may be relevant to the fact that impairment of striatum plays a key role in Parkinsonism, and also supported by the previous result that the striatum has the highest activity of phosphatidylethanolamine PENMT (Lee and Charlton, 2001). The acute administration of lyso-PTC decreased the turnover of 5-HT in the striatum, whereas successive doses showed a slight increase of 5-HT turnover, suggesting that the serotonergic nervous system is responding in a similar manner to the dopaminergic system and could also be affected by lyso-PTC administration. The effect of lyso-PTC on dopaminergic neurotransmission show a very similar pattern to the effects of MPP+ in which the acute administration of MPP+ induced the accumulation of DA and decreased its metabolites and caused Parkinsonism in long term treatments (Burns et al., 1983; Chiueh et al., 1984; Desole et al., 1993; Miele et al., 1995). The result that MPP+ increased the formation of lyso-PTC indicates that lyso-PTC might play a role in MPTP neurotoxicity. The acute administration of lyso-PTC appears to cause the accumulation of DA, and the reduction of the levels of DA metabolites, DOPAC and HVA, in the striatum. This initial occurrence by the single dose of lyso-PTC suggests that lyso-PTC may interfere with dopaminergic neurotransmission process such as DA release, binding to postsynaptic receptors, uptake or metabolism, resulting in retarding DA turnover and inducing
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bradykinesia in animals. All these results indicate that lyso-PTC which is increased from S-adenosylmethionine (SAM)-mediated excessive phospholipid methylation or MPP+ might play a role, at least in part, in their actions on the dopaminergic system. It has been reported that lyso-PTC decreased DA D2 binding activity in caudate microsomal membranes (Oliveira et al., 1984). In their study, the effects of phospholipase A2 (PLA2) and lyso-PTC as a product of PLA2 on dopamine receptor binding activity were tested to determine which substance, PLA2 or lysoPTC, is actually responsible for the inhibition of dopamine receptor binding activity. It was found that the phospholipid hydrolysis products (lyso-PTC) rather than PLA2 are directly involved in the alteration of dopamine receptor binding activity, implicating the potential role of lyso-PTC. The results from our laboratory showed that lyso-PTC decreased DA D1 and D2 binding activities in rat striatal membranes (Lee et al., 2004b) and increased DA release and inhibited DA uptake in PC12 cells, implicating that lyso-PTC indeed impedes several steps of DA neurotransmission in the striatum. Several published studies that lyso-PTC is involved in exocytosis (Vogel et al., 1993) and vesicle fusion during the process of neurotransmitter release (Martin and Ruysschaert, 1995) strongly supports the role of lyso-PTC on the neurotransmission of catecholaminergic neurons. The fact that the DA content and the formation of lyso-PTC are highest in the striatum compared to other regions in the brain may explain the susceptibility of dopaminergic neurons and the selective damage of the nigrostriatal pathways. The reduction of dopaminergic neurotransmission induced by lyso-PTC in the rat striatum might be closely related to the effect of lyso-PTC on the impairment of locomotor activities in rats. Lyso-PTC is considered as the most potent substance among the secondary products of phospholipid methylation to alter dopaminergic action and basal ganglia activity and, therefore, it was attempted to block SAM-induced PD-like changes by the inhibition of lyso-PTC formation using quinacrine. The data obtained in the present study showed that quinacrine alleviated SAM-induced impairment of locomotor activity as indicated by compensating the decreased values of movement parameters such as movement time, total distances and the number of movement. The results that SAM and SAH levels were elevated in the striatum after the injections of SAM indicate that the inhibitory effects of quinacrine on SAM actions were not by decreasing SAM levels directly. It has been reported that quinacrine protected MPTP or 6-hydroxy dopamine (6-OHDA)-
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induced Parkinsonism in rodents (Tariq et al., 2001), suggesting the possible role of lyso-PTC in Parkinsonism. Quinacrine is a lipophilic compound and crosses the blood–brain barrier (Dubin et al., 1982; O’Regan et al., 1995). This lipophilicity of quinacrine might be an important characteristic to be used systemically as a therapeutic agent. PD is an age-related disorder and methylation reactions increase in aging (Sellinger et al., 1988) in which lyso-PTC is elevated as a consequence of excessive methylation of PTE to PTC. This may lead to the slow failure of striatal dopaminergic neurotransmission and result in hypokinesia. Moreover, the potent detergentlike cytotoxic effect of lyso-PTC could damage the dopaminergic nerve endings and impair dopaminergic functions. In conclusion, lyso-PTC, which is one of the products resulting from excessive methylation and MPP+, caused hypokinesia and biochemical changes that resemble PD. These results provide additional evidence that hypermethylation and lyso-PTC may be involved in the pathogenesis of PD and the MPTP neurotoxicity. The current findings may suggest the therapeutic potential of quinacrine as a PLA2 inhibitor for the treatment of PD. ACKNOWLEDGMENTS The authors would like to thank Ms. X. Liu for her excellent laboratory assistance. This work was supported by grants received from the National Institutes of Health (RO1 28432, RR 03020, and GM 08111). REFERENCES Aoyama K, Matsubara K, Okada K, Fukushima S, Shimizu K, Yamaguchi S, Uezono T, Satomi M, Hayase N, Ohta S, Shiono H, Kobayashi S. N-methylation ability for azaheterocyclic amines is higher in Parkinson’s disease: nicotinamide loading test. J Neural Transm 2000;107:985–95. Aoyama K, Matsubara K, Kondo M, Murakawa Y, Suno M, Yamashita K, Yamaguchi S, Kobayashi S. Nicotinamide-N-methyltransferase is higher in the lumbar cerebrospinal fluid of patients with Parkinson’s disease. Neurosci Lett 2001;298:78–80. Arias HR. The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor. Biochim Biophys Acta 1997;1347:9–22. Burns RS, Chiueh CC, Markey SP, Ebert MH, Jacobowitz DM, Kopin IJ. A primate model of Parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Nat Acad Sci USA 1983;80:4546–50.
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