- Email: [email protected]
N-methylisoquinolinium ion as an inhibitor of tyrosine hydroxylase, aromatic l -amino acid decarboxylase and monoamine oxidase
Neurochem. Int. Vol. 15, No. 3, pp. 315 320, 1989 Printed in Great Britain. All rights reserved
0197-0186/89 $3.00 + 0.00 Copyright ~:~ 1989 Pergamon Press plc
N-METHYLISOQUINOLINIUM ION AS AN INHIBITOR OF TYROSINE HYDROXYLASE, AROMATIC L-AMINO ACID DECARBOXYLASE AND MONOAMINE OXIDASE MAKOT0 NAOI ~, TSUTOMU TAKAHASHI2, HASAN PARVEZ3, RYOSUKE KABEYAj, EriCH TAGUCHIl, KEIKO YAMAGUCHI~YOKO HIRATA4, MASAYASUMINAMI4 and TOSHIHARU NAGATSU~ Department of Biochemistry, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan, 2Department of Food and Nutrition, Konan Women's College, Konan, Japan, 3Unite de Neuropharmacologie, Universit6 de Paris Xl, Orsay Cedex, France and 4Department of Hygiene and Public Health, Nippon Medical School, Tokyo, Japan (Received 21 December 1988; accepted 12 April 1989) Abstract--The effect of the N-methylisoquinolinium ion (NMIQ +) on the activity of enzymes related to
the metabolism of dopamine was studied using a rat clonal pheochromocytoma PC12h cell line. The activities of tyrosine hydroxylase (TH), aromatic L-amino acid deearboxylase (AADC) and monoamine oxidase (MAO) were inhibited by NMIQ +, but the mechanism of inhibition of these enzymes differed from each other. TH activity in the cells was inhibited by NMIQ + with an ICs0 of about 75/~M. Aromatic L-amino acid decarboxylase (AADC) was also inhibited by NMIQ + but in competition with a co-factor, pyridoxal-5-phosphate, and the K~value was 90 #M. MAO was inhibited by NMIQ + in competition with a substrate, kynuramine, and the K, value was 20 #M. In vivo effects ofNMIQ + on these enzymes in PCI2h cells were examined by culture of the cells in the presence of I00 nM 1 mM NMIQ + for 6 days. After 6 days culture, TH activity was reduced in cells cultured with NMIQ + at concentrations higher than I0 #M, but the activities of AADC and MAO were reduced only in cells cultured with 1 mM NMIQ +. In addition, NMIQ + was transported into the cells by a transport system specific for dopamine. These data suggest that NMIQ + may perturb the catecholamine metabolism of a dopaminergic system in the brain, as a naturally-occurring compound.
Discovery of N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) as a neurotoxin, which degenerates the nigro-striatal system in humans and elicits symptoms very similar to parkinsonism (Davis et al., 1979), suggests to us that there may be similar such exogenous or endogenous compounds in human brains that may cause degeneration in some specific
Address all correspondence and reprint requests to: Dr Makoto Naoi, Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan. Abbreviations: NMIQ +, N-methyl-isoquinolinium ion; NMTIQ, N-methyl-l,2,3,4-tetrahydroisoqainoline; TH, tyrosine hydroxylase; MAO, monoamine oxidase; AADC, aromatic L-amino acid decarboxylase; MPTP, N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine; MPP +, N-methyl-4-phenyl-pyridinium ion; HPLC, high performance liquid chromatography; DA, dopamine; L-DOPA, 3,4-dihydroxy-L-phenylalanine; 5-HTP, 5hydroxytryptophan; 5-HT, 5-hydroxytryptamine; PLP, pyridoxal-5-phosphate; (6R)BH4, (6R)-L-erythro5,6,7,8-tetrahydrobiopterin; ECD, electrochemical detector; COMT, catechol-O-methyltransferase. 315
neurons in the brain. Recently in our laboratory, tetrahydroisoquinoline and 2-methyl-tetrahydroquinoline were found in human brains and the former c o m p o u n d was found to increase in a parkinsonian brain (Niwa et al., 1987). Similar quinoline derivatives were also detected in rat brains (Kohno et al., 1986). By comparison of chemical structure with an oxidative product of MPTP, the N-methyl4-phenylpyridinium ion (MPP+), the N-methylisoquinilinium ion ( N M I Q +) was proposed as a neutrotoxin candidate and in vitro activity of enzymes related to catecholamine metabolism was found to be inhibited by N M I Q +. N M I Q + inhibited tyrosine hydroxylase [tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2, TH] in slices of the nigro-striatal system of rat brains (Hirata et al., 1986). In addition, N M I Q + inhibits type A monoamine oxidase [monoamine: oxygen oxidoreductase (deaminating), EC 1.4.3.4, MAO] in human brain synaptosomal and placental mitochondria (Naoi et al., 1987a). Recently, we found that N M I Q + is produced by M A O in human brain
316
MAKOTO NAOI et al.
s y n a p t o s o m e s from N - m e t h y l - l , 2 , 3 , 4 - t e t r a h y d r o i s o quinoline ( N M T I Q ) (Naoi et al., 1989). E n z y m a t i c p r o d u c t i o n o f the oxidative form o f N M T I Q m a y be c o m p a t i b l e to t h a t o f M P P + from M P T P by m o n o a m i n e oxidase ( C h i b a et al., 1964). These d a t a suggest t h a t N M I Q ÷ m a y be a true toxin to degenerate d o p a m i n e r g i c neurons, which m a y be synthesized from T I Q in h u m a n subjects. To examine in vitro and in vivo effects of N M I Q ÷ on the enzymes participating in the biosynthesis a n d m e t a b o l i s m of catecholamines, a clonal rat p h e n o c h r o m o c y t o m a PC12h cell line was used, because the cells mainly produce d o p a m i n e (DA) as a n e u r o t r a n s m i t t e r ( H a t a n a k a , 1981). M A O in P C 1 2 h cells is of type A (Naoi et al., 1987b; Y o u d i m et al., 1986). This p a p e r describes in vitro a n d in vivo effects o f N M I Q ÷ on the enzyme activities o f TH, M A O a n d a r o m a t i c L-amino acid decarboxylase (aromatic L-amino acid carboxy-lyase, E C 4.1.1.28, A A D C ) in P C 1 2 h cells. N M I Q + was f o u n d to inhibit A A D C activity in a d d i t i o n to T H a m d M A O activity. The cells were cultured for 6 days in the presence of 100 n M - I m M N M I Q ÷ a n d the effects o n the enzyme activity in the cells were examined. EXPERIMENTAL PROCEDURES
Materials NMIQ ÷ was synthesized as reported previously (Hirata et al., 1986). L-Tyrosine, 4-bromo-3-hydroxybenzyloxyamine (NSD-1055), an inhibitor of AADC, 5-hydroxy-L-tryptophan (5-HTP), DA, 3,4-dihydroxy-L-phenylalanine (LDOPA) and pyridoxyal-5-phosphate (PLP) were purchased from Naealai tesque (Kyoto, Japan); kynuramine from Sigma (St Louis, Mo., U.S.A.): 5 hydroxytryptamine (5-HT) from Merck (Darmstadt, F.R.G.); sodium-l-octanesulfonate from Aldrich (Milwaukee, Wis., U.S.A.). Clorgyline, a specific inhibitor of type A MAO, was kindly donated by May & Baker (Dagenham, U.K.). (6R)-L-erythro-5,6,7,8Tetrahydrobiopterin [(6R)BH4] was kindly donated by Dr S. Matsuura, Department of Chemistry, College of General Education, Nagoya University (Nagoya, Japan); and 5(4-chlorophenyl)-2,5-dihydro[3H]imidazo(2, l-~)isoindol-5ol (Mazindol), and inhibitor of DA uptake, was donated by Sandoz (Basel, Switzerland). PC12h cells were cultured as described previously (Naoi et al., 1988c), using a reduced concentration of fetal calf serum and so-called "serum-free" culture medium. The cells were cultured to become confluent, harvested and centrifuged at 600g for 10min. The cells (c. 10~ cells) were cultured in a 25 cm 2 culture dish with the following culture medium: Cosmedium-001 (Cosmo Bio, Tokyo, Japan) containing 5% fetal bovine serum (Gibco Life Technologies, Grand Island, N.Y.U.S.A.), in the absence or presence of 100 n M - l mM NMIQ +. After 3 days culture the culture medium was changed and after 6 days the cells were harvested, washed twice with phosphate-buffered saline and suspended in l0 mM potassium phosphate buffer, pH 7.4. The cell suspension was sonicated in a Branson sonicator for
30 s at a power level of 20. The sonicated cells were kept frozen at - 8 0 ° C and used for the assay of activity of TH, AADC and MAO. Assay for enzyme activity o f TH, A A D C and M A O TH activity in the sonicated cells was assayed in the presence of 200#M L-tyrosine, l mM (6R)BH4, l mM NSD-1055, an inhibitor of AADC and 10#g catalase in 100#1 of 100mM sodium acetate buffer, pH6.0, as described previously (Naoi et al., 1988b). TH activity was determined by measurement of enzymatic conversion of L-tryosine into L-DOPA, which was quantitatively assayed using high performance liquid chromatography (HPLC) with a Coulochem 5100A electrochemical detector (ECD) and a Coulochem 5011 analytical cell (ESA, Bedford, Mass., U.S.A.). AADC activity was measured as reported previously (Naoi et al., 1988a). A PC12h cell suspension (1(~50 pg protein) was incubated with l mM L-DOPA in 100/~1 of 20 mM sodium phosphate buffer, pH 7.2, containing 5 # M pyridoxial-5-phosphate, 0.34mM ascorbic acid and 10/IM clorgyline, a type A MAO inhibitor. After precipitation of protein, DA formed by AADC was quantitatively assayed using PHLC and ECD. MAO activity was measured tluorimetrically using kynuramine as substrate, according to a slightly modified method of Kraml (1965) as reported previously (Naoi et al., 1986b). Kinetical data on the activity of these three enzymes and the effect of NMIQ + were calculated by plotting the data according to Lineweaver and Burke using eight different concentrations of substrate or co-factor or, according to Dixon, using six different concentrations of NMIQ + and three different concentrations of substrate or co-factor. TH activity in the cells was also measured without addition of substrate and co-factor, and in the presence of an AADC inhibitor, NSD-1055. The intact cells (30 #g protein) were suspended in 100 pl of the modified Krebs Ringer solution composed of 126 mM NaC1, 4.75 mM KCI, 1.27 mM CaCI 2, 1.42 mM MgSO 4, 10mM D-glucose, l mM ascorbic acid in 15raM sodium phosphate buffer, pH 7.4, in the presence of 1 mM NSD-1055 and 1 # M - I mM NMIQ +. After 15 min incubation at 37°C, the cell suspension was mixed with 100 pl of 180 mM sodium acetate-70 mM citric acid buffer, pH 4.35, containing 260pM disodium EDTA and 260 # M sodium octanesulfonate, to which methanol was added to 21%. After mixing, the sample was centrifuged at 15,000g for 10min, and the supematant was filtered through a Millipore HV filter. L-DOPA produced by TH in the cells was assayed by HPLC, as described in one of our papers (Naoi et al., 1988c). The amount of protein was measured according to Bradford using bovine y-globulin as standard (Bradford, 1976). Assay o f N M I Q + taken up into PCI2h cells Uptake of NMIQ + into PCI2h cells was examined, using cell suspension in the modified Krebs-Ringer solution. The cell suspension (60/~g protein) in 100 #1 of the Krebs Ringer solution was incubated with 100 nM 1 mM NMIQ + at 37°C for 20 min, then centrifuged at 600g for I0 min. The cells were washed twice with 1.5 ml of phosphate-buffered saline and then suspended in 50/~1 of distilled water. After mixing with 50/~1 of 0.I M perchloric acid containing 0.4 mM sodium metabisulfite and 0.1 mM disodium EDTA, the sample was centrifuged at 15,000g for 10min and filtered. The amount of NMIQ + taken up into cells was quantitatively determined fluorimetrically by HPLC and
N-Methylisoquinolinium ion inhibits dopamine metabolism
317
Table 1. Kinetic data of TH, AADC and MAO in PC12h cells and effect of NMIQ ÷ TH Toward L-tyrosine g m (taM) VmaX (pmol/min/mg protein) Toward (6R)BH a Kin1 (taM) Vma,t (pmol/min/mg protein) Kin2 (taM) Vma~2(pmol/min/mg protein) IC50 of NMIQ +*
19.5 + 6.9 264 + 68 361 + 510 + 87.5 + 267 + 75 +
AADC Toward L-DOPA, using 10taM PLP Km (/2 M) Vmax (nmol/min/mg protein) Toward PLP, using 1 mM L-DOPA as substratet Km (ta M) lima~ (pmol/min/mg protein) Ki of NMIQ ÷ in terms of PLP, using 1 mM L-DOPA MAO Toward kynuramine Km (taM) Vm.. (pmol/min/mg protein) K~ of NMIQ + (taM)
21.4+_ 1.6 1.31 _+ 0.03 0.62_+0.10 436 + 58 89.9 _+ 7.5 taM
58 38 6.4 74 50#M + 1 mM NMIQ ÷ 26.5_+ 1.1 1.48 + 0.22 + 100taM NMIQ 1.31 _+0.27 689 _+ 121
17.8 _+0.9 869 + 30 20.0 _+ 6.5
*lCs0 is the concentration of NMIQ + required to yield 50% inhibition of TH activity in the intact cells. tKinetic data were obtained from the enyzme activity increased by addition of PLP. Each value represents the mean and SD of triplicate measurements of two experiments, using eight different concentrations.
fluorescence detection. An HPLC apparatus, Shimadzu LC4A HPLC (Kyoto, Japan), was used connected to a Shimadzu fluorescence detector RF-500LCA. The column used was a Shimadzu pre-packed reverse-phase column STR
iI A
loo
O D S - H (4 mm i.d. x 150 mm). The sample was eluted at a rate of 0.8 ml per min with 90 mM sodium acetate-35 mM citric acid buffer, pH4.35, containing 1 3 0 p M disodium E D T A and I 1 5 # M sodium octanesulfonate, to which methanol was added to 10.5%. Fluorescence intensity at 3 8 0 n m was monitored with excitation at 3 4 0 n m in a Shimadzu integrator CR3A, and quantitation of N M I Q + was carried out by comparison of the peak area with that of the standard.
/ RESULTS
.>_
"4 o
50
-//
6
5
4
3
- L o g [NM'rQ +] (M) Fig. 1. Effect of N M I Q ÷ on T H activity in intact P C I 2 h cells. P C I 2 h ceils ( 3 0 # g protein) were w a s h e d and susp e n d e d in K r e b s - R i n g e r solution. The cells were i n c u b a t e d for 20 m i n at 37°C in the presence o f 1 m M N S D - 1 0 5 5 a n d in the absence a n d presence o f 1 p M - 1 m M N M I Q +. After t e r m i n a t i o n o f the reaction, the s a m p l e was treated as described in E x p e r i m e n t a l Procedures. E a c h p o i n t represents the m e a n a n d S D o f d u p l i c a t e m e a s u r e m e n t s o f two experiments.
Table 1 summarizes the value of the Michaelis constant (Km) and the maximal velocity (Vmax) of the enzymes used in this experiment. Toward (6R)BH4, T H in PC12h cells was found to have two different types of kinetic properties; one is with a low affinity ( K m l ) and a high activity (Vmaxl) and another was a high affinity (Kin2) and a low activity (Vm,,2). Figure 1 shows that N M I Q + inhibited T H activity in the intact cells in a dose-dependent way, and ICs0 was calculated to be 75 # M . However, when T H activity in the sonicated cells was assayed in the presence of exogenously added L-tyrosine and (6R)BH 4, N M I Q + did not inhibit T H activity. N M I Q + inhibited A A D C activity toward L-DOPA and the inhibition was competitive to a co-factor, PLP, as shown in Fig. 2. In the presence of 1 0 # M PLP, the value of Km and Vmaxof A A D C in terms of L-DOPA were not affected by the presence of even 1 m M N M I Q + (Table 1). The
318
MAKOTO NAOI et al. ReLative uptake of NMIQ (%) 50
1[
o
~-
15
.=_ E io
c
I
Control
E
E
100
i
I
+ L - DOPA (1 raM) I
+ DA (1 m M ) 5
+ 5-HTP (1 m M )
0
!
I
5
10
+ 5-HT (1 rnM) 4- NA (IOOFM)
(/.,/,M -1 )
1/[PLP]
Fig. 2. Effect of PLP concentration and NMIQ + on AADC activity of L-DOPA. PC12h cells (17/~g protein) were incubated with 1 mM L-DOPA in the absence and presence of 250 # M NMIQ +. The reciprocal of the reaction velocity was plotted against that of the PLP concentration, according to Lineweaver and Burk. Curve I: control; Curve II: in the presence of NMIQ +. Each point represents the mean of triplicate measurements of two experiments. inhibitor c o n s t a n t (K~) value o f N M I Q + in terms of P L P was o b t a i n e d to be 89.9 4-7.5 # M , as s h o w n in Table 1. N M I Q + inhibited M A O in the cells and the inhibition was competitive to a substrate, k y n u r a m i n e , as s h o w n in Fig. 3. The K~ value was 20.0 4- 6.5/~M, which is very similar to the Km value with k y n u r a m i n e of 17.8 + 0.9 # M .
54 o
Q.
][
E" 3
t:
E
L o
E
2
0
' 10
' 20
' 30
%
4
I
+ Mozindo L (IOOFM)
Fig. 4. Effects of catecholamines, related compounds and Mazindol on the uptake of NMIQ + in PC12-h cells. PC 12h cells (60 #g protein) were incubated with 1 mM NMIQ + in the presence of 1 mM L-DOPA, DA, 5-hydroxytryptophan (5-HTP) and 5-HT, or 100#M noradrenaline (NA) and Mazindol. After incubation at 37'~C for 20 min, the cells were washed and treated as described in Experimental Procedures. Each value represents the mean and SD of triplicate measurements of two experiments.
N M I Q + was f o u n d to be taken up into P C I 2 h cells at c o n c e n t r a t i o n s higher t h a n 1 0 0 # M . Effects of biogenic amines, the related c o m p o u n d s , a n d Mazindol, an inhibitor of D A uptake system, on N M I Q + uptake are s h o w n in Fig. 4. The uptake was inhibited by L-DOPA, DA, 5-HT a n d Mazindol, but neither 5 - H T P n o r n o r a d r e n a l i n e inhibited the uptake. Effects of the presence of N M I Q ÷ in the culture m e d i u m on the enzyme activities of TH, A A D C a n d M A O in PC12h cells were examined after 6 days culture with 100 n M - i m M o f N M I Q +. Figure 5 shows that the enzyme activity o f T H was reduced in the presence of N M I Q + c o n c e n t r a t i o n s higher t h a n 10#M, a n d the activity was a b o u t 50% of t h a t o f the control. But the activity of A A D C a n d M A O was reduced only in the presence o f 1 m M N M I Q +.
' 60 DISCUSSION
[NMIO +] (FcM)
Fig. 3. Effect of kynuramine concentration and NMIQ + on MAO activity in PC12h cells. MAO activity in PCI2h cells (87 #g protein) was measured in the presence of 33-3 and 50 # M kynuramine and various concentrations of NMIQ +. The reciprocal of the reaction velocity was plotted against NMIQ + concentration, according to Dixon. Curve I: 50#M; Curve II: 33.3#M of kynuramine. Each point represents the mean and SD of triplicate measurements of two experiments.
T I Q a n d o t h e r tetrahydroisoquinolines have been confirmed to be p r o d u c e d from m o n o a m i n e s by c o n d e n s a t i o n o f a ~ - a r y e t h y l a m i n e with a c a r b o n y l c o m p o u n d by a n o n - e n y z m a t i c Pictet-Spengler reaction (Whaley a n d G o v i n d a c h a r i , 1951). These comp o u n d s are considered to be synthesized in vivo in h u m a n subjects at significant rates (Collins, 1980). Elevation of their levels was reported in alcoholics
N-Methylisoquinolinium ion inhibits dopamine metabolism I .,.
Ill
. - , ' / ./.,"----k-'l~ 0.6
o o.
.=_ 0.5
0.4
>
0.3
0
0.2
"o
0.1
g
o ~
0
.=_. E 4
T p--
I
0
f
< //
I 7
-
Log
I 6 [NMIQ
I 5
I 4 +]
I 3 (M)
Fig. 5. Effect of NMIQ + on the activities of TH, AADC and MAO in PCI2h cells cultured in the presence of NMIQ +. PCI2h cells were cultured in the presence of 100 nM 1 mM NMIQ ÷ for 6 days. At day 3, the culture medium was changed with the medium containing the same concentrations of NMIQ +. Curve I: TH activity; Curve II: AADC activity to L-DOPA; and Curve IlI: MAO activity. Each point represents the mean and SD of triplicate measurements of three experiments.
(Collins et al., 1979), phenylketonurics (Lasala and Coscia, 1979), and patients with parkinsonism treated with L-DOPA (Collins, 1980). Out of the tetrahydroquinolines, salsolinol and tetrahydropapaveroline were reported to inhibit in vivo and in vitro activity of MAO and catechol-O-methyl transferase (COMT) in rat brain (Giovine et al., 1976), and norlaudanosolinecarboxylic acids inhibit in vitro activity of TH, dopamine-#-hydroxylase and COMT (Coscia et al., 1980). The data presented in this paper show that NMIQ + inhibits in vitro enzyme activity of TH, AADC and MAO in PC12h cells. TH activity in the intact cells, measured without addition of exogenous substrate or co-factor, was inhibited by NMIQ +, while TH activity in sonicated cells was not inhibited in the presence of high concentrations of the reduced form of a co-factor, (6R)BH4, as under the reaction conditions commonly used for the assay of TH activity. The discrepancy may be due to the inhibition of dihydropteridine reductase (NADH: 6,7-dihydropteridine oxidoreductase, EC !.6.99.10) by NMIQ + in the intact cells, as in the case of Y,4'deoxynorlaudanosolinecarboxylic acid or other DA-derived tetrahydroisoquinolines, which inhibit dihydropteridine reductase (Shen et al., 1982). AADC was inhibited by NMIQ + and the inhibition
319
was competitive to PLP, which was observed with MPP + (Naoi et al., 1988a). Type A MAO in PC12h cells was inhibited by NMIQ + competitively with a substrate, kynuramine, and the inhibition was reversible, as reported previously using human brain synaptosomal mitochondria (Naoi et al., 1987a). In the cells cultured with NMIQ +, TH activity was reduced with NMIQ + of low concentrations, while the activities of the other two enzymes, AADC and MAO, were reduced by NMIQ + only at high concentrations. The difference of the effect of NMIQ + on the in vitro and in vivo activities of these enyzmes may be due to difference in the type of inhibition and sensitivity of these enzymes to NMIQ +. MPP + was found to be taken up into the nigrostriatal system by the DA uptake system (Javitch and Snyder, 1985), which was further confirmed using PCI2h cells (Takahashi et al., 1987). NMIQ + was also taken up into PC12h cells by the uptake system common to that of DA, which was confirmed by competition of the uptake with DA and L-DOPA, and by inhibition by Mazindol, an inhibitor of DA uptake. The uptake of NMIQ + was also inhibited by 5-HT, but at present the mechanism of inhibition has not been well elucidated, even though a similar inhibition by 5-HT was observed on the uptake of MPP + into PC12h cells (Takahashi et al., 1987). These results suggest that NMIQ + may be transported and accumulated in dopaminergic neurons, and may perturb the catecholamine metabolism in some specific regions of the human brain. From these points of view, NMIQ + effects catecholamine metabolism in PCI2h cells in a very similar way to MPP +. However, NMIQ + did not cause cell death, which indicates that the mechanism of cell degeneration by MPP + may be different from the mechanism of inhibition of catecholamine metabolism by MPP + and NMIQ +. Acknowledgements--This work was supported by a Grant-
in-Aid for ScientificResearch on Priority Areas, Ministry of Education, Science and Culture, Japan. We wish to thank Ms Hitoe Itoh for her technical assistance. REFERENCES
Bradford M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein using the principle of protein dye binding. Analyt. Biochem. 72, 248-254. Chiba K., Trevor A. and Castagnoli Jr. N. (1984) Metabolism of the neurotroxic tertiary amine, MPTP, by brain monoamine oxidase. Biochem. biophys. Res. Commun. 120, 574-578. Collins M. A. (1980) Neuroamine condensations in human subjects. Adv. exp. reed. Biol. 126, 87 102.
320
MAKOTO NAOI et al.
Collins M. A., Num W. P., Borge G. F., Teas G. and Goldfarb C. (1979) Dopamine-related tetrahydroisoquinolines; significant urinary excretion by alcoholics after alcohol consumption. Science 206, 1184-1186. Coscia C. J., Burke W. J., Galloway M. P., Kosloff A. H., Lasala J. M., McFarlane J., Mitchel J. S., O'Toole M. M. and Roth B. L. (1980) Effects of norlaudanosolinecarboxylic acids on enzymes of catecholamine metabolism. J. Pharmae. exp. Ther. 212, 91 96. Davis G. C., Williams A. C., Markey S. P., Ebert M. H., Caine E. D., Reichert C. M. and Kopin I. J. (1979) Chronic parkinsonism secondary to intravenous injection of meperidine analogues. Psychiat. Res. 1, 249 254. Giovine A., Renis M. and Bertolino A. (1976) In vivo and in vitro studies on the effect of tetrahydopapaverine and salsolinol on COMT and MAO activity in rat brain. Pharmacology 14, 8(~94. Hatanaka H. (1981) Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line. Brain Res. 222, 225-233. Hirata Y., Sugimura H., Takei H. and Nagatsu T. (1986) The effect of pyridinium salts, structurally related compounds of l methyl-4-phenylpyridinium ion (MPP ÷), on tyrosine hydroxylation in rat striatal tissue slices. Brain Res. 397, 341-344. Javitch J. A. and Snyder S. H. (1984) Uptake of MPP + by dopamine neurons explains selectivity of parkinosonisminducing neurotoxin, MPTP. Eur. J. Pharmac. 106, 455-456. Kohno M., Ohta S. and Hirobe M. (1986) Tetrahydroisoquinoline and 1-methyltetrahydroisoquinoline as novel endogenous amines in rat brain. Biochem. biophys. Res. Commun. 140, 448 154. Kraml M. (1965) A rapid microfluorimetric determination of monoamine oxidase. Biochem. Pharmac. 14, 1683-1685. Lasala J. M. and Coscia C. J. (1979) Accumulation of a tetrahydroisoquinoline in phenylketonuria. Science 203, 283-284. Naoi M. and Nagatsu T. (1986a) Specific binding of glycosylated fl-galactosidase to bovine brain synaptic membrane. J. Neurochem. 46, 655-657. Naoi M. and Nagatsu T. (1986b) Inhibition of monoamine oxidase by 3,4-dihydroxyphenylserine. J. Neurochem. 47, 604 607. Naoi M., Hirata Y. and Nagatsu T. (1987a) Inhibition
of monoamine oxidase by N-methylisoquinolinium ion. J. Neurochem. 48, 709 712. Naoi M., Suzuki H., Takahashi T., Shibahara K. and Nagatsu T. (1987b) Ganglioside GM1 causes expression of type B monoamine oxidase in a rat clonal pheochromocytoma cell line, PC12h. J. Neurochem. 49, 1602-1605. Naoi M., Takahashi T., Ichinose H. and Nagatsu T. (1988a) Inhibition of aromatic L-amino acid decarboxylase in clonal pheochromocytoma PCI2h cells by N-methyl-4phenylpyridinium ion (MPP+). Biochem. biophys. Res. Commun. 152, 15 21. Naoi M., Takahashi T. and Nagatsu T. (1988b) Simple assay procedure for tyrosine hydroxylase activity by high-performance liquid chromatography employing coulometric detection with minimal sample preparation. J. Chromat. 427, 229 238. Naoi M., Takahashi T. and Nagatsu T. (1988c) Effect of l-methyl-4-phenylpyridinium ion (MPP ÷) on catecholamine levels and activity of related enzymes in colonal rat pheochromocytoma PCI2h cells. Life Sci. 43, 1485 1491. Naoi M., Matsuura A., Parvez H., Takahashi T., Hirata Y., Minami M. and Nagatsu T. (1989) Oxidation of Nmethyl-l,2,3,4-tetrahydroisoquinoline into the N-methylisoquinolinium ion by monoamine oxidase. J. Neurochem. 52, 653 655. Niwa T., Takeda N., Kaneda N., Hashizume Y. and Nagatsu T. (1987) Presence of tetrahydroisoquinoline and 2-methyl-tetrahydroquinoline in a parkinsonian and normal human brains. Biochem. biophys. Res. Commun. 144, 1084-1089. Shen R., Smith R. V., Davis P. J., Brubaker A. and Abell C. W. (1982) Dopamine-derived tetrahydroisoquinolines. Novel inhibitors of dihydropteridine reductase. J. biol. Chem. 257, 7294-7297. Takahashi T., Naoi M. and Nagatsu T. (1987) Uptake of N-methyl-4-phenylpyridinium ion (MPP ÷) into PC12h pheochromocytoma cells. Neurochem. Int. 11, 89-93. Whatley W. M. and Govindachari T. R (1951) The PictetSpengler synthesis of tetrahydroisoquinolines and related compounds. Org. React. 6, 151-190. Youdim M. B. H., Heldman E., Pollard H. B., Fleming P. and McHugh E. (1986) Contrasting monoamine oxidase activity and tyramine induced catecholamine release in PC12 and chromaffin ceils. Neuroscience 19, 1311-1318.
0197-0186/89 $3.00 + 0.00 Copyright ~:~ 1989 Pergamon Press plc
N-METHYLISOQUINOLINIUM ION AS AN INHIBITOR OF TYROSINE HYDROXYLASE, AROMATIC L-AMINO ACID DECARBOXYLASE AND MONOAMINE OXIDASE MAKOT0 NAOI ~, TSUTOMU TAKAHASHI2, HASAN PARVEZ3, RYOSUKE KABEYAj, EriCH TAGUCHIl, KEIKO YAMAGUCHI~YOKO HIRATA4, MASAYASUMINAMI4 and TOSHIHARU NAGATSU~ Department of Biochemistry, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan, 2Department of Food and Nutrition, Konan Women's College, Konan, Japan, 3Unite de Neuropharmacologie, Universit6 de Paris Xl, Orsay Cedex, France and 4Department of Hygiene and Public Health, Nippon Medical School, Tokyo, Japan (Received 21 December 1988; accepted 12 April 1989) Abstract--The effect of the N-methylisoquinolinium ion (NMIQ +) on the activity of enzymes related to
the metabolism of dopamine was studied using a rat clonal pheochromocytoma PC12h cell line. The activities of tyrosine hydroxylase (TH), aromatic L-amino acid deearboxylase (AADC) and monoamine oxidase (MAO) were inhibited by NMIQ +, but the mechanism of inhibition of these enzymes differed from each other. TH activity in the cells was inhibited by NMIQ + with an ICs0 of about 75/~M. Aromatic L-amino acid decarboxylase (AADC) was also inhibited by NMIQ + but in competition with a co-factor, pyridoxal-5-phosphate, and the K~value was 90 #M. MAO was inhibited by NMIQ + in competition with a substrate, kynuramine, and the K, value was 20 #M. In vivo effects ofNMIQ + on these enzymes in PCI2h cells were examined by culture of the cells in the presence of I00 nM 1 mM NMIQ + for 6 days. After 6 days culture, TH activity was reduced in cells cultured with NMIQ + at concentrations higher than I0 #M, but the activities of AADC and MAO were reduced only in cells cultured with 1 mM NMIQ +. In addition, NMIQ + was transported into the cells by a transport system specific for dopamine. These data suggest that NMIQ + may perturb the catecholamine metabolism of a dopaminergic system in the brain, as a naturally-occurring compound.
Discovery of N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) as a neurotoxin, which degenerates the nigro-striatal system in humans and elicits symptoms very similar to parkinsonism (Davis et al., 1979), suggests to us that there may be similar such exogenous or endogenous compounds in human brains that may cause degeneration in some specific
Address all correspondence and reprint requests to: Dr Makoto Naoi, Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan. Abbreviations: NMIQ +, N-methyl-isoquinolinium ion; NMTIQ, N-methyl-l,2,3,4-tetrahydroisoqainoline; TH, tyrosine hydroxylase; MAO, monoamine oxidase; AADC, aromatic L-amino acid decarboxylase; MPTP, N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine; MPP +, N-methyl-4-phenyl-pyridinium ion; HPLC, high performance liquid chromatography; DA, dopamine; L-DOPA, 3,4-dihydroxy-L-phenylalanine; 5-HTP, 5hydroxytryptophan; 5-HT, 5-hydroxytryptamine; PLP, pyridoxal-5-phosphate; (6R)BH4, (6R)-L-erythro5,6,7,8-tetrahydrobiopterin; ECD, electrochemical detector; COMT, catechol-O-methyltransferase. 315
neurons in the brain. Recently in our laboratory, tetrahydroisoquinoline and 2-methyl-tetrahydroquinoline were found in human brains and the former c o m p o u n d was found to increase in a parkinsonian brain (Niwa et al., 1987). Similar quinoline derivatives were also detected in rat brains (Kohno et al., 1986). By comparison of chemical structure with an oxidative product of MPTP, the N-methyl4-phenylpyridinium ion (MPP+), the N-methylisoquinilinium ion ( N M I Q +) was proposed as a neutrotoxin candidate and in vitro activity of enzymes related to catecholamine metabolism was found to be inhibited by N M I Q +. N M I Q + inhibited tyrosine hydroxylase [tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2, TH] in slices of the nigro-striatal system of rat brains (Hirata et al., 1986). In addition, N M I Q + inhibits type A monoamine oxidase [monoamine: oxygen oxidoreductase (deaminating), EC 1.4.3.4, MAO] in human brain synaptosomal and placental mitochondria (Naoi et al., 1987a). Recently, we found that N M I Q + is produced by M A O in human brain
316
MAKOTO NAOI et al.
s y n a p t o s o m e s from N - m e t h y l - l , 2 , 3 , 4 - t e t r a h y d r o i s o quinoline ( N M T I Q ) (Naoi et al., 1989). E n z y m a t i c p r o d u c t i o n o f the oxidative form o f N M T I Q m a y be c o m p a t i b l e to t h a t o f M P P + from M P T P by m o n o a m i n e oxidase ( C h i b a et al., 1964). These d a t a suggest t h a t N M I Q ÷ m a y be a true toxin to degenerate d o p a m i n e r g i c neurons, which m a y be synthesized from T I Q in h u m a n subjects. To examine in vitro and in vivo effects of N M I Q ÷ on the enzymes participating in the biosynthesis a n d m e t a b o l i s m of catecholamines, a clonal rat p h e n o c h r o m o c y t o m a PC12h cell line was used, because the cells mainly produce d o p a m i n e (DA) as a n e u r o t r a n s m i t t e r ( H a t a n a k a , 1981). M A O in P C 1 2 h cells is of type A (Naoi et al., 1987b; Y o u d i m et al., 1986). This p a p e r describes in vitro a n d in vivo effects o f N M I Q ÷ on the enzyme activities o f TH, M A O a n d a r o m a t i c L-amino acid decarboxylase (aromatic L-amino acid carboxy-lyase, E C 4.1.1.28, A A D C ) in P C 1 2 h cells. N M I Q + was f o u n d to inhibit A A D C activity in a d d i t i o n to T H a m d M A O activity. The cells were cultured for 6 days in the presence of 100 n M - I m M N M I Q ÷ a n d the effects o n the enzyme activity in the cells were examined. EXPERIMENTAL PROCEDURES
Materials NMIQ ÷ was synthesized as reported previously (Hirata et al., 1986). L-Tyrosine, 4-bromo-3-hydroxybenzyloxyamine (NSD-1055), an inhibitor of AADC, 5-hydroxy-L-tryptophan (5-HTP), DA, 3,4-dihydroxy-L-phenylalanine (LDOPA) and pyridoxyal-5-phosphate (PLP) were purchased from Naealai tesque (Kyoto, Japan); kynuramine from Sigma (St Louis, Mo., U.S.A.): 5 hydroxytryptamine (5-HT) from Merck (Darmstadt, F.R.G.); sodium-l-octanesulfonate from Aldrich (Milwaukee, Wis., U.S.A.). Clorgyline, a specific inhibitor of type A MAO, was kindly donated by May & Baker (Dagenham, U.K.). (6R)-L-erythro-5,6,7,8Tetrahydrobiopterin [(6R)BH4] was kindly donated by Dr S. Matsuura, Department of Chemistry, College of General Education, Nagoya University (Nagoya, Japan); and 5(4-chlorophenyl)-2,5-dihydro[3H]imidazo(2, l-~)isoindol-5ol (Mazindol), and inhibitor of DA uptake, was donated by Sandoz (Basel, Switzerland). PC12h cells were cultured as described previously (Naoi et al., 1988c), using a reduced concentration of fetal calf serum and so-called "serum-free" culture medium. The cells were cultured to become confluent, harvested and centrifuged at 600g for 10min. The cells (c. 10~ cells) were cultured in a 25 cm 2 culture dish with the following culture medium: Cosmedium-001 (Cosmo Bio, Tokyo, Japan) containing 5% fetal bovine serum (Gibco Life Technologies, Grand Island, N.Y.U.S.A.), in the absence or presence of 100 n M - l mM NMIQ +. After 3 days culture the culture medium was changed and after 6 days the cells were harvested, washed twice with phosphate-buffered saline and suspended in l0 mM potassium phosphate buffer, pH 7.4. The cell suspension was sonicated in a Branson sonicator for
30 s at a power level of 20. The sonicated cells were kept frozen at - 8 0 ° C and used for the assay of activity of TH, AADC and MAO. Assay for enzyme activity o f TH, A A D C and M A O TH activity in the sonicated cells was assayed in the presence of 200#M L-tyrosine, l mM (6R)BH4, l mM NSD-1055, an inhibitor of AADC and 10#g catalase in 100#1 of 100mM sodium acetate buffer, pH6.0, as described previously (Naoi et al., 1988b). TH activity was determined by measurement of enzymatic conversion of L-tryosine into L-DOPA, which was quantitatively assayed using high performance liquid chromatography (HPLC) with a Coulochem 5100A electrochemical detector (ECD) and a Coulochem 5011 analytical cell (ESA, Bedford, Mass., U.S.A.). AADC activity was measured as reported previously (Naoi et al., 1988a). A PC12h cell suspension (1(~50 pg protein) was incubated with l mM L-DOPA in 100/~1 of 20 mM sodium phosphate buffer, pH 7.2, containing 5 # M pyridoxial-5-phosphate, 0.34mM ascorbic acid and 10/IM clorgyline, a type A MAO inhibitor. After precipitation of protein, DA formed by AADC was quantitatively assayed using PHLC and ECD. MAO activity was measured tluorimetrically using kynuramine as substrate, according to a slightly modified method of Kraml (1965) as reported previously (Naoi et al., 1986b). Kinetical data on the activity of these three enzymes and the effect of NMIQ + were calculated by plotting the data according to Lineweaver and Burke using eight different concentrations of substrate or co-factor or, according to Dixon, using six different concentrations of NMIQ + and three different concentrations of substrate or co-factor. TH activity in the cells was also measured without addition of substrate and co-factor, and in the presence of an AADC inhibitor, NSD-1055. The intact cells (30 #g protein) were suspended in 100 pl of the modified Krebs Ringer solution composed of 126 mM NaC1, 4.75 mM KCI, 1.27 mM CaCI 2, 1.42 mM MgSO 4, 10mM D-glucose, l mM ascorbic acid in 15raM sodium phosphate buffer, pH 7.4, in the presence of 1 mM NSD-1055 and 1 # M - I mM NMIQ +. After 15 min incubation at 37°C, the cell suspension was mixed with 100 pl of 180 mM sodium acetate-70 mM citric acid buffer, pH 4.35, containing 260pM disodium EDTA and 260 # M sodium octanesulfonate, to which methanol was added to 21%. After mixing, the sample was centrifuged at 15,000g for 10min, and the supematant was filtered through a Millipore HV filter. L-DOPA produced by TH in the cells was assayed by HPLC, as described in one of our papers (Naoi et al., 1988c). The amount of protein was measured according to Bradford using bovine y-globulin as standard (Bradford, 1976). Assay o f N M I Q + taken up into PCI2h cells Uptake of NMIQ + into PCI2h cells was examined, using cell suspension in the modified Krebs-Ringer solution. The cell suspension (60/~g protein) in 100 #1 of the Krebs Ringer solution was incubated with 100 nM 1 mM NMIQ + at 37°C for 20 min, then centrifuged at 600g for I0 min. The cells were washed twice with 1.5 ml of phosphate-buffered saline and then suspended in 50/~1 of distilled water. After mixing with 50/~1 of 0.I M perchloric acid containing 0.4 mM sodium metabisulfite and 0.1 mM disodium EDTA, the sample was centrifuged at 15,000g for 10min and filtered. The amount of NMIQ + taken up into cells was quantitatively determined fluorimetrically by HPLC and
N-Methylisoquinolinium ion inhibits dopamine metabolism
317
Table 1. Kinetic data of TH, AADC and MAO in PC12h cells and effect of NMIQ ÷ TH Toward L-tyrosine g m (taM) VmaX (pmol/min/mg protein) Toward (6R)BH a Kin1 (taM) Vma,t (pmol/min/mg protein) Kin2 (taM) Vma~2(pmol/min/mg protein) IC50 of NMIQ +*
19.5 + 6.9 264 + 68 361 + 510 + 87.5 + 267 + 75 +
AADC Toward L-DOPA, using 10taM PLP Km (/2 M) Vmax (nmol/min/mg protein) Toward PLP, using 1 mM L-DOPA as substratet Km (ta M) lima~ (pmol/min/mg protein) Ki of NMIQ ÷ in terms of PLP, using 1 mM L-DOPA MAO Toward kynuramine Km (taM) Vm.. (pmol/min/mg protein) K~ of NMIQ + (taM)
21.4+_ 1.6 1.31 _+ 0.03 0.62_+0.10 436 + 58 89.9 _+ 7.5 taM
58 38 6.4 74 50#M + 1 mM NMIQ ÷ 26.5_+ 1.1 1.48 + 0.22 + 100taM NMIQ 1.31 _+0.27 689 _+ 121
17.8 _+0.9 869 + 30 20.0 _+ 6.5
*lCs0 is the concentration of NMIQ + required to yield 50% inhibition of TH activity in the intact cells. tKinetic data were obtained from the enyzme activity increased by addition of PLP. Each value represents the mean and SD of triplicate measurements of two experiments, using eight different concentrations.
fluorescence detection. An HPLC apparatus, Shimadzu LC4A HPLC (Kyoto, Japan), was used connected to a Shimadzu fluorescence detector RF-500LCA. The column used was a Shimadzu pre-packed reverse-phase column STR
iI A
loo
O D S - H (4 mm i.d. x 150 mm). The sample was eluted at a rate of 0.8 ml per min with 90 mM sodium acetate-35 mM citric acid buffer, pH4.35, containing 1 3 0 p M disodium E D T A and I 1 5 # M sodium octanesulfonate, to which methanol was added to 10.5%. Fluorescence intensity at 3 8 0 n m was monitored with excitation at 3 4 0 n m in a Shimadzu integrator CR3A, and quantitation of N M I Q + was carried out by comparison of the peak area with that of the standard.
/ RESULTS
.>_
"4 o
50
-//
6
5
4
3
- L o g [NM'rQ +] (M) Fig. 1. Effect of N M I Q ÷ on T H activity in intact P C I 2 h cells. P C I 2 h ceils ( 3 0 # g protein) were w a s h e d and susp e n d e d in K r e b s - R i n g e r solution. The cells were i n c u b a t e d for 20 m i n at 37°C in the presence o f 1 m M N S D - 1 0 5 5 a n d in the absence a n d presence o f 1 p M - 1 m M N M I Q +. After t e r m i n a t i o n o f the reaction, the s a m p l e was treated as described in E x p e r i m e n t a l Procedures. E a c h p o i n t represents the m e a n a n d S D o f d u p l i c a t e m e a s u r e m e n t s o f two experiments.
Table 1 summarizes the value of the Michaelis constant (Km) and the maximal velocity (Vmax) of the enzymes used in this experiment. Toward (6R)BH4, T H in PC12h cells was found to have two different types of kinetic properties; one is with a low affinity ( K m l ) and a high activity (Vmaxl) and another was a high affinity (Kin2) and a low activity (Vm,,2). Figure 1 shows that N M I Q + inhibited T H activity in the intact cells in a dose-dependent way, and ICs0 was calculated to be 75 # M . However, when T H activity in the sonicated cells was assayed in the presence of exogenously added L-tyrosine and (6R)BH 4, N M I Q + did not inhibit T H activity. N M I Q + inhibited A A D C activity toward L-DOPA and the inhibition was competitive to a co-factor, PLP, as shown in Fig. 2. In the presence of 1 0 # M PLP, the value of Km and Vmaxof A A D C in terms of L-DOPA were not affected by the presence of even 1 m M N M I Q + (Table 1). The
318
MAKOTO NAOI et al. ReLative uptake of NMIQ (%) 50
1[
o
~-
15
.=_ E io
c
I
Control
E
E
100
i
I
+ L - DOPA (1 raM) I
+ DA (1 m M ) 5
+ 5-HTP (1 m M )
0
!
I
5
10
+ 5-HT (1 rnM) 4- NA (IOOFM)
(/.,/,M -1 )
1/[PLP]
Fig. 2. Effect of PLP concentration and NMIQ + on AADC activity of L-DOPA. PC12h cells (17/~g protein) were incubated with 1 mM L-DOPA in the absence and presence of 250 # M NMIQ +. The reciprocal of the reaction velocity was plotted against that of the PLP concentration, according to Lineweaver and Burk. Curve I: control; Curve II: in the presence of NMIQ +. Each point represents the mean of triplicate measurements of two experiments. inhibitor c o n s t a n t (K~) value o f N M I Q + in terms of P L P was o b t a i n e d to be 89.9 4-7.5 # M , as s h o w n in Table 1. N M I Q + inhibited M A O in the cells and the inhibition was competitive to a substrate, k y n u r a m i n e , as s h o w n in Fig. 3. The K~ value was 20.0 4- 6.5/~M, which is very similar to the Km value with k y n u r a m i n e of 17.8 + 0.9 # M .
54 o
Q.
][
E" 3
t:
E
L o
E
2
0
' 10
' 20
' 30
%
4
I
+ Mozindo L (IOOFM)
Fig. 4. Effects of catecholamines, related compounds and Mazindol on the uptake of NMIQ + in PC12-h cells. PC 12h cells (60 #g protein) were incubated with 1 mM NMIQ + in the presence of 1 mM L-DOPA, DA, 5-hydroxytryptophan (5-HTP) and 5-HT, or 100#M noradrenaline (NA) and Mazindol. After incubation at 37'~C for 20 min, the cells were washed and treated as described in Experimental Procedures. Each value represents the mean and SD of triplicate measurements of two experiments.
N M I Q + was f o u n d to be taken up into P C I 2 h cells at c o n c e n t r a t i o n s higher t h a n 1 0 0 # M . Effects of biogenic amines, the related c o m p o u n d s , a n d Mazindol, an inhibitor of D A uptake system, on N M I Q + uptake are s h o w n in Fig. 4. The uptake was inhibited by L-DOPA, DA, 5-HT a n d Mazindol, but neither 5 - H T P n o r n o r a d r e n a l i n e inhibited the uptake. Effects of the presence of N M I Q ÷ in the culture m e d i u m on the enzyme activities of TH, A A D C a n d M A O in PC12h cells were examined after 6 days culture with 100 n M - i m M o f N M I Q +. Figure 5 shows that the enzyme activity o f T H was reduced in the presence of N M I Q + c o n c e n t r a t i o n s higher t h a n 10#M, a n d the activity was a b o u t 50% of t h a t o f the control. But the activity of A A D C a n d M A O was reduced only in the presence o f 1 m M N M I Q +.
' 60 DISCUSSION
[NMIO +] (FcM)
Fig. 3. Effect of kynuramine concentration and NMIQ + on MAO activity in PC12h cells. MAO activity in PCI2h cells (87 #g protein) was measured in the presence of 33-3 and 50 # M kynuramine and various concentrations of NMIQ +. The reciprocal of the reaction velocity was plotted against NMIQ + concentration, according to Dixon. Curve I: 50#M; Curve II: 33.3#M of kynuramine. Each point represents the mean and SD of triplicate measurements of two experiments.
T I Q a n d o t h e r tetrahydroisoquinolines have been confirmed to be p r o d u c e d from m o n o a m i n e s by c o n d e n s a t i o n o f a ~ - a r y e t h y l a m i n e with a c a r b o n y l c o m p o u n d by a n o n - e n y z m a t i c Pictet-Spengler reaction (Whaley a n d G o v i n d a c h a r i , 1951). These comp o u n d s are considered to be synthesized in vivo in h u m a n subjects at significant rates (Collins, 1980). Elevation of their levels was reported in alcoholics
N-Methylisoquinolinium ion inhibits dopamine metabolism I .,.
Ill
. - , ' / ./.,"----k-'l~ 0.6
o o.
.=_ 0.5
0.4
>
0.3
0
0.2
"o
0.1
g
o ~
0
.=_. E 4
T p--
I
0
f
< //
I 7
-
Log
I 6 [NMIQ
I 5
I 4 +]
I 3 (M)
Fig. 5. Effect of NMIQ + on the activities of TH, AADC and MAO in PCI2h cells cultured in the presence of NMIQ +. PCI2h cells were cultured in the presence of 100 nM 1 mM NMIQ ÷ for 6 days. At day 3, the culture medium was changed with the medium containing the same concentrations of NMIQ +. Curve I: TH activity; Curve II: AADC activity to L-DOPA; and Curve IlI: MAO activity. Each point represents the mean and SD of triplicate measurements of three experiments.
(Collins et al., 1979), phenylketonurics (Lasala and Coscia, 1979), and patients with parkinsonism treated with L-DOPA (Collins, 1980). Out of the tetrahydroquinolines, salsolinol and tetrahydropapaveroline were reported to inhibit in vivo and in vitro activity of MAO and catechol-O-methyl transferase (COMT) in rat brain (Giovine et al., 1976), and norlaudanosolinecarboxylic acids inhibit in vitro activity of TH, dopamine-#-hydroxylase and COMT (Coscia et al., 1980). The data presented in this paper show that NMIQ + inhibits in vitro enzyme activity of TH, AADC and MAO in PC12h cells. TH activity in the intact cells, measured without addition of exogenous substrate or co-factor, was inhibited by NMIQ +, while TH activity in sonicated cells was not inhibited in the presence of high concentrations of the reduced form of a co-factor, (6R)BH4, as under the reaction conditions commonly used for the assay of TH activity. The discrepancy may be due to the inhibition of dihydropteridine reductase (NADH: 6,7-dihydropteridine oxidoreductase, EC !.6.99.10) by NMIQ + in the intact cells, as in the case of Y,4'deoxynorlaudanosolinecarboxylic acid or other DA-derived tetrahydroisoquinolines, which inhibit dihydropteridine reductase (Shen et al., 1982). AADC was inhibited by NMIQ + and the inhibition
319
was competitive to PLP, which was observed with MPP + (Naoi et al., 1988a). Type A MAO in PC12h cells was inhibited by NMIQ + competitively with a substrate, kynuramine, and the inhibition was reversible, as reported previously using human brain synaptosomal mitochondria (Naoi et al., 1987a). In the cells cultured with NMIQ +, TH activity was reduced with NMIQ + of low concentrations, while the activities of the other two enzymes, AADC and MAO, were reduced by NMIQ + only at high concentrations. The difference of the effect of NMIQ + on the in vitro and in vivo activities of these enyzmes may be due to difference in the type of inhibition and sensitivity of these enzymes to NMIQ +. MPP + was found to be taken up into the nigrostriatal system by the DA uptake system (Javitch and Snyder, 1985), which was further confirmed using PCI2h cells (Takahashi et al., 1987). NMIQ + was also taken up into PC12h cells by the uptake system common to that of DA, which was confirmed by competition of the uptake with DA and L-DOPA, and by inhibition by Mazindol, an inhibitor of DA uptake. The uptake of NMIQ + was also inhibited by 5-HT, but at present the mechanism of inhibition has not been well elucidated, even though a similar inhibition by 5-HT was observed on the uptake of MPP + into PC12h cells (Takahashi et al., 1987). These results suggest that NMIQ + may be transported and accumulated in dopaminergic neurons, and may perturb the catecholamine metabolism in some specific regions of the human brain. From these points of view, NMIQ + effects catecholamine metabolism in PCI2h cells in a very similar way to MPP +. However, NMIQ + did not cause cell death, which indicates that the mechanism of cell degeneration by MPP + may be different from the mechanism of inhibition of catecholamine metabolism by MPP + and NMIQ +. Acknowledgements--This work was supported by a Grant-
in-Aid for ScientificResearch on Priority Areas, Ministry of Education, Science and Culture, Japan. We wish to thank Ms Hitoe Itoh for her technical assistance. REFERENCES
Bradford M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein using the principle of protein dye binding. Analyt. Biochem. 72, 248-254. Chiba K., Trevor A. and Castagnoli Jr. N. (1984) Metabolism of the neurotroxic tertiary amine, MPTP, by brain monoamine oxidase. Biochem. biophys. Res. Commun. 120, 574-578. Collins M. A. (1980) Neuroamine condensations in human subjects. Adv. exp. reed. Biol. 126, 87 102.
320
MAKOTO NAOI et al.
Collins M. A., Num W. P., Borge G. F., Teas G. and Goldfarb C. (1979) Dopamine-related tetrahydroisoquinolines; significant urinary excretion by alcoholics after alcohol consumption. Science 206, 1184-1186. Coscia C. J., Burke W. J., Galloway M. P., Kosloff A. H., Lasala J. M., McFarlane J., Mitchel J. S., O'Toole M. M. and Roth B. L. (1980) Effects of norlaudanosolinecarboxylic acids on enzymes of catecholamine metabolism. J. Pharmae. exp. Ther. 212, 91 96. Davis G. C., Williams A. C., Markey S. P., Ebert M. H., Caine E. D., Reichert C. M. and Kopin I. J. (1979) Chronic parkinsonism secondary to intravenous injection of meperidine analogues. Psychiat. Res. 1, 249 254. Giovine A., Renis M. and Bertolino A. (1976) In vivo and in vitro studies on the effect of tetrahydopapaverine and salsolinol on COMT and MAO activity in rat brain. Pharmacology 14, 8(~94. Hatanaka H. (1981) Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line. Brain Res. 222, 225-233. Hirata Y., Sugimura H., Takei H. and Nagatsu T. (1986) The effect of pyridinium salts, structurally related compounds of l methyl-4-phenylpyridinium ion (MPP ÷), on tyrosine hydroxylation in rat striatal tissue slices. Brain Res. 397, 341-344. Javitch J. A. and Snyder S. H. (1984) Uptake of MPP + by dopamine neurons explains selectivity of parkinosonisminducing neurotoxin, MPTP. Eur. J. Pharmac. 106, 455-456. Kohno M., Ohta S. and Hirobe M. (1986) Tetrahydroisoquinoline and 1-methyltetrahydroisoquinoline as novel endogenous amines in rat brain. Biochem. biophys. Res. Commun. 140, 448 154. Kraml M. (1965) A rapid microfluorimetric determination of monoamine oxidase. Biochem. Pharmac. 14, 1683-1685. Lasala J. M. and Coscia C. J. (1979) Accumulation of a tetrahydroisoquinoline in phenylketonuria. Science 203, 283-284. Naoi M. and Nagatsu T. (1986a) Specific binding of glycosylated fl-galactosidase to bovine brain synaptic membrane. J. Neurochem. 46, 655-657. Naoi M. and Nagatsu T. (1986b) Inhibition of monoamine oxidase by 3,4-dihydroxyphenylserine. J. Neurochem. 47, 604 607. Naoi M., Hirata Y. and Nagatsu T. (1987a) Inhibition
of monoamine oxidase by N-methylisoquinolinium ion. J. Neurochem. 48, 709 712. Naoi M., Suzuki H., Takahashi T., Shibahara K. and Nagatsu T. (1987b) Ganglioside GM1 causes expression of type B monoamine oxidase in a rat clonal pheochromocytoma cell line, PC12h. J. Neurochem. 49, 1602-1605. Naoi M., Takahashi T., Ichinose H. and Nagatsu T. (1988a) Inhibition of aromatic L-amino acid decarboxylase in clonal pheochromocytoma PCI2h cells by N-methyl-4phenylpyridinium ion (MPP+). Biochem. biophys. Res. Commun. 152, 15 21. Naoi M., Takahashi T. and Nagatsu T. (1988b) Simple assay procedure for tyrosine hydroxylase activity by high-performance liquid chromatography employing coulometric detection with minimal sample preparation. J. Chromat. 427, 229 238. Naoi M., Takahashi T. and Nagatsu T. (1988c) Effect of l-methyl-4-phenylpyridinium ion (MPP ÷) on catecholamine levels and activity of related enzymes in colonal rat pheochromocytoma PCI2h cells. Life Sci. 43, 1485 1491. Naoi M., Matsuura A., Parvez H., Takahashi T., Hirata Y., Minami M. and Nagatsu T. (1989) Oxidation of Nmethyl-l,2,3,4-tetrahydroisoquinoline into the N-methylisoquinolinium ion by monoamine oxidase. J. Neurochem. 52, 653 655. Niwa T., Takeda N., Kaneda N., Hashizume Y. and Nagatsu T. (1987) Presence of tetrahydroisoquinoline and 2-methyl-tetrahydroquinoline in a parkinsonian and normal human brains. Biochem. biophys. Res. Commun. 144, 1084-1089. Shen R., Smith R. V., Davis P. J., Brubaker A. and Abell C. W. (1982) Dopamine-derived tetrahydroisoquinolines. Novel inhibitors of dihydropteridine reductase. J. biol. Chem. 257, 7294-7297. Takahashi T., Naoi M. and Nagatsu T. (1987) Uptake of N-methyl-4-phenylpyridinium ion (MPP ÷) into PC12h pheochromocytoma cells. Neurochem. Int. 11, 89-93. Whatley W. M. and Govindachari T. R (1951) The PictetSpengler synthesis of tetrahydroisoquinolines and related compounds. Org. React. 6, 151-190. Youdim M. B. H., Heldman E., Pollard H. B., Fleming P. and McHugh E. (1986) Contrasting monoamine oxidase activity and tyramine induced catecholamine release in PC12 and chromaffin ceils. Neuroscience 19, 1311-1318.