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Inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity and ATP synthesis by tetrahydroisoquinoline
Neuroscience Letters, 86 (1988) 105-108
105
Elsevier Scientific Publishers Ireland Ltd. NSL 05175
Inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity and ATP synthesis by tetrahydroisoquinoline Keiji Suzuki, Yoshikuni Mizuno and Mitsuo Yoshida Department of Neurology, Jichi Medical School, Tochigi-ken (Japan) (Received 9 November 1987; Accepted 24 November 1987)
Key words: Tetrahydroisoquinoline; NADH-ubiquinone oxidoreductase (complex I); Adenosine triphosphate (ATP); Mitochondrion; Mitochondrial respiration; Parkinson's disease Effects of tetrahydroisoquinoline (TIQ) on mitochondrial respiration, NADH-ubiquinone oxidoreductase (complex I) activity and on adenosine triphosphate (ATP) synthesis were studied using mitochondria prepared from mouse brains. Tetrahydroisoquinoline significantly inhibited mitochondrial respiration supported by glutamate + malate, pyruvate + malate or ct-ketoglutarate. Activity of complex I and synthesis of ATP were also significantly inhibited by TIQ. Mitochondrial respiration supported by succinate and subsequent ATP synthesis were not inhibited at all by 5 mM of TIQ. Our study has revealed a novel action of TIQ, which has been proposed as a candidate for an endogenous substance that may induce Parkinson's disease.
The etiology of Parkinson's disease remains unknown. It has been proposed that some environmental or endogenous substances may induce this disease. 1-Methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP) was found to produce Parkinson-like syndrome [5]. It has been shown that an oxidation product of MPTP, 1-methyl-4phenylpyridinium ion (MPP +) [4], is responsible for degeneration of the nigrostriatal dopaminergic neurons [3, 5]. It has also been reported that MPP ÷ inhibits the activity of NADH-ubiquinone oxidoreductase (complex I) [7] and the synthesis of adenosine triphosphate (ATP) in mouse brains [8]. Recently, a great effort has been paid to find an endogenous substance which is toxic to nigrostriatal dopaminergic neurons. 1,2,3,4-Tetrahydroisoquinoline (TIQ) has emerged as one of such examples [9]. Although this substance has been identified in human brains [10], its pharmacological properties have not been elucidated yet. We have studied effects of TIQ on mitochondrial respiration, complex I activity and on ATP synthesis in mouse brains. T w o - t o 3 - m o n t h - o l d m a l e C 5 7 / B L m i c e w e r e u s e d . A f t e r s a c r i f i c e b y c e r v i c a l dis-
Correspondence." K. Suzuki, Department of Neurology, Jichi Medical School, Tochigi-ken, Japan 329-04. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
106 location, m i t o c h o n d r i a l suspensions were p r e p a r e d from whole b r a i n s a c c o r d i n g to the m e t h o d o f O z a w a et al [11]. Po pellets were suspended in a m e d i u m c o n t a i n i n g (in m M ) : m a n n i t o l 300, KC1 10, p o t a s s i u m p h o s p h a t e buffer p H 7.5 10, Tris-HC1 buffer p H 7.4 10, E D T A d i s o d i u m salt 0.2, MgSO4 4 a n d 0.2% bovine serum a l b u m i n . Oxygen c o n s u m p t i o n was m e a s u r e d p o l a r o g r a p h i c a l l y using a C l a r k - t y p e oxygen electrode with g l u t a m a t e + m a l a t e , p y r u v a t e + m a l a t e , ~ - k e t o g l u t a r a t e o r succinate (with r o t e n o n e ) as a s u b s t r a t e at a final c o n c e n t r a t i o n o f 5 m M . A d e n o s i n e d i p h o s p h a t e ( A D P ) was a d d e d at a final c o n c e n t r a t i o n o f 0.375 m M except in the case o f succinate. In the latter case, 0.225 m M was used. T e t r a h y d r o i s o q u i n o l i n e was a d d e d s i m u l t a n e o u s l y with each s u b s t r a t e at a final c o n c e n t r a t i o n o f 5 m M . A t the end o f p o l a r o g r a p h y , an a l i q u o t o f the suspension was transferred to a l - m l cuvette, a n d activity o f c o m p l e x I was assayed a c c o r d i n g to t h e m e t h o d o f Hatefi [1]. A n o t h e r aliq u o t was used for the assay o f A T P c o n t e n t a c c o r d i n g to the m e t h o d o f J a w o r e k and Welsch [2] with a m o d i f i c a t i o n as described before [8]. The e q u i p m e n t used was a S h i m a d z u UV-120 s p e c t r o p h o t o m e t e r with a n i s o t h e r m a l cell-holder a n d an a u t o matic recorder. M o l a r extinction coefficient o f N A D H o f 6270 was used for the calculation. P r o t e i n was a s s a y e d a c c o r d i n g to the m e t h o d o f L o w r y et al. [6]. Results were analyzed statistically using the S t u d e n t ' s t-test. Results are s u m m a r i z e d in T a b l e I. State 3 r e s p i r a t i o n s u p p o r t e d by g l u t a m a t e + m a l a t e was significantly inhibited to 44% o f the c o n t r o l b y T I Q at a final c o n c e n t r a tion o f 5 m M . State 3 r e s p i r a t i o n s u p p o r t e d by p y r u v a t e + m a l a t e was inhibited to a p p r o x i m a t e l y 40% o f control, a n d that by ~ - k e t o g l u t a r a t e to 39%. R e s p i r a t i o n supp o r t e d by succinate was slightly inhibited w i t h o u t statistical significance. However, TABLE 1 EFFECTS OF TETRAHYDROISOQUINOLINE (TIQ) Mean _+S.E.M. (n = 4). Units: state 3, nmol of oxygen/min mg protein; ATP, nmol of ATP formed after incubation of mitochondria with the substrate and ADP with or without TIQ (amount of ADP added = 750 nmol); complex I, nmol of NADH oxidized/min mg protein. Substrates were added at a final concentration of 5 mM. TIQ was added simultaneously with the substrate at a final concentration of 5 mM. Non-paired t-test as compared with respective controls, *P < 0.001, **P < 0.01. Substrate Glutamate + malate Control TIQ Pyruvate + malate Control TIQ ct-Ketoglutarate Control TIQ Succinate Control TIQ
State 3
ATP
Complex 1
101.8_+ 7.8 56.8+ 5.1"
581-t- 51 478-t- 7**
167.7_+13.5 117.0_+10.1"
91.0_+ 10.7 54.2_+ 7.5*
n.d. n.d.
n.d. n.d.
39.6+ 5.1 24.1 _+ 8.9**
580-+ 67 265_+ 128"*
133.0-+ 6.3 83.4_+ 10.2"
94.1 _+20.6 77.3_+22.5
395 ± 26 377+ 36
n.d. n.d.
107 with a higher dose of T I Q (10 mM), oxygen consumption of state 3 respiration was significantly inhibited to 71% (data not shown). Synthesis of A T P was significantly inhibited when glutamate + m a l a t e or ~-ketoglutarate was used as substrate. However, synthesis of ATP was not inhibited when succinate was used as a substrate. Activity of complex I was also significantly inhibited by T I Q when glutamate + malate or ~-ketoglutarate was used. Tetrahydroisoquinoline, having a structure closely related to M P P ÷, has been proposed as a candidate for an endogenous toxic substance which may induce Parkinson's disease, since it was found in the brain of a patient with Parkinson's disease in an increased amount [10] and also showed a toxic effect on the nigrostriatal dopaminergic system in experimental animals [9]. It appears to be important to elucidate pharmacological properties of TIQ. In the present study, we showed inhibition of mitochondrial respiration and A T P synthesis by TIQ. Inhibition o f complex I by T I Q appeared to be one of the reasons for the inhibition o f mitochondrial respiration. Since the magnitude of inhibition of state 3 respiration by T I Q was larger than that of inhibition of complex I activity, we could not rule out the presence of other sites of action of TIQ. The effects of T I Q on mitochondrial respiratory functions were quite similar to those of M P P ÷. We previously reported inhibition o f mitochondrial respiration, A T P synthesis and N A D H ubiquinone oxidoreductase activity by MPP ÷ [7, 8] and postulated energy crisis as a pathogenetic mechanism of MPTP-induced parkinsonism. One o f the differences between M P P ÷ and T I Q was the concentration of those substances which could induce significant inhibition of mitochondrial respiration. Although M P P ÷ was effective at 0.05 m M concentration, 5 m M of T I Q was necessary to produce the inhibition. Therefore, presence of another more sensitive site of action of TIQ cannot be ruled out. Also, causal relationship between T I Q and Parkinson's disease has not been established yet. Nonetheless, it appears to be important to continue efforts to identify endogenous substances which have pharmacological and toxicological properties similar to M P P ÷. Studies on effects on mitochondrial respiration may serve as one of the screening methods to find such substances. We greatly appreciate the technical assistance of Miss Michiko Hashimoto. The study was supported by a Grant-in-Aid for Priority Areas from the Ministry of Education, Science and Culture, Japan.
1 Hatefi, Y., Preparation and properties of NADH: ubiquinone oxidoreductase (complex I), EC 1.6.5.3. In S. Fleischer and L. Packer (Eds.), Methods in Enzymology,Vol. 53, Academic, New York, 1978, pp. ll 14. 2 Jaworek, D. and Welsch, J., Adenosine 5'-triphosphate, UV-method with phosphoglycerate kinase. In H.U. Bergmeyer,J. Bergmeyerand M. Grassl (Eds.), Methods of Enzymatic Analysis, Vol. VII, VCH, Weinheim, 1985, pp. 340-346. 3 Jenner, P., Rupniak, N.M.J., Rose, S., Kelly, E., Kilpatrick, G.L., Lees, A. and Marsden, C.D., lMethyl-4-phenyl-l,2,3,6-tetrahydropyridine-induced parkinsonism in the common marmoset, Neurosci. Lett., 50 (1984) 85-90. 4 Langston, J.W., Irwin, I., Langston, E.B. and Forno, L.S., l-Methyl-4-phenylpyridiniumion (MPP*):
108
5 6 7 8 9
10
11
identification of metabolite of MPTP, a toxin selective to the substantia nigra, Neurosci. Lett., 48 (1984) 87-92. Langston, J.W., MPTP neurotoxicity: an overview and characterization of phases of toxicity, Life Sci., 36 (1985) 201 206. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 258 (1951) 2285-2292. Mizuno, Y., Saitoh, T. and Sone, N., Inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity by l-methyl-4-phenylpyridinium ion, Biochem. Biophys. Res. Commun., 143 (1987) 294-299. Mizuno, Y., Suzuki, K., Sone, N. and Saitoh, T., Inhibition of ATP synthesis by 1-methyl-4-phenylpyridinium ion (MPP ÷) in isolated mitochondria from mouse brains, Neurosci. Lett., in press. Nagatsu, T. and Hirata, Y., Inhibition of tyrosine hydroxylase system by MPTP, l-methyl-4-phenylpyridinium ion (MPP ÷) and the structurally related compounds in vitro and in vivo, Eur. Neurol., 26 Suppl. 1 (1987) 11--15. Niwa, T., Takeda, N., Kaneda, N., Hashizume, Y. and Nagatsu, T., Presence of tetrahydroisoquinoline and 2-methyl-tetrahydroquinoline in parkinsonian and human brains, Biochem. Biophys. Res. Commun., 144 (1987) 1084-1089. Ozawa, K., Seta, K., Takeda, H., Ando, K., Handa, H. and Araki, C., On the isolation of mitochondria with high respiratory control from rat brain, J. Biochem., 59 (1966) 501 510.
105
Elsevier Scientific Publishers Ireland Ltd. NSL 05175
Inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity and ATP synthesis by tetrahydroisoquinoline Keiji Suzuki, Yoshikuni Mizuno and Mitsuo Yoshida Department of Neurology, Jichi Medical School, Tochigi-ken (Japan) (Received 9 November 1987; Accepted 24 November 1987)
Key words: Tetrahydroisoquinoline; NADH-ubiquinone oxidoreductase (complex I); Adenosine triphosphate (ATP); Mitochondrion; Mitochondrial respiration; Parkinson's disease Effects of tetrahydroisoquinoline (TIQ) on mitochondrial respiration, NADH-ubiquinone oxidoreductase (complex I) activity and on adenosine triphosphate (ATP) synthesis were studied using mitochondria prepared from mouse brains. Tetrahydroisoquinoline significantly inhibited mitochondrial respiration supported by glutamate + malate, pyruvate + malate or ct-ketoglutarate. Activity of complex I and synthesis of ATP were also significantly inhibited by TIQ. Mitochondrial respiration supported by succinate and subsequent ATP synthesis were not inhibited at all by 5 mM of TIQ. Our study has revealed a novel action of TIQ, which has been proposed as a candidate for an endogenous substance that may induce Parkinson's disease.
The etiology of Parkinson's disease remains unknown. It has been proposed that some environmental or endogenous substances may induce this disease. 1-Methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP) was found to produce Parkinson-like syndrome [5]. It has been shown that an oxidation product of MPTP, 1-methyl-4phenylpyridinium ion (MPP +) [4], is responsible for degeneration of the nigrostriatal dopaminergic neurons [3, 5]. It has also been reported that MPP ÷ inhibits the activity of NADH-ubiquinone oxidoreductase (complex I) [7] and the synthesis of adenosine triphosphate (ATP) in mouse brains [8]. Recently, a great effort has been paid to find an endogenous substance which is toxic to nigrostriatal dopaminergic neurons. 1,2,3,4-Tetrahydroisoquinoline (TIQ) has emerged as one of such examples [9]. Although this substance has been identified in human brains [10], its pharmacological properties have not been elucidated yet. We have studied effects of TIQ on mitochondrial respiration, complex I activity and on ATP synthesis in mouse brains. T w o - t o 3 - m o n t h - o l d m a l e C 5 7 / B L m i c e w e r e u s e d . A f t e r s a c r i f i c e b y c e r v i c a l dis-
Correspondence." K. Suzuki, Department of Neurology, Jichi Medical School, Tochigi-ken, Japan 329-04. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
106 location, m i t o c h o n d r i a l suspensions were p r e p a r e d from whole b r a i n s a c c o r d i n g to the m e t h o d o f O z a w a et al [11]. Po pellets were suspended in a m e d i u m c o n t a i n i n g (in m M ) : m a n n i t o l 300, KC1 10, p o t a s s i u m p h o s p h a t e buffer p H 7.5 10, Tris-HC1 buffer p H 7.4 10, E D T A d i s o d i u m salt 0.2, MgSO4 4 a n d 0.2% bovine serum a l b u m i n . Oxygen c o n s u m p t i o n was m e a s u r e d p o l a r o g r a p h i c a l l y using a C l a r k - t y p e oxygen electrode with g l u t a m a t e + m a l a t e , p y r u v a t e + m a l a t e , ~ - k e t o g l u t a r a t e o r succinate (with r o t e n o n e ) as a s u b s t r a t e at a final c o n c e n t r a t i o n o f 5 m M . A d e n o s i n e d i p h o s p h a t e ( A D P ) was a d d e d at a final c o n c e n t r a t i o n o f 0.375 m M except in the case o f succinate. In the latter case, 0.225 m M was used. T e t r a h y d r o i s o q u i n o l i n e was a d d e d s i m u l t a n e o u s l y with each s u b s t r a t e at a final c o n c e n t r a t i o n o f 5 m M . A t the end o f p o l a r o g r a p h y , an a l i q u o t o f the suspension was transferred to a l - m l cuvette, a n d activity o f c o m p l e x I was assayed a c c o r d i n g to t h e m e t h o d o f Hatefi [1]. A n o t h e r aliq u o t was used for the assay o f A T P c o n t e n t a c c o r d i n g to the m e t h o d o f J a w o r e k and Welsch [2] with a m o d i f i c a t i o n as described before [8]. The e q u i p m e n t used was a S h i m a d z u UV-120 s p e c t r o p h o t o m e t e r with a n i s o t h e r m a l cell-holder a n d an a u t o matic recorder. M o l a r extinction coefficient o f N A D H o f 6270 was used for the calculation. P r o t e i n was a s s a y e d a c c o r d i n g to the m e t h o d o f L o w r y et al. [6]. Results were analyzed statistically using the S t u d e n t ' s t-test. Results are s u m m a r i z e d in T a b l e I. State 3 r e s p i r a t i o n s u p p o r t e d by g l u t a m a t e + m a l a t e was significantly inhibited to 44% o f the c o n t r o l b y T I Q at a final c o n c e n t r a tion o f 5 m M . State 3 r e s p i r a t i o n s u p p o r t e d by p y r u v a t e + m a l a t e was inhibited to a p p r o x i m a t e l y 40% o f control, a n d that by ~ - k e t o g l u t a r a t e to 39%. R e s p i r a t i o n supp o r t e d by succinate was slightly inhibited w i t h o u t statistical significance. However, TABLE 1 EFFECTS OF TETRAHYDROISOQUINOLINE (TIQ) Mean _+S.E.M. (n = 4). Units: state 3, nmol of oxygen/min mg protein; ATP, nmol of ATP formed after incubation of mitochondria with the substrate and ADP with or without TIQ (amount of ADP added = 750 nmol); complex I, nmol of NADH oxidized/min mg protein. Substrates were added at a final concentration of 5 mM. TIQ was added simultaneously with the substrate at a final concentration of 5 mM. Non-paired t-test as compared with respective controls, *P < 0.001, **P < 0.01. Substrate Glutamate + malate Control TIQ Pyruvate + malate Control TIQ ct-Ketoglutarate Control TIQ Succinate Control TIQ
State 3
ATP
Complex 1
101.8_+ 7.8 56.8+ 5.1"
581-t- 51 478-t- 7**
167.7_+13.5 117.0_+10.1"
91.0_+ 10.7 54.2_+ 7.5*
n.d. n.d.
n.d. n.d.
39.6+ 5.1 24.1 _+ 8.9**
580-+ 67 265_+ 128"*
133.0-+ 6.3 83.4_+ 10.2"
94.1 _+20.6 77.3_+22.5
395 ± 26 377+ 36
n.d. n.d.
107 with a higher dose of T I Q (10 mM), oxygen consumption of state 3 respiration was significantly inhibited to 71% (data not shown). Synthesis of A T P was significantly inhibited when glutamate + m a l a t e or ~-ketoglutarate was used as substrate. However, synthesis of ATP was not inhibited when succinate was used as a substrate. Activity of complex I was also significantly inhibited by T I Q when glutamate + malate or ~-ketoglutarate was used. Tetrahydroisoquinoline, having a structure closely related to M P P ÷, has been proposed as a candidate for an endogenous toxic substance which may induce Parkinson's disease, since it was found in the brain of a patient with Parkinson's disease in an increased amount [10] and also showed a toxic effect on the nigrostriatal dopaminergic system in experimental animals [9]. It appears to be important to elucidate pharmacological properties of TIQ. In the present study, we showed inhibition of mitochondrial respiration and A T P synthesis by TIQ. Inhibition o f complex I by T I Q appeared to be one of the reasons for the inhibition o f mitochondrial respiration. Since the magnitude of inhibition of state 3 respiration by T I Q was larger than that of inhibition of complex I activity, we could not rule out the presence of other sites of action of TIQ. The effects of T I Q on mitochondrial respiratory functions were quite similar to those of M P P ÷. We previously reported inhibition o f mitochondrial respiration, A T P synthesis and N A D H ubiquinone oxidoreductase activity by MPP ÷ [7, 8] and postulated energy crisis as a pathogenetic mechanism of MPTP-induced parkinsonism. One o f the differences between M P P ÷ and T I Q was the concentration of those substances which could induce significant inhibition of mitochondrial respiration. Although M P P ÷ was effective at 0.05 m M concentration, 5 m M of T I Q was necessary to produce the inhibition. Therefore, presence of another more sensitive site of action of TIQ cannot be ruled out. Also, causal relationship between T I Q and Parkinson's disease has not been established yet. Nonetheless, it appears to be important to continue efforts to identify endogenous substances which have pharmacological and toxicological properties similar to M P P ÷. Studies on effects on mitochondrial respiration may serve as one of the screening methods to find such substances. We greatly appreciate the technical assistance of Miss Michiko Hashimoto. The study was supported by a Grant-in-Aid for Priority Areas from the Ministry of Education, Science and Culture, Japan.
1 Hatefi, Y., Preparation and properties of NADH: ubiquinone oxidoreductase (complex I), EC 1.6.5.3. In S. Fleischer and L. Packer (Eds.), Methods in Enzymology,Vol. 53, Academic, New York, 1978, pp. ll 14. 2 Jaworek, D. and Welsch, J., Adenosine 5'-triphosphate, UV-method with phosphoglycerate kinase. In H.U. Bergmeyer,J. Bergmeyerand M. Grassl (Eds.), Methods of Enzymatic Analysis, Vol. VII, VCH, Weinheim, 1985, pp. 340-346. 3 Jenner, P., Rupniak, N.M.J., Rose, S., Kelly, E., Kilpatrick, G.L., Lees, A. and Marsden, C.D., lMethyl-4-phenyl-l,2,3,6-tetrahydropyridine-induced parkinsonism in the common marmoset, Neurosci. Lett., 50 (1984) 85-90. 4 Langston, J.W., Irwin, I., Langston, E.B. and Forno, L.S., l-Methyl-4-phenylpyridiniumion (MPP*):
108
5 6 7 8 9
10
11
identification of metabolite of MPTP, a toxin selective to the substantia nigra, Neurosci. Lett., 48 (1984) 87-92. Langston, J.W., MPTP neurotoxicity: an overview and characterization of phases of toxicity, Life Sci., 36 (1985) 201 206. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 258 (1951) 2285-2292. Mizuno, Y., Saitoh, T. and Sone, N., Inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity by l-methyl-4-phenylpyridinium ion, Biochem. Biophys. Res. Commun., 143 (1987) 294-299. Mizuno, Y., Suzuki, K., Sone, N. and Saitoh, T., Inhibition of ATP synthesis by 1-methyl-4-phenylpyridinium ion (MPP ÷) in isolated mitochondria from mouse brains, Neurosci. Lett., in press. Nagatsu, T. and Hirata, Y., Inhibition of tyrosine hydroxylase system by MPTP, l-methyl-4-phenylpyridinium ion (MPP ÷) and the structurally related compounds in vitro and in vivo, Eur. Neurol., 26 Suppl. 1 (1987) 11--15. Niwa, T., Takeda, N., Kaneda, N., Hashizume, Y. and Nagatsu, T., Presence of tetrahydroisoquinoline and 2-methyl-tetrahydroquinoline in parkinsonian and human brains, Biochem. Biophys. Res. Commun., 144 (1987) 1084-1089. Ozawa, K., Seta, K., Takeda, H., Ando, K., Handa, H. and Araki, C., On the isolation of mitochondria with high respiratory control from rat brain, J. Biochem., 59 (1966) 501 510.