Naunyn-Schmiedeberg's

Archivesof Pharmacology

Naunyn-Schmiedeberg's Arch Pharmacol (1989) 339:424-432

© Springer-Verlag 1989

Inhibition of biopterin synthesis and DOPA production in PC-12 pheochromocytoma cells induced by 6-aminonicotinamide W. Jung and H. Herken Institut fiir Pharmakologie, Freie Universitfit Berlin, Thielallee 69/73, D-1000 Berlin 33

Summary. Pheochromocytoma cells (clone PC-12) were treated with 6-aminonicotinamide. Tetrahydrobiopterin content and DOPA production of the cells were determined by reverse-phase ItPLC and subsequent electrochemical detection. The same chromatographic system was used to determine total biopterin (tetrahydrobiopterin, dihydrobiopterin and quinoide dihydrobiopterin) by fluorescence detection. Tetrahydrobiopterin plays a decisive role as cofactor of tyrosine hydroxylase for the biosynthesis of DOPA and dopamine. Addition of 6-aminonicotinamide to the culture medium resulted in the accumulation of 6-phosphogluconate, suggesting that PC-12 cells synthesize 6-aminonicotinamideadenine-dinucleotide-phosphate (6-ANADP) by a glycohydrolase localized in the endoplasmic reticulum. This substance is known to be a strong inhibitor of 6phosphogluconate dehydrogenase and leads to a blockade of the pentose phosphate pathway. In our experiments, the synthesis of biopterins was depressed after application of 6aminonicotinamide. The decrease of intracellular tetrahydrobiopterin and total biopterin by 6-aminonicotinamide at different concentrations was strongly correlated with a reduced cellular DOPA production. The decreased content of biopterin cofactor was compensated by addition of the precursor sepiapterin, indicating that the NADPH2-dependent reductases in biopterin synthesis are not inhibited by the antimetabolite. However, DOPA production rermained suppressed at the same time. After application of NADH2, we observed an increased DOPA production though the decreased biopterin levels remained almost unchanged. The results imply that the first step in the synthesis of biopterin from GTP as well as the recycling pathway of the oxidized cofactor might be the site of action of the antimetabolite. Restriction of the synthesis of the biopterin cofactor might be a pathogenetically important disorder at the initial phase of Parkinson's disease. Key words: 6-Aminonicotinamide - Biopterin synthesis DOPA production - Dihydropteridine reductase Sepiapterin reductase

Introduction Parkinson's disease is characterized biochemically by a disturbance of dopamine metabolism in the nigrostriatal system, the degree of neuropathy being determined by the destruction of nerve cells in the compact layer of the substantia Send offprint requests to H. Herken at the above address

nigra. Analyses of such functional disorders led to the conclusion that Parkinson's disease is a striatal dopamine deficiency syndrome revealed by increasing muscular rigidity (Hornykiewicz 1962, 1972; Hassler 1972; Bernheimer et al. 1973). Tetrahydrobiopterin plays a decisive role as cofactor of tyrosine hydroxylase in the functional system responsible for the biosynthesis of DOPA and dopamine. This compound supplies the reduction equivalents necessary for activation of the molecular oxygen. According to reports by Kaufman (1975) this results in the intermediate formation of 4-ahydroperoxy-H4pterin, which participates in the hydroxylation of the substrate, tyrosine, by tyrosine-hydroxylase. Tetrahydrobiopterin is the cofactor of various mixed function oxygenases participating in the synthesis of tyrosine, catecholamines and serotonin (Kaufman 1959; Nagatsu et al. 1964; Lovenberg et al. 1967). This cofactor is found in all nerve cells and chromaffin cells producing catecholamines or serotonin. In the meantime there exist findings that nerve cells that synthesize neurotransmitters of the type mentioned are also able to de novo synthesize the cofactor tetrahydrobiopterin from guanosine-triphosphate as precursor via different intermediate steps (rev. see Nichol et al. 1985). Little is as yet known about the susceptibility of this system to lesions or diseases possibly leading to a Parkinson syndrome by disturbance of the DOPA and dopamine synthesis. There exist reports of Nagatsu (1981) and Le Witt et al. (1984) stating that the tetrahydrobiopterin values in the brain of patients with Parkinson's disease are much lower than normal. In order to analyse the possible participation of the biopterin system in conditions leading to dopamine deficiency we have studied the effect of an antimetabolite of nicotinamide using PC-12 pheochromocytoma cells as an experimental model. The antimetabolite 6-aminonicotinamide (6-AN), which is effective in the production of such changes in rats differs from the natural product nicotinamide only by substitution with an amino group in position C6. The syndrome is characterized by a decrease in dopamine content and a significant slow-down in the utilization of this transmitter in the cells of the corpus striatum (Kehr et al. 1978; Loos et al. 1979). In the animals, a longlasting pathological muscular rigidity was registered that, as this was the case with Parkinson's disease, was abolished by L-DOPA or by the dopamine agonists, bromocriptine or lisuride (Loos et al. 1977). The neurotoxic effect occurred after a latency period of several hours, which implies that the effective compound is synthesized by the metabolism of nerve cells. A glycohydrolase (EC 3.2.2.6) located in the endoplasmic reticulum (Kaplan and Ciotti 1954, 1956; Kaplan et al. 1954)

425 hydrolyzes the link between ribose and pyridine N in the substrates NAD and NADP. By an exchange reaction, 6AN is transferred to adenine-dinucleotide(-P) instead of nicotinamide according to the principle of a competitive antagonism. This reaction results in the formation of the neurotoxic substance 6-aminonicotinamide-adenine-dinucleotide-phosphate (6-ANADP), which was isolated from the brains of rats treated with 6-AN (Coper and Herken 1963). 6-ANADP is a highly potent inhibitor of both dehydrogenases in the oxidative part of the pentose phosphate pathway. 6-Phosphogluconate dehydrogenase is the most sensitive enzyme for 6-ANADP. The inhibition constant, Ki, for the enzyme isolated from rat brain was 1.3 x 10 -7 M (Herken et al. 1969; Herken 1971). This explains the enormous accumulation of 6-phosphogluconate and the reduction of NADPH2 synthesis in vivo found in the course of studies on the brains of rats treated with 6-AN (Herken et al. 1969, 1973, 1974). The most severe lesions occur in those brain regions, in which the highest accumulation of 6-phosphogluconate is found. Thus, accumulated 6phosphogluconate is a reliable marker for 6-ANADP synthesis in the cells. These findings, in particular the inhibition of the 6-phosphogluconate dehydrogenase in the brains of rats treated with 6-AN, were in the meantime confirmed by several authors (Kauffman 1970; Kauffman and Johnson 1974; Krieglstein and Stock 1975; Deshpande et al. 1978). The effect of 6-AN does not selectively concern a defined cell type but the loss of function of the dopaminergic cells is, not doubt, the most prominent sign of the neurotoxic effect. These findings, in particular the decreased NADPH2 synthesis caused by the blockade of the pentose phosphate pathway, led to the investigations of tetrahydrobiopterin synthesis and its role as cofactor of tyrosine hydroxylase. The reductase participating in the synthesis requires NADPH2 (rev. see Nichol et al. 1985). NADPH2-dependent sepiapterin reductase, called biopterin synthase by Switchenko et al. 1984, catalyzes the last step in tetrahydrobiopterin de novo synthesis (main pathway), reducing H 4 pterin 2 or lactoyl H 4 pterin, respectively, to tetrahydrobiopterin (Milstien and Kaufman 1983, 1985; Smith and Nichol 1984; Switchenko et al. 1984; Nichol et al. 1985). In a so-called salvage pathway, sepiapterin reductase reduces lactoyl H2 pterin ( = sepiapterin) to dihydrobiopterin, the latter being reduced to tetrahydrobiopterin by NADPH2-dependent dihydrofolate reductase (DHFR). The utilization of sepiapterin for tetrahydrobiopterin synthesis by chromaffin cells in culture has already been proved by others for other reasons (AbouDonia et al. 1986). In our case, we added sepiapterin to the culture medium of PC-12 cells to clarify, whether the blockade of the pentose phosphate pathway induced by 6AN leads to a significant lack in reduction equivalents in synthesis and function of the biopterin cofactor. As intact dopaminergic nerve cells from the brain are hard to obtain in sufficient quantities, experiments for the elucidation of the molecular basis of the above-mentioned disturbances in the dopamine metabolism were carried out with a clonal cell line (PC-12), whose chromaffin cells were obtained from a rat pheochromocytoma by cloning (Greene and Tischler 1976). This cell line possesses the ability of identical reproduction throughout numerous subcultivations. These cells synthesize all the enzymes for catecholamine synthesis, including the cofactor tetrahydrobiopterin

(Herken 1983; Br/iutigam et al. 1984, 1985). PC-12 cells contain considerable amounts of biopterin. Simultaneous determination of tetrahydrobiopterin and total biopterin confirmed that the biopterin fraction consisted almost completely of tetrahydrobiopterin (Buff and Dairman 1975; Fukushima and Nixon 1980; Brfiutigam et al. 1984). Firstly, the still open question had to be elucidated, whether PC-12 cells possess the glycohydrolase responsible for the exchange of 6-aminonicotinamide for nicotinamide in NADP. This can be found out by determination of the accumulation of 6-phosphogluconate following addition of 6-AN to the culture medium. The following experiments were carried out: 1) Experiments for the indirect proof of the synthesis of 6-ANADP by determining the accumulation of 6-phosphogluconate in the cells after the action of 6-AN. 2) Effect of 6-ANADP synthesis on intracellular content of tetrahydrobiopterin and of total biopterin. Dependence of DOPA production on the biopterin content of the cells under various experimental conditions. 3) Investigation of NADPH2-dependent reductases by studies on the catalysis of sepiapterin to tetrahydrobiopterin by sepiapterin reductase and dihydrofolate reductase. Materials and methods

Cell culture conditions and experimental incubation Rat pheochromocytoma cells, clone PC-12, a gift of Drs. Sutter and Zimmermann, Berlin, were cultured as monolayers in Dulbecco's modification of Eagle's minimum essential medium (DMEM) containing 4.5 g/1 glucose and 3.7 g/1 NaHCO3 (Biochrom, Berlin). The medium was supplemented with 250 U/1 penicillin, 250 ~tg/1 streptomycin, 10ml/1 NEAA (non-essential amino acid concentrate), 4 mM L-glutamine (all obtained from Biochrom, Berlin), 10% (v/v) fetal calf serum and 5% horse serum (Gibco, Eggenstein, FRG). Cultivation was performed in Costar plastic flasks (75 cm 2, 250 ml; Tecnomara Deutschland GmbH, Fernwald, FRG) in 20 ml of medium at an atmosphere containing 10% COzat 37°C. The medium was changed after 2 and 5 days of cultivation. At the last change of medium, the culture was found to be in a stationary phase referring to growth. After 7 days, the cells were subcultivated. The experiments were carried out in the stationary phase. 6-aminonicotinamide (Sigma, Deisenhofen, FRG) was added to the culture medium. Incubation of treated and untreated cells was carried out under growth conditions, the incubation time depending on the experimental conditions. DOPA decarboxylase was blocked with m-hydroxybenzylhydrazine (NSD 1015; Sigma, Deisenhofen, FRG). The medium was changed I h before the end of the experiment, and NSD 1015 (Sigma, Deisenhofen, FRG) and was added with fresh culture medium. After i h of incubation, DOPA production was measured in the medium. In special experiments, L-sepiapterin (Schircks, Jona, Switzerland) or NADH2 (Boehringer Mannheim GmbH, Mannheim, FRG), respectively, was added to the medium I h before the end of the experiment. At the end of the experiment, the cells were scraped off with 2 x 300 ~tl perchloric acid containing 10 mM dithioerythritol (Sigma, Deisenhofen, FRG). The standard assay

426 Cell monolayers in stationary phase. Add 6-AN to the medium. Dosage and incubation time depending on experimental conditions

I

1 h before end of experiment, wash cell monolayers with 2 x 20 ml DMEM. 37° C. Add 6 ml fresh culture medium with 5 x 10-4 M NSD 1015 and 6-AN concentration as before. Depending on the experiment, add sepiapterin

I

After 1 h of incubation, take 1 ml medium for DOPA assay

I Wash cell monolayers twice with 20 ml ice-cold 0.9% NaC1 Scrape off cells with 2 x 300 ILlice-cold 0.2 M perchloric acid containing 10 mM dithioerythritol

I

Sonicate suspension 3 × 10 s, 40 W, on ice

I

Centifuge homogenate 15 rain at 6500 x g at 4°C --

Take I00 gl supernatant for direct H4 biopterin determination (HPLC with ELCD, 300 mV)

Take 150 gl of supernatant and add 25 gl of 2.5 % acidified iodine-solution --Take 100 btl supernatant for determination of 6-phosphogluconate Keep mixture at room temperature for 1- 1.5 h and gluconate according to Kolbe (1974) Add 25 btl of 5% ascorbic acid - - Take pellet and determine cell protein according to Lowry et al. Centrifuge, separate biopterin with HPLC, determine by fluores(1951) (bovine albumin as standard) cence detection Fig. 1. Assay procedure for simultaneous determination of H4 biopterin, total biopterin, DOPA, 6-phosphogluconate and gluconate in PC12-cells

procedure for the simultaneous determination of tetrahydrobiopterin, biopterin, DOPA, 6-phosphogluconate, gluconate and protein is shown in Fig. 1.

HPLC separation and determination of tetrahydrobiopterin, total biopterin and DOPA Tetrahydrobiopterin, biopterin and DOPA were separated with reversed-phase HPLC. Hewlett-Packard HPLC-systerns 1082B and 1084B (B6blingen, FRG)were used; separation was achieved by 4.6 x 125 mm column filled with ODS hypersil, 5 btm (Shandon, Frankfurt, FRG). As mobile phase, two isocratic systems with the following compositions were used: First system: 33 mM citric acid and 66 mM Na-acetate, pH 4.3, containing 0.5 mM 1-octanesulfonic acid monohydrate, 0.5 mM DL-n-butylamine, 0.1 mM EDTA and 3.5% methanol for determination of tetrahydrobiopterin and total biopterin. Second system: 56 mM citric acid and 44 mM Na-acetate, pH4.3, containing 0 . 5 m M 1-octanesulfonic acid monohydrate, 0.5 mM DL-n-butylamine, 0.1 mM EDTA and 3.5% methanol for determination of DOPA.

mined after iodine oxidation according to Fukushima and Nixon (1980) as modified by Br/iutigam et al. (1984). The spectrofluorimeter systems Kratos GM/FS 970 (Weiterstadt, FRG) was used. In our system, the excitation wave length was set at 280 nm with an emission cut-off filter (370 nm). Because of a hitherto unknown fraction X interfering with the biopterin in peak after application of 6-AN, the mobile phase composition was changed. With the mobile phase as described above and a flow rate of 0.5 ml/min, separation of biopterin and fraction X was achieved.

Identification of the eluted material as tetrahydrobiopterin and biopterin. Coelution of tetrahydrobiopterin and biopterin standards with the presumed tetrahydrobiopterin and biopterin from cells elicited neither double peaks nor shoulders in the chromatogram. The ratio of applied oxidation potentials (mV) to detector response (current nA) showed identical values of intracellular tetrahydrobiopterin and tetrahydrobiopterin standard as well as did the fluorescence spectrum of intracellular biopterin and biopterin standard. Biopterins were purchased from Schircks (Jona, Switzerland).

DOPA was determined in the medium after blockade of DOPA-decarboxylase with m-hydroxybenzylhydrazine added to the medium (final concentration 5 x 10 -4 M) 1 h before the end of the experiment. DOPA content in the medium was assayed after purification with aluminium oxide (Anton and Sayre 1964) and detected by ELCD using an oxidation potential of + 800 mV (Brfiutigam et al. 1984, 1985).

Tetrahydrobiopterin was determined directly in the supernatant by electrochemical detection (ELCD) according to Br/iutigam and Dreesen (1982) and Br/iutigam et al. (1982 b). For a better separation of tetrahydrobiopterin and dithioerythritol peaks, the mobile phase composition was changed as described above. The flow rate was 0.8 ml/min and the oxidation potential of the glassy carbon working electrode was set at + 300 mV. An ELCD apparatus Waters M 460 (Millipore, Berlin) was used.

determined enzymatically in a recording Gilford spectrophotometer as described elsewhere (Lange et al. 1970).

Total biopterin. 7,8-dihydrobiopterin (BH2), quinoid-dihydrobiopterin (qBH2) and tetrahydrobiopterin were deter-

Protein was determined according to Lowry et al. (1951) with bovine albumin as standard.

6-Phosphogluconate and gluconate contents in the cells were

427 Table 1. 6-AN-dependent increase of intracellular 6-phosphogluconate and gluconate in PC-12-cells

,-.?

"6

•

A Dose response after 48 h of incubation with 6-AN 200.

Metabolite [nmoles/mg protein] Controls

1

10

100

119.1_+13.2 tt6.9-+18.1

247.3_+20.8 271.1_+ 5.4 316.8_+18.7 439.0_+13.6 (n = 3, means -+ SD)

E

Q. o -~ 100-

,=_ a~

'~

]I, ,

N 0" 0

0 1 8 16 24 32 40 48

1

Incubation time 6-phosphogluconate

Gluconate [nmoles/mg prot.]

<0.5 0 . 7 ± 0.8 10.8_+ 0.9 269.0 + 12.7 340.0 ± 12.6 414.0 ± 24.2 377.6 ± 17.4 368.1 _+ 15.9

1.3 ± <0.5 15.0-+ 467.0 ± 583.1 ± 673.8 ± 657.3 ± 682.1 ±

o

l~O00

g

B Time response with 100 gg 6-AN/ml medium Hours

1600o

o E

[gg 6-AN/ml medium]

6-pg 4.0_+0.5 Gluconate 1.3_+0.5

DOPA

I---I Hi+. biopterin biop,eNn

10 6-AN [ p g / m t ]

2000 g o

100

0.4 6.8 18.9 20.0 38.0 38.0 36.4

-6000

o

200

2

o E

(n = 4, means _+SD) Cells were incubated under growth conditions as described in Methods. Assay procedure for determination of 6-phosphogluconate (6-pg) and gluconate see Fig. 1. The values of controls (A) and after 1 h incubation time (B) range at the lower level of detection of the method applied

}

NHNI

100"

6-Phosphogluconate and gluconate The neurotoxic effect o f the antimetabolite 6 - A N on d o p a mine-producing cells requires the synthesis o f 6 - A N A D P , which leads to an accumulation o f 6-phosphogluconate. PC12 cells possess a glycohydrolase catalyzing the exchange o f nicotinamide and 6 - A N in 6 - A N A D P molecules. Table I shows the accumulation o f 6-phosphogluconate and gluconate in PC-12 cells depending on the applied concentration o f 6 - A N (Table 1 A). The increase o f metabolites becomes visible after a latency period o f several hours (Table 1 B). The increase o f 6-phosphogluconate is limited by enzymatic d e p h o s p h o r y l a t i o n to gluconate, which is released into the culture m e d i u m (Keller et al. 1976). The content o f 6-phosphogluconate in untreated cells ranges at the lower level o f detection of the m e t h o d applied. Cells treated with 100 lag o f 6 - A N / m l m e d i u m show an e n o r m o u s increase o f the metabolite after 16 h. This implies that 6A N A D P is synthesized by the glycohydrolase reaction in the endoplasmic reticulum o f PC-12 cells. This result corresponds to our earlier findings in other neuronal and neuroglial cell lines (Kolbe et al. 1977).

Biopterin content and DOPA production Figure 2 shows the effect o f 6 - A N on intracellular biopterin levels and D O P A p r o d u c t i o n o f PC-12 cells. After incub a t i o n with 6 - A N for 48 h, a dose-dependent decrease o f

E o. o.

"

E oJ

}

0 Results

~000

'I

8

16

2#

32

"2 000

~0

or ~

~8

~ime of incubation [hrs.] Fig. 2A, B. 6-AN-dependent intracellular content of tetrahydrobiopterin and total biopterin (bars), and cellular DOPA production within t h (points), the latter measured in the medium. (A) Dose response after 48 h of incubation with 6-AN (n = 3 - 1 2 , means ± SD). (B) Time response with 100 gg 6-AN/ml medium (n = 3 - 4 , means _+ SD). 6-AN was added to the culture medium. Ceils were incubated under growth conditions as described in Methods. The medium was changed I h before the end of the experiment, m-Hydroxybenzylhydrazine (final concentration 5 x 10 -4 M) was added to the fresh medium for final incubation of I h. Assay procedure for simultaneous determination of the metabolites see Fig. 1

b o t h t e t r a h y d r o b i o p t e r i n and total biopterin in the cells was found (Fig. 2A). According to the time-dependent accumulation of 6-phosphogluconate described above, the effect o f the antimetabolite occurs after a latency period o f several hours (Fig. 2B). After 32 h o f incubation with 100 gg o f 6A N / m l medium, biopterin content in the cells is reduced to less than 1/3 as c o m p a r e d with controls. D u r i n g the determination o f total biopterin we found a substance, which we called fraction X. This substance was not detectable in the controls and showed a dose-dependent increase in cells treated with 6-AN. This effect was also observed in glial cells which do not synthesize biopterins. U n d e r the applied H P L C conditions, the retention time o f fraction X differed from that o f the total biopterin fraction. The substance showed a fluorescence spectrum which is iden-

428 Table 2. Effect of sepiapterin on intracellular content of tetra-

Table 3. Effect of NADH2 on intracellular content of tetra-

hydrobiopterin and total hiopterin, and on cellular DOPA production (n = 4, means _+ SD)

hydrobiopterin and total biopterin, and on cellular DOPA production within 1 h. (n = 4, means _+ SD)

[pmoles/mg protein] H4-biopterin

total biopt.

[pmoles/mg protein] DOPA

H4-biopterin

total biopt.

DOPA

146.3 _ 20.1" 105.8 + 7.9*

180.6 + 16.8"* 149+ 5.7**

3487 + 345 11031 + 1209

No 6-AN + sepiapterin

133.8 + 20.9 149.7_+ 13.2 2976 + 25* 462.1 ___62.3** 614.8 + 40.8** 5698 + 968*

No 6-AN + NADH2

lpg6-AN/ml + sepiapterin

74.1+11.3 462.4 _+ 10.0

99.1+ 7.7 582.1_+ 11.9

1869_+303 3460 _ 119

lgg6-AN/ml + NADH2

89.5_+ 1.7 94.9 _ 6.3

117.3+ 1.9 120.8+ 8.8

2048+ 132 6572 + 1072

10 gg 6-AN/ml + sepiapterin

30.1 _+ 2.1 513.8 _+ 32.2

34.4_+ 2.6 604.2 + 27.2

1206_+ 50* 2490 _+219"

10 gg 6-AN/ml + NADH2

25.9-+ 5.0 38.5 + 6.3

30.5_+ 8.8 58.8 + 10.2

1210 + 80 3353 + 321

100 ~tg 6-AN/ml + sepiapterin

19.2 + 1.3 16.6 + 1.3 398.1 _+27.6** 546.8_+25.9**

997 + 71 2194_+117

100 gg 6-AN/ml + NADH2

21.3 ___ 3.3 26.8 + 2.1

28.9 + 3.1 47.5_+ 4.6

1428 _+ 138 3437_ 133

Cell monolayers were incubated with 6-AN for 48 h. The medium was changed after 47 h. Sepiapterin (final concentration 2 x 1 0 - S M ) was added to the fresh medium containing mhydroxybenzylhydrazine (final concentration 5 x 10 -4 M) and 6AN concentration as before. Final incubation with sepiapterin for 1 h. Assay procedure for simultaneous determination of the metabolites see Fig. t. Biopterin values represent intracellular concentrations, DOPA was measured in the medium. Groups marked with the same figure were compared according to Student t-test (*P < 0.05 = significant, **P > 0.05 = not significant

Cell monolayers were incubated with 6-AN for 48 h. After 24 h, NADH2 (final concentration 1 x 10 -3 M) was added to the medium. The medium was changed after 47 h. Containing mhydroxybenzylhydrazine (final concentration 5 × 10 .4 M) and 6AN and NADH2 concentration as before. Final incubation with NADH2 for 1 h. Assay procedure for simultaneous determination of the metabolites see Fig. 1. Biopterin values represent intracellular concentrations, DOPA was measured in the medium. Groups marked with the same figure were compared according to Student t-test (*P < 0.05 = significant, **P > 0.05 = not significant)

tical with that o f the 6 - A N analogues o f N A D and N A D P (Herken and N e u h o f f 1964). This implies that fraction X mainly consists o f the 6 - A N analogue o f adenine-dinucleotide-phosphate synthesized by the glycohydrolase and does not contain biopterin. D O P A p r o d u c t i o n was measured in the m e d i u m at the end of the total experimental incubation period. One hour before, the m e d i u m was changed a n d m-hydroxybenzylhydrazine was a d d e d to block D O P A - d e c a r b o x y l a s e . U n d e r these conditions, m o r e than 90% o f the accumulating D O P A is released into the m e d i u m (Vaccaro et al. 1980; Erny et al. 1981 ; Brfiutigam et al. 1982a, b). Figure 2 shows that D O P A p r o d u c t i o n is correlated with the biopterin levels. These results imply that the antimetabolite, 6-AN, or the 6 - A N - c o n t a i n i n g analogue of N A D P leads to a longlasting i m p a i r m e n t o f biopterin synthesis. The decrease in D O P A p r o d u c t i o n evidently is a consequence o f this process.

ever, there is no indication for a direct inhibition of tyrosine hydroxylase caused by the antimetabolite. Table 3 shows a considerable increase o f D O P A production in cells treated with 6 - A N after application o f N A D H 2 to the culture medium, though the decreased biopterin levels caused by 6 - A N remained almost unchanged. The decrease o f t e t r a h y d r o b i o p t e r i n values after application o f N A D H 2 observed in the controls cannot yet be explained. The only enzyme in t e t r a h y d r o b i o p t e r i n synthesis which requires N A D H 2 is dihydropteridine reductase, which catalyzes the reduction of the oxidized biopterin cofactor in the recycling p a t h w a y (see Fig. 3). This implies that a blockade o f the N A D H 2 - d e p e n d e n t recycling p a t h w a y for t e t r a h y d r o b i o p t e r i n regeneration is compensated by N A D H 2 . The blockade o f cofactor utilization by the 6-ANcontaining analogue o f N A D by competitive antagonism or direct link is possible but remains to be proved. In the brains of rats treated with 6-AN, the ratio o f 6 - A N A D to N A D was considerably lower than that o f 6 - A N A D P to N A D P (Brunnemann et al. 1964). In contrast to 6 - A N A D P , a specific enzyme which is inhibited by 6 - A N A D in the presence o f the natural nucleotide was not found (Coper and N e u b e r t 1964). D i h y d r o p t e r i d i n e reductase has not yet been studied in this context.

Sepiapterin reductase, dihydrofolate reductase, dihydropteridine reductase Table 2 shows biopterin and D O P A values o f cells treated with 6 - A N and o f controls after application o f sepiapterin 1 h before the end o f the incubation with 6 - A N (total incub a t i o n time 48 h). Both 6 - A N - t r e a t e d and untreated cells show increased t e t r a h y d r o b i o p t e r i n and total biopterin fractions after application o f sepiapterin without significant differences between the t e t r a h y d r o b i o p t e r i n or total biopterin values o f b o t h treated and untreated cells. This indicates that b o t h the sepiapterin reductase and the dihydrofolate reductase reaction in t e t r a h y d r o b i o p t e r i n synthesis are n o t impaired by 6-AN. Unexpectedly, the increase o f D O P A p r o d u c t i o n in cells treated simultaneously with 6 - A N and sepiapterin does not follow the increased biopterin values. This discrepancy is obviously dependent on the concentration o f 6-AN. How-

Discussion

Biopterin synthesis A discussion o f the blockade of biopterin synthesis and D O P A p r o d u c t i o n in PC-12 cells caused by 6 - A N requires a short description o f the present knowledge on the synthesis p a t h w a y (see Fig. 3). The first and at the same time rate-limiting enzyme in de novo synthesis of t e t r a h y d r o b i o p t e r i n from G T P is G T P cyclohydrolase (EC 3.5.4.16), which catalyzes the opening

429

GiP

DE NOVO PATHWAY

EnzymeA

Cyclohydrolase F Pyd P3 Enzyme B H2Neopterin-P 3 PPH4Synthase Mg**

l

6-PyruvoylH4Pterin

PPH4Reductase ( Sepiapterin Reductase ? )

SALVAGE PATHWAY

NADPH2

6-Lactoyl H2 Pterin (= Sepiapterin )

6-Lactoyl H4 Pterin H4 Pterin-2 Sepiapterin Reductase

1 NADPH2

Sepiapterin Reductase

NADPH2

Dihydrofolate Reductase H4 Biopterin Tyr " x ~ /

, ~ D i h y d r opteridlnA~PH2

/NAP% DOPA

H2 Biopterin

~

RECYCLING PATHWAY

J

q BH2

of the imidazol ring, followed by the formation of a pyrazine ring (Ref. see Nichol et al. 1985). The first pterin derivative is D-erythro 7,8-dihydroneopterin-triphosphate. In the opinion of Gill and coworkers (Gill et al. 1978; G/tl 1982), the formation of D-erythro 7,8-dihydroneopterin-triphosphate is not catalyzed by a single enzyme. These authors characterized two enzymes, cyclohydrolase A (I, II) and enzyme B. The first hydrolyzes GTP to 2-amino 6-(5triphosphoribosyl)-amino 5- or 6-formamido 6-hydroxypyrimidine (FPydP3), while the latter cyclizes FPydP3 to Derythro 7,8-dihydroneopterin-triphosphate. This statement is of importance for our studies, because enzyme B is regarded to be the site of action of inhibitors of biopterin synthesis. The next step of tetrahydrobiopterin synthesis is the formation of 6-pyruvoyl-tetrahydropterin by dephosphorylation and a change in the side chain of the substrate (Takakiwa et al. 1986; rev. see Nichol et al. 1985). Thus the formation of the basic pterin structure is terminated. This first phase of biopterin synthesis does not require NADPH2. The second phase does require NADPH2 for the reduction of the two keto groups in the side chain of 6pyruvoyl-tetrahydropterin. Depending on. the position of the keto group, which is reduced first, two intermediates are described in the literature: lactoyl-H4-pterin (Switchenko et al. 1984; Milstien and Kaufman 1983, 1985), and H4-pterin 2 (Smith and Nichol 1984; Nichol et al. 1985). In any case, sepiapterin reductase catalyzes the last step in the de novo synthesis, the reduction of the second keto group. The enzyme is obviously not very specific. The possibility that sepiapterin reductase can also catalyze both keto groups of

Fig. 3. Biosynthesis of tetrahydrobiopterin

6-pyruvoyl-tetrahydropterin was suggested by the finding that it has diketo-reductase activity (Katoh and Sueoka 1984). In the so-called salvage pathway, the same enzyme catalyzes the reduction of lactoyl-H2-pterin to dihydrobiopterin, which in turn is reduced to tetrahydrobiopterin by the NADPH2-dependent dihydrofolate reductase (Milstien and Kaufman 1983; Smith and Nichol 1984). The decrease of tetrahydrobiopterin synthesis in PC-12 cells after application of 6-AN gave rise to the presumption that this process is caused by the blockade of the pentose phosphate pathway leading to a reduced supply with NADPH2 as described above. Jansson et al. (1977), who confirmed our results, as well as the other authors quoted assumed that the function of tyrosine-hydroxylase is possibly disturbed by decreased enzymic reduction of the biopterin cofactor and might, thereby, cause the decreased DOPA production. Our experiments with sepiapterin show that this is not the case. Application of sepiapterin to the culture medium of 6-AN-treated and untreated cells resulted in an unexpected increase both of tetrahydrobiopterin and total biopterin. Utilization of sepiapterin for tetrahydrobiopterin synthesis requires two NADPH2-dependent reductases, sepiapterin reductase and dihydrofolate reductase. Our results imply that there is no indication for a lack of NADPH2 disturbing the reducing steps in tetrahydrobiopterin synthesis. Therefore, the simultaneous decrease of tetrahydrobiopterin and total biopterin to levels of less than 1/3 of the controls, depending on the concentration of 6AN, can only be the result of a blockade in the first phase of the cofactor synthesis, the NADPH2-independent formation of the pterin molecule. The latency period of this reaction,

430 correlation with the accumulation of 6-phosphogluconate, implies that the effect is caused by the inhibitor, 6-ANADP. Since there is no indication for a blockade of 6-pyruvoyltetrahydropterin-synthase, the formation of I>erythro 7,8dihydroneopterin-triphosphate seems to be the step that is inhibited by the antimetabolite. Gill and coworkers (Gill et al. 1978; Gall and Whitacre 1981; Gill 1982)demonstrated that 2;4-diamino 6-hydropyrimidine and the 2-deoxy compound of the guanine nucleotide FPydP3 (dFPydP3) are powerful inhibitors of enzyme B both in vivo and in vitro. Application of the inhibitors resulted in an accumulation of FPydP3 and a decrease of biopterin synthesis. The clone PC-12 also shows a decrease of the cofactor content after application of 2,4-diamino 6-hydropyrimidine (Br/iutigam et al. 1984). However, ICso of 2,4-diamino 6hydropyrimidine had to be much higher than that of 6ANADP. The question of whether 6-ANADP inhibits the same enzyme still remains to be elucidated. For technical reasons, we were not yet able to determine an intermediate, in particular not FPydPa.

DOPA production and biopterin recycling pathway Tetrahydrobiopterin as cofactor of hydroxylases provides electrons to reduce molecular oxygen and is, in turn, oxidized to the quinoide dihydrobiopterin. The activation of molecular oxygen results in the formation of 4-ahydroperoxy-H4pterin, which participates in the hydroxylation of the substrate (Kaufman 1975). Table 2 shows that 1 pmol tetrahydrobiopterin catalyzes the hydroxylation of a multiple amount of tyrosine. This function cannot be sustained without an effective recycling system, which prevents the early depletion of the tetrahydrobiopterin pool. Gill and Whitacre (1981) calculated the capacity of this system determinating the content of cofactor in the brains of rats. The authors showed that a decreased tetrahydrobiopterin content induced by the inhibitor, 2,4-diamino 6hydropyrimidine, did not necessarily result in the decrease of neurotransmitter synthesis as long as the function of the recycling pathway was maintained. Our experiments with sepiapterin added to 6-AN-treated cells showed that the compensation of cofactor content was not followed by a compensation of DOPA production. This might indicate that the recycling system is inhibited by 6AN. As shown in Fig. 3, the reduction of quinoide dihydrobiopterin is catalyzed by dihydrobiopterin-reductase, which is NADH2-dependent. In our earlier investigations, we did not yet find an enzyme that was inhibited by 6-ANAD in the presence of the cofactor NAD. The comparison of the DOPA values of treated and untreated cells indicate that the increase of DOPA production, particularly found in 6-ANtreated cells after application of high concentrations of NADH2, may be explained by a competitive antagonism versus the presumed inhibitor 6-ANAD. The recycling pathway is the mode of action of the pyridine derivative, l-methyl 4-phenyl 1,2,5,6-tetrahydropyridine (MPTP) or its metabolite, MPP +, a specifically potent neurotoxin causing Parkinsonism. Blair et al. (1984) showed that MPTP is a competitive inhibitor of dihydrobiopterin-reductase with respect to N A D H (Ki = 4.6 x 10 -5 tool/l). The stimulation of DOPA production in cells that were not treated with 6-AN is a very interesting result, which

requires further investigation. It remains to be elucidated, whether this process is connected with the increasing formation of a reactive 4-a-hydro-peroxy-H4pterin. The results reported in this paper explain the reasons for the decrease of DOPA content and its utilization in corpus striatum of rat brains resulting in the Parkinson-like syndrome induced by 6-AN. Restriction of the synthesis of the biopterin cofactor might be a pathogenetically important disorder at the initial phase of Parkinson's disease. Nagatsu (1981) found a decrease of total biopterin by almost two thirds of control values in autopsy preparations of the nucleus caudatus of patients who died of Parkinson's disease. The first steps of biosynthesis from GTP to 7,8-neopterintriphosphate seem to be particularly sensitive towards the action of exogenous substances. The fact that biopterin and folic acid possess the same basal pterin structure should be of importance for the disturbance of cellular metabolism. It is remarkable and possibly indicates a connection between the function of both substances that the addition of 6-AN to the culture medium during the growth phase of the PC12 clone causes a clearly decreased cell division. This, of course, does not allow to draw definite conclusions concerning the genesis of Parkinson's disease.

Acknowledgement. The authors wish to thank Dr. M. Brfiutigam and Dr. K. Keller for helpful discussions, Miss S. Mueller and Miss B. D6ring for expert technical assistance and Mrs. R. Kr[iger for help with the manuscript. The work was supported by a grant from the Bundesminister ffir Forschung und Technologie. References

Abou-Donia MM, Wilson SP, Zimmerman TP, Nichol CA, Viveros OH (1986) Regulation of guanosine triphosphate cyclohydrolase and tetrahydrobiopterin levels and the role of the cofactor in tyrosine hydroxylation in primary cultures of adrenomedullary chromaffin cells. J Neurochem 461190 - 1199 Anton AH, Sayre DF (1964) The distribution of dopamin and DOPA in various animals and a method for their determination in diverse biological material. J Pharmacol Exp Ther 145: 326336 Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F (1973) Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol Sci 20:415- 455 Blair JA, Parveen H, Barford PA, Leeming RJ (1984) Aetiology of Parkinson's disease. Lancet I:167 Br/~utigam M, Dreesen R (1982) Determination of L-erythro-tetrahydrobiopterin in biological tissues by high pressure liquid chromatography and electrochemical detection. Hoppe-Seyler's Z Physiol Chem 363 : 1203- 1207 Brfiutigam M, Dreesen R, Flosbach C-W, Herken H (1982a) Mouse neuroblastoma clone N I E-115: A suitable model for studying the action of dopamine agonists on tyrosine hydroxylase activity. Biochem Pharmacol 31 : 1279-1283 Brfiutigam M, Dreesen R, Herken H (1982b) Determination of reduced biopterins by high pressure liquid chromatography and electrochemical detection. Hoppe-Seyler's Z Physiol Chem 363 : 341 -- 343 Br/iutigam M, Dreesen R, Herken H (1984) Tetrahydrobiopterin and total biopterin content of neuroblastoma (N1E-115, N2A) and pheochromocytoma (PC-12) clones and the dependence of catecholamine synthesis on tetrahydrobiopterin concentration. J Neurochem 42: 390-- 396 Brfiutigam M, Kittner B, Herken H (1985) Evaluation of neurotropic drug actions on tyrosine hydroxylase activity and dopamine metabolism in clonal cell lines. Arzneimittelforschung 35 : 277 - 284

431 Brunnemann A, Coper H, Neubert D (1964) Biosynthese und Wirkung des 6-Aminonicotinamidadenidinucleotids (6ANAD). Naunyn-Schmiedebergs Arch Pharmacol 246:437451 Buff K, Dairman W (1975) Biosynthesis of biopterin by two clones of mouse neuroblastoma. Mol Pharmacol 11 : 87-- 93 Coper H, Herken H (1963) Sch/idigung des Zentralnervensystems durch Antimetaboliten des NikotinsS.ureamids. Ein Beitrag zur Molekularpathologie der Pyridinukleotide. Dtsch Med Wochenschr 88 : 2025 - 2036 Coper H, Neubert D (1964) EinfluB von NADP-Analogen auf die Reaktionsgeschwindigkeit einiger NADP-bed/irftiger Oxidoreduktasen. Biochim Biophys Acta 89 : 23 - 32 Deshpande SS, Albuquerque EX, Kauffman FC, Guth L (1978) Physiological, biochemical and histological changes in skeletal muscle, neuromuscularjunction and spinal cord of rats rendered paraplegic by subarachnoidal administration of 6-aminonicotinamide. Brain Res 140: 89-109 Erny RE, Berezo MW, Perlman RL (1981) Activation of tyrosine 3-monooxygenase in pheochromocytoma cells by adenosine. J Biol Chem 256:1335-1339 Fukushima T, Nixon JC (1980) Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 102:176188

Gill EM (1982) Biosynthesis and function of unconjugated pterins in mammalian tissues. Adv Neurochem 4:83-148 Gill EM, Whitacre DH (1981) Biopterin. VII. Inhibition of synthesis of reduced biopterins and its bearing on the function of cerebral tryptophan 5-hydroxylase in vivo. Neurochem Res 6 : 233 - 241 G~I EM, Nelson SM, Sherman AD (1978) Biopterin. III. Purification and characterization of enzymes involved in the cerebral synthesis of 7,8-dihydrobiopterin. Neurochem Res 3 : 6 9 - 8 8 Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73: 2424- 2428 Hassler R (1972) Physiopathology of rigidity. In: Siegfried J (ed) Parkinson's disease, vol I. Huber, Bern Stuttgart Toronto, pp 19 - 4 5 Herken H (1971) Antimetabolic action of 6-aminonicotinamideon the pentose phosphate pathway in the brain. In: Aldridge WN (ed) Mechanisms of toxicity, Macmillan, London, pp 189- 203 Herken H (1983) Clonale Nervenzellinien in der Kultur. Modelle zum Studium molekularer Grundlagen neuropharmakologischer Wirkungen. Klin Wochenschr 61 : 1 - 1 6 Herken H, Neuhoff V (1964) Spektrofluorometrische Bestimmung des Einbaus von 6-Aminonicotinsfiureamid in die oxydierten Pyridinnukleotide der Niere. Naunyn-Schmiedebergs Arch Pharmacol 247 : 187- 201 Herken H, Lange K, Kolbe H (1969) Brain disorders induced by pharmacological blockade of the pentose phosphate pathway. Biochem Biophys Res Commun 36:93-100 Herken H, Keller K, Kolbe H, Lange K, Schneider H (1973) Experimentelle Myelopathie. Biochemische Grundlagen ihrer cellul/iren Pathogenese. Klin Wochenschr 51:644-657 Herken H, Lange K, Kolbe H, Keller K (1974) Antimetabolicaction on the pentose phosphate pathway in the central nervous system induced by 6-aminonicotinamide. In: Genazzani E, Herken H (eds) Central nervous system - studies on metabolic regulation and function. Springer, Berlin Heidelberg New York, pp 41 54 Hornykiewicz O (1962) Dopamin (3-Hydroxytryptamin) im Zentralnervensystem und seine Beziehung zum ParkinsonSyndrom des Menschen. Dtsch Med Wochenschr 87:18071810 Hornykiewicz O (1972) Neurochemistry of Parkinsonism. In: Lajtha A (ed) Handbook of neurochemistry, vol 7. Plenum Press, New York, pp 465-501 Jansson SE, Gripenberg J, H/irk6nen M (1977) The effect of 6aminonicotinamideblockade of the pentose phosphate pathway on catecholamines in the rat adrenal medulla, superior cervical

ganglion, hypothalamus and synaptosome fractions. Acta Physiol Scand 99 : 467 - 475 Kaplan NO, Ciotti MM (1954) The 3-acetylpyridine analog ofDPN. J Am Chem Soc 76:1713-1714 Kaplan NO, Ciotti MM (1956) Chemistry and properties of the 3acetylpyridine analog of diphosphopyridine nucleotide. J Biol Chem 221 : 823 - 832 Kaplan NO, Goldin A, Humphreys SR, Ciotti MM, Venditti JM (1954) Significance of enzymatically catalyzed exchange reactions in chemotherapy. Science 120: 437 - 440 Katoh S, Sueoka T (1984) Sepiapterin reductase exhibits a NADPHdependent dircarbonyl reductase. Biochem Biophys Res Commun 118:859-869 Kauffman FC (1970) Effects of 6-aminonicotinamideon NADP +dependent enzymes and metabolism in mouse brain. Pharmacologist 12 :222 Kauffman FC, Johnson EC (1974) Cerebral energy reserves and glycolysis in neural tissue of 6-aminonicotinamide-treatedmice. J Neurobiol 5 : 379 - 392 Kaufman S (1959) Studies on the mechanism of the enzymatic conversion of phenylalanine to tyrosine. J Biol Chem 234: 2677- 2682 Kaufman S (1975) Studies on the mechanism of phenylalanine hydroxylase: Detection of an intermediate. In: Pfleiderer W (ed) Chemistry and biology of pteridines, de Gruyter, Berlin New York, pp 291-301 Kehr W, Halbhfibner K, Loos D, Herken H (1978) Impaired dopamine function and muscular rigidity induced by 6-aminonicotinamide in rats. Naunyn-Schmiedebergs'sArch Pharmacol 304:317--319 Keller K, Kolbe H, Herken H (1976) Glycolysis and glycogen metabolism after inhibition of hexose monophosphate pathway in C6 glial cells. Naunyn-Schmiedeberg's Arch Pharmacol 294:213--215 Kolbe H, Keller K, Lange K, Herken H, with techn assistance of Gaisser H, Monden I (1977) Glucose metabolism in C-1300 neuroblastoma cells after inhibition of hexose monophosphate pathway. Naunyn-Schmiedeberg's Arch Pharmacol 296:123130 Krieglstein J, Stock R (1975) Decreased glycolytic flux rate in the isolated perfused rat brain after pretreatment with 6-aminonicotinamide in rats. Naunyn-Schmiedeberg'sArch Pharmacol 290 : 323 - 327 Lange K, Kolbe H, Keller K, Herken H (1970) Der Kohlehydratstoffwechsel des Gehirns nach Blockade des Pentose-PhosphatWeges durch 6-Aminonicotinsfiureamid. Hoppe-Seyler's Z Physiol Chem 351 : 1241 - 1252 Le Witt PA, Miller LP, Newman, RP, Burns RS, Insel T, Levine RA, Lovenberg W, Calne DB (1984) Tyrosine hydroxylase cofactor (tetrahydrobiopterin) in parkinsonism. In: Hassler RG, Christ JF (eds) Adv in neurology, vo140. Raven, New York, pp 4 5 9 462 Loos D, Halbhiibner K, Herken H (1977) Lisuride, a potent drug in the treatment of muscular rigidity in rats. Naunyn-Sehmiedeberg's Arch Pharmacol 300:195-198 Loos D, Halbhiibuer K, Kehr W, Herken H (1979) Actio of dopamine agonists on Parkinson-like muscle rigidity induced by 6-aminonicotinamide.Neuroscience 4: 667- 676 Lovenberg B, Jequier E, Sjoersdma A (1967) Tryptophan hydroxylation: Measurement in pineal gland, brain stem and carcinoid tumor. Science 155: 217- 219 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193 : 265 - 275 Milstien S, Kaufman S (1983) Tetrahydro-sepiapterin is an intermediate in BH4 synthesis. Biochem Biophys Res Commun 115:888-893 Milstien S, Kaufman S (1985) Synthesis of tetrahydrobiopterin: Conversion of dihydroneopterin triphosphate to tetrahydropterin intermediates. Biochem Biophys Res Commun 128 : 1099 -- 1107

432 Nagatsu T (1981) Biopterin cofactor and regulation of monoaminesynthesizing mono-oxygenases. Trends Pharmacol Sci 2 : 2 7 6 279 Nagatsu T, Levitt M, Udenfriend S (1964) Tyrosine hydroxylase. The initial step in norepinephrine biosynthesis. J Biol Chem 239:2910-2917 Nichol CA, Smith GK, Duch DS (1985) Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem 54: 729 -- 764 Smith GK, Nichol CA (1984) Two new tetrahydropterin intermediates in the adrenal medullary de novo biosynthesis of tetrahydrobiopterin. Biochem Biophys Res Commun 120:761- 766

Switchenko AC, Primus JP, Brown GM (1984) Intermediates in the enzymatic synthesis in Drosophila melanogaster. Biochem Biophys Res Commun 120 : 7 5 4 - 760 Takakiwa SI, Curtius HC, Redweik U, Leimbacher W, Ghisla S (1986) Biosynthesis of tetrahydrobiopterin. Purification of 6pyruvoyl tetrahydropterin synthase from human liver. Eur J Biochem 161:295-302 Vaccaro KK, Liang BT, Perelle BA, Perlman RL (1980) Tyrosine 3-monooxygenase regulates cateeholamine synthesis in pheochromocytoma cells. J Biol Chem 255:6539-6541 Received August 15, 1988/Accepted January 4, 1989