The role of 6R-tetrahydrobiopterin in the nervous system

Progress in Neurobiology 61 (2000) 415±438

www.elsevier.com/locate/pneurobio

The role of 6R-tetrahydrobiopterin in the nervous system Kunio Koshimura*, Yoshio Murakami, Junko Tanaka, Yuzuru Kato First Division, Department of Medicine, Shimane Medical University, 89-1 Enya-cho, Izumo 693-8501, Japan Received 13 September 1999

Abstract In addition to its cofactor activities for aromatic L-amino acid hydroxylases and nitric oxide synthase (NOS), 6Rtetrahydrobiopterin (6R-BH4) shows diverse actions on neurons. Dopamine release from the rat striatum or PC12 cells was stimulated by 6R-BH4. The action of 6R-BH4 was independent of its cofactor activities and stereospeci®c. Ca2+ channels in rat brain and PC12 cells were activated by 6R-BH4 via cAMP-protein kinase A pathway. Membrane potential of PC12 cells was deplorized by 6R-BH4. Thus, it is assumed that 6R-BH4 acts on its speci®c action site (possibly outside of the cell membrane) to stimulate dopamine release by activating Ca2+ channels. Apoptosis induced by depletion of serum and nerve growth factor in PC12 cells was prevented by 6R-BH4. The cell surviving e€ect of 6R-BH4 was also mediated by activation of Ca2+ channels and cAMP-protein kinase A pathway. However, since 6R-BH4 did not activate mitogen activated protein kinase, it did not support neuronal di€erentiation. Nitric oxide (NO)-induced cell death was prevented by 6R-BH4 in PC12 cells. NOS activity was not changed by exogenous 6R-BH4, but NO metabolites in culture medium were decreased by 6R-BH4. When endogenous 6R-BH4 was reduced by inhibition of biosynthesis, cell death was induced in PC12 cells. Superoxide is observed to be generated during autoxidation of 6R-BH4. Superoxide producing system mimicked the cell protective action of 6R-BH4 against NO toxicity. Thus, it is considered that 6R-BH4 protects PC12 cells against NO toxicity by generating superoxide during its autoxidation. These results raised the possibility that 6R-BH4 is a self-protective factor against NO toxicity in NO producing neurons. Our ®ndings indicate that 6R-BH4 regulates neuronal activities in the brain and that 6R-BH4 can be a promising drug for neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. # 2000 Elsevier Science Ltd. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416

2.

E€ect on dopamine release from rat brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416

3.

E€ect on Ca2+ channels in rat brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420

4.

E€ect on dopamine release and Ca2+ channels in PC12 cells . . . . . . . . . . . . . . . . . . . . . . . 422

5.

E€ect on survival of PC12 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424

6.

E€ect on cytotoxicity of NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

Abbreviations: DAHP, 2,3-diamino-6-hydroxypyrimidine; DMNX, dorsal motor nucleus of the vagus; icv, intracerebroventricular; L-NAME, L-nitro-arginine methyl ester; MAP, mitogen activated protein; 6-MPH4, 6-methyl-tetrahydropterin; a-MT, a-methyl-p-tyrosine; NGF, nerve growth factor; NO, nitric oxide; NOS, nitric oxide synthase; PACAP, pituitary adenylate cyclase activating polypeptide; 6R-BH4, 6R-tetrahydrobiopterin; 6S-BH4, 6S-tetrahydrobiopterin; SNP, sodium nitroprusside; TPA, phorbol 12-myrisate 13-acetate. * Corresponding author. Tel.: +81-853-23-2111; fax: +81-853-23-8650. E-mail address: [email protected] (K. Koshimura). 0301-0082/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 1 - 0 0 8 2 ( 9 9 ) 0 0 0 5 9 - 3

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7.

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

1. Introduction 6R-Tetrahydrobiopterin (6R-BH4) is a natural cofactor for aromatic L-amino acid hydroxylases such as phenylalanine hydroxylase (Kaufman, 1959; Brenneman and Kaufman, 1964), tryptophan hydroxylase (Lovenberg et al., 1967) and tyrosine hydroxylase (Nagatsu et al., 1964). 6R-BH4 is also known to be a cofactor for nitric oxide (NO) synthase (NOS) (Kwon et al., 1989; Mayer et al., 1990, 1991). 6R-BH4 is synthesized from GTP (Fig. 1). The ®rst step as conversion of GTP to dihydroneopterin triphosphate catalyzed by GTP cyclohydrolase I is rate-limiting. Then, dihydroneopterin triphosphate is converted to 6R-BH4 by the action of subsequent enzymes of the 6R-BH4 biosynthesis pathway, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase (Fig. 1). There is a diastereoisomer of 6R-BH4, 6S-tetrahydrobiopterin (6S-BH4) (Fig. 2), which has a cofactor activity for aromatic L-amino acid hydroxylases (Hasegawa et al., 1979; Bailey et al., 1991; Minami et al., 1992) but not for NOS (Klatt et al., 1994). In tissues, most of the biopterin exists as a reduced form (predominantly 6RBH4) (Fukushima and Nixon, 1980). In the brain, distribution of 6R-BH4 is well correlated with tyrosine hydroxylase activity or catecholamine content (Bullard et al., 1978), showing that 6R-BH4 is present exclusively in monoaminergic neurons in the brain. Several lines of evidence indicate that congenital deficit in 6R-BH4-related enzymes causes atypical phenylketonuria (Smith et al., 1975; Nagatsu et al., 1981; Niederwieser et al., 1984, 1985). 6R-BH4 content in the cerebrospinal ¯uid or brain tissue decreases in patients with Parkinson's disease (Lovenberg et al., 1979; Fujishiro et al., 1990; Furukawa et al., 1991), Alzheimer's disease (Williams et al., 1980; Kay et al., 1986), dystonia (Williams et al., 1979; LeWitt et al., 1988) and depression (LeWitt et al., 1988). Treatment with 6R-BH4 was e€ective in some of these patients (Curtius et al., 1983; Nagatsu et al., 1984). Abnormal metabolism of monoamine is suggested in infantile autism and treatment with 6R-BH4 was e€ective (Naruse et al., 1987). It has been reported that 6R-BH4 content in cerebrospinal ¯uid decreases in aged people (Levine, 1988), suggesting a role of 6R-BH4 for aging. These

data raise the possibility that 6R-BH4 plays a critical role for neuronal activity in the brain. To clarify the action of 6R-BH4 in the brain, we have investigated the e€ects of 6R-BH4 on neurons using in vivo and in vitro study. In this article, we show some of our results and discuss the role of 6RBH4 in the brain. 2. E€ect on dopamine release from rat brain Since Km value of 6R-BH4 for tyrosine hydroxylase (Numata et al., 1977; Oka et al., 1981) is lower than the tissue content of 6R-BH4 (Fukushima and Nixon, 1980; Levine et al., 1981; Nagatsu et al., 1981), it is considered that tyrosine hydroxylase is not saturated with 6R-BH4 in the brain. Therefore, exogenous 6RBH4 may stimulate dopamine synthesis by activating tyrosine hydroxylase. In the rat striatum, exogenous 6R-BH4 was shown to increase tyrosine hydroxylase activity (Miwa et al., 1985). Enhancement of dopamine synthesis, in turn, may stimulate dopamine release. To investigate the e€ect of 6R-BH4 on dopamine release in vivo, we measured dopamine release from the striatum of Wistar male rats by means of brain microdialysis (Koshimura et al., 1990, 1991, 1994, 1995). Under anesthesia, a probe for microdialysis was inserted into the striatum stereotactically (Fig. 3). After insertion of the dialysis probe, the rats were allowed to move freely during the experiments. The dialysis probe was continuously perfused with Ringer solution. In the striatum, extracellular dopamine comes into the dialysis probe through the semipermeable membrane (Fig. 3). The perfused ¯uid (dialysate) was collected at 20-min interval. Several hours after insertion of the probe, dopamine levels in dialysates reached a steady state which was maintained for 6 h. After collecting ®ve fractions of dialysates as a basal level, drugs were administered to the rats by intracerebroventricular (icv) injection or addition to the perfusion ¯uid. Dopamine in dialysates was extracted with alumina and measured with high performance liquid chromatography (HPLC) combined with an electrochemical detector. When 6R-BH4 was administered by icv injection ipsilateral to the examined striatum, dopamine

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Fig. 1. Biosynthetic pathway of tetrahydrobiopterin. GTP-CH: GTP cyclohydrolase I; H2NEOPTERIN-PPP: dihydroneopterin triphosphate; PYRUVOYL-H4PTERIN SYNTHASE: pyruvoyl-tetrahydropterin synthase; 6-PYRUVOYL-H4PTERIN: 6-pyruvoyl-tetrahydropterin; 6-(L-1'HYDROXY-2-OXOPROPYL)-H4PTERIN: 6-(L-1'-hydroxy-2-oxoprophyl)-tetrahydropterin; 6-LACTOYL-H4PTERIN: 6-lactoyl-tetrahhydropterin; H4BIOPTERIN: tetrahydrobiopterin.

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Fig. 2. Pteridines.

levels increased. In contrast, icv injection of 6R-BH4 contralateral to the examined striatum had no e€ect on dopamine levels in the dialysates (Fig. 4). These results show that 6R-BH4 acted only in the vicinity of the injected point. When 6R-BH4 was added to the perfusion ¯uid, dopamine levels in the dialysates increased in a dose-related manner (Fig. 5). Dopamine levels in the dialysates were stimulated by 6R-BH4, an active form as a cofactor but not biopterin, an inactive form (Fig. 6). Dopamine levels in the dialysates were increased by 6S-BH4, a diastereoisomer of 6R-BH4, or 6-methyl-tetrahydropterin (6-MPH4), a synthetic cofactor. However, the e€ects of these pteridines were much weaker than that of 6R-BH4 (Fig. 7). These results indicate that the action of 6R-BH4 had stereospeci®city. Dopamine levels in the dialysates were markedly decreased by a Na+ channel blocker, tetrodotoxin or a Ca2+ channel blocker, NKY-722 (Ohue et al., 1991a), either before or after administration of 6R-BH4 (Figs. 8 and 9). Thus, it is considered that most of the dopamine in the dialysates was released from neurons. The recovery rate of dopamine through the dialysis membrane was calculated to be about 3% (Fig. 10). Thus, 6R-BH4 seemed to be e€ective at 7.5 mM. Since the

Fig. 3. In vivo brain microdialysis. DA: dopamine.

Fig. 4. E€ect of intracereboventricular administration of 6R-BH4 on dopamine release from the rat striatum (Koshimura et al., 1990). w: control; *: 6R-BH4 administered ipsilateral to the dialyzed striatum; R: 6R-BH4 administered contralateral to the dialyzed striatum.

concentration of 6R-BH4 in the rat striatum is assumed to be 100 mM (Levine et al., 1981), 6R-BH4 may stimulate dopamine release from the striatum at

Fig. 5. E€ect of 6R-BH4 on dopamine release from rat striatum (Koshimura et al., 1995). A: 6R-BH4 was perfused for 60 min as shown by a bar. w: control; W: 0.25 mM; Q: 0.5 mM; *: 1 mM; R: 5 mM. B: Increments of dopamine release above the control value after di€erent doses of 6R-BH4 are shown.

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Fig. 6. E€ects of 6R-BH4 and biopterin on dopamine release from the rat striatum (Koshimura et al., 1990).

physiological concentrations. Since 6R-BH4 is a cofactor for tyrosine hydroxylase, 6R-BH4-induced dopamine release may be due to enhancement of tyrosine hydroxylase activity. This is not likely because 6RBH4-induced increase in dopamine release persisted in the presence of a-methyl-p-tyrosine (a-MT), an inhibitor of tyrosine hydroxylase. In contrast to 6R-BH4, dopamine releases induced by 6S-BH4 or 6-MPH4 were blunted by a-MT (Fig. 11). These data demon-

419

strate that 6R-BH4-induced dopamine release was mostly independent of its cofactor activity for tyrosine hydroxylase, whereas 6S-BH4ÿ or 6-MPH4ÿ induced dopamine release by was dependent on their cofactor activities for tyrosine hydroxylase. That is, in contrast to 6R-BH4, dopamine releases induced by 6S-BH4 or 6-MPH4 might be due to enhanced activity of tyrosine hydroxylase. Moreover, 6R-BH4, is also a cofactor for NOS and NO is reported to stimulate dopamine release (Zhu and Luo, 1992; Liang and Kaufman, 1998). We examined the e€ect of 6R-BH4 in the presence of NOS inhibitors and demonstrated that dopamine-releasing action of 6R-BH4 was independent of its cofactor activity for NOS (Fig. 12). When 6S-BH4 or 6-MPH4 was coadministered with 6R-BH4, 6RBH4-induced dopamine release was blunted (Fig. 13). These results indicate that 6S-BH4 and 6-MPH4 acted as antagonists for dopamine-releasing action of 6RBH4. These data taken together, raise the possibility that 6R-BH4 stimulates dopamine release by acting on the speci®c site, where 6S-BH4 or 6-MPH4 acts as an antagonist (Klatt et al., 1994). Released dopamine is mostly retaken into the nerve terminals. Therefore, 6R-BH4-induced increase in dopamine levels in the dialysates may be due to an inhibition of dopamine uptake. This is again not likely because 6R-BH4induced increase in dopamine levels in the dialysates persisted in the presence of nomifensine, an inhibitor for dopamine uptake system. (Fig. 14). To further investigate the site of 6R-BH4 action on dopamine release, we examined the e€ects of sepiapterin, a precursor of 6R-BH4, on dopamine release from rat striatum. Sepiapterin is membrane permeable and is converted to 6R-BH4 by sepiapterin reductase and dihydrofolate reductase in the neurons (Figs. 15 and 16). When sepiapterin was added to the perfusion

Fig. 7. E€ects of derivatives of 6R-BH4 on dopamine release from rat striatum in vivo (Koshimura et al., 1991, 1995).

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6R-BH4 (Mataga et al., 1991; Wolf et al., 1991). However, it remains to be clari®ed whether the stimulating e€ect of 6R-BH4 is independent of its cofactor activity. It was observed that acetylcholine release from the hippocampus was stimulated by 6R-BH4 (Ohue et al., 1991a, 1991b, 1992a, 1992b). The e€ect of 6R-BH4 on acetylcholine release was mediated by monoaminergic neurons. Glutamate release from the striatum was also stimulated by 6R-BH4, whereas the stimulating e€ect was abolished by depletion of dopaminergic input (Mataga et al., 1991). Therefore, it is suggested that 6R-BH4 has a stimulating e€ect only on dopamine release in the brain. 6R-BH4 is considered to modulate the release of various neurotransmitters by enhancing dopamine release in the brain. Fig. 8. E€ect of tetrodotoxin (TTX) on pteridine-induced dopamine release in vivo (Koshimura et al., 1990).

3. E€ect on Ca2+ channels in rat brain

¯uid, dopamine release considerably increased (Fig. 17). Administration of sepiapterin increased 6R-BH4 content in the striatal tissues (Fig. 18), but had little e€ect on extracellular 6R-BH4 concentration (Fig. 19). Sepiapterin-induced dopamine release was abolished by a-MT (Fig. 17), showing that sepiapterin-derived 6R-BH4 enhanced tyrosine hydroxylase activity to induce dopamine release. These results indicate that intracellular 6R-BH4 stimulated dopamine release by enhancing tyrosine hydroxylase activity as a cofactor. Therefore, it is assumed that 6R-BH4 stimulates dopamine release independent of its cofactor activity by acting from the outside of neurons. The e€ect of 6R-BH4 on the release of other neurotransmitters has been reported by several laboratories. Serotonin release from the striatum was stimulated by

To further investigate the mechanism of dopaminereleasing action of 6R-BH4, we examined the e€ect of 6R-BH4 on ion channels. First, we examined ion channel currents in the dopaminergic neurons in the dorsal motor nucleus of the vagus (DMNX) of 10±14 day old Wistar rats using a slice-patch study (Fig. 20) (Shiraki et al., 1996). After the rat brain was removed, the brain was placed in ice-cold Krebs solution which was gassed with 95% O2±5% CO2, and cut into 120 mm thick slices for patch clamp study. After the slices were incubated in oxygenated Krebs bu€er at 378C for 1 or 2 h, the slice was placed in the chamber of microscope with Nomarski optics and perfused with oxygenated Krebs solution. Under cell-attached recording, 6R-BH4 induced inward currents in the neurons of DMNX (Fig. 21). To investigate the 6R-BH4-induced inward current, we examined the e€ect of 6R-BH4 on ion currents in the neurons of DMNX by whole-cell record-

Fig. 9. E€ect of NKY 722 on pteridine-induced dopamine release in vivo.

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Fig. 10. Recovery of 6R-BH4 through the dialysis membrane.

ing. From the current after administration of 6R-BH4 (Fig. 22 right upper panel), outward K+ current (Fig. 22 left lower panel) was subtracted. The resulting current (Fig. 22 right lower panel) was assumed to be induced by 6R-BH4. Since 6R-BH4-induced current was compatible to Ca2+ currents, we then examined the e€ects of 6R-BH4 on isolated Ca2+ channel currents. The Ca2+ channel currents were measured by whole cell voltage clamp recording (Fig. 23). The Ca2+ channel currents recorded in DMNX were sensitive to o-conotoxin and Cd2+, and resistant to dihydropyridine (nicardipine) (Fig. 24), indicating that the Ca2+ channels examined are classi®ed as N-type (Table 1). Administration of 6R-BH4 to the perfusion ¯uid increased Ca2+ channel currents in DMNX neurons of rats, which was reversible (Fig. 25A). In contrast to 6R-BH4, 6S-BH4 had little e€ect on the Ca2+ channel current (Fig. 25B). These data show that the activating e€ect of 6R-BH4 on the Ca2+ channel was

stereospeci®c, which was compatible to the dopamine releasing e€ect of 6R-BH4. Following the administration of L-DOPA, a product of tyrosine hydroxyl-

Fig. 11. E€ect of a-methyl-p-tyrosine (a-MT) on pteridine-induced dopamine release (Koshimura et al., 1990, 1991). Pteridines were perfused at 1 mM for 60 min as shown by a bar and a-MT was intraperitoneally injected at 250 mg/kg as shown by an arrow.

Fig. 13. E€ect of 6S-BH4 or 6-MPH4 on 6R-BH4-induced dopamine release (Koshimura et al., 1995). Pteridines (1 mM) were perfused for 60 min as shown by a bar and a-MT (250 mg/kg) was intraperitoneally injected as shown by an arrow.

Fig. 12. E€ects of NOS inhibitors on 6R-BH4-induced dopamine release (Koshimura et al., 1994). NOS inhibitors and 6R-BH4 were perfused for 60 min as shown by bars. w: control; *: 6R-BH4 (1 mM); R: N-nitro-arginine (LNA) (3 mM) + 6R-BH4 (1 mM); Q: LNG-monomethyl-arginine (LNMMA) (3 mM) 6R-BH4 (1 mM). : p < 0.01 vs. corresponding control value.

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K. Koshimura et al. / Progress in Neurobiology 61 (2000) 415±438 Table 1 Characterization of Ca2+ channels in DMNX neurons by blockers

L-type N-type T-type P-type

Sensitive

Resistant

Dihydrooyridine o-conotoxin, Cd2+ o-conotoxin o-agatoxin

Dihydropyridine Cd2+, dihydropyridine o-conotoxin, dihydropyridine

ase or NOS. These data were also compatible to the dopamine releasing action of 6R-BH4. These results taken together demonstrate that 6R-BH4 activates neuronal Ca2+ channels independent of its cofactor activity, and raise the possibility that 6R-BH4 stimulates dopamine release by activating Ca2+ channels in the brain. Fig. 14. E€ect of nomifensine, an inhibitor of dopamine uptake, on 6R-BH4-induced dopamine release in vivo (Koshimura et al., 1990). Nomifensine was intraperitoneally injected at 20±200 mg/kg as shown by an arrow and 6R-BH4 was perfused as shown by a bar. w: : nomifensine 20 mg/kg; u: nomifensine 100 mg/kg; a: control; nomifensine 200 mg/kg; *: nomifensine 100 mg/kg +6R-BH4 (1 mM).

ation, to the perfusion ¯uid, Ca2+ channel currents were unchanged (Fig. 26A). When sodium nitroprusside (SNP), an NO generator, was added to the perfusion ¯uid, Ca2+ channel currents were not a€ected (Fig. 26B). These results indicate that the stimulating e€ect of 6R-BH4 on Ca2+ channel currents was independent of its cofactor activity for tyrosine hydroxyl-

Fig. 15. Action of 6R-BH4. qBH2: quinonoid dihydrobiopterin; DA: dopamine.

4. E€ect on dopamine release and Ca2+ channels in PC12 cells To further investigate the mechanism of the dopamine releasing action of 6R-BH4, we studied the dopamine-releasing action of 6R-BH4 in the brain using di€erentiated PC12 cells (Koshimura et al., 1999b). PC12 cells were cultured with Dulbecco's modi®ed Eagle's medium supplemented with 10% fetal calf serum and 10% horse serum, and di€erentiated with 100 ng/ml nerve growth factor (NGF) for 5±7 days. For dopamine release study, PC12 cells were incubated with Krebs±Hepes bu€er containing test drugs for 10 min. When 6R-BH4 was added to the incubation buffer, dopamine release from PC12 cells increased in a dose-related manner (Fig. 27A). The dopamine-releasing action of 6R-BH4 persisted in the presence of aMT or L-nitro-arginine methyl ester (L-NAME), an in-

Fig. 16. Sepiapterin: a precursor of 6R-BH4.

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Fig. 17. E€ect of a-MT on sepiapterin-induced dopamine release (Koshimura et al., 1994). Pteridines were perfused as shown by bars and a-MT was intraperitoneally injected at 250 mg/kg as shown by arrows.

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Fig. 19. E€ects of sepiapterin and 6R-BH4 on extracellular 6R-BH4 concentration (Koshimura et al., 1994).

hibitor of NOS (Fig. 27A), but was blunted by nicardipine. The dopamine releasing action of 6R-BH4 was not mimicked by 6S-BH4 or sepiapterin (Fig. 27B). These results show that the dopamine releasing action of 6R-BH4 in the brain can be simulated in di€erentiated PC12 cells. Then, we examined the e€ect of 6RBH4 on Ca2+ channel activity in PC12 cells (Koshimura et al., 1999a). For this purpose, we measured 2‡ 45 Ca uptake into PC12 cells, an index for Ca2+ channel activity. After PC12 cells were incubated with 2‡ Krebs±Hepes bu€er containing 45 Ca and test drugs, incubation bu€er was removed. Then, cells were washed and the radioactivity in the cells was measured. 2‡ uptake reached plateau at 40 s and The 45 Ca remained constant up to 120 s (Fig. 28). Therefore, 2‡ 45 Ca uptake for 1 min is considered as an index for

activated Ca2+ channel population. When 6R-BH4 2‡ was added to the incubation bu€er, 45 Ca uptake into PC12 cells increased in a dose-related manner (Fig. 29). Intracellular Ca2+ concentration monitored by fura-2 was increased by 6R-BH4 (Fig. 30A). Thus 6RBH4 activated Ca2+ channels to increase intracellular Ca2+ concentration in PC12 cells. 6R-BH4-induced increase in Ca2+ uptake persisted in the presence of aMT or L-NAME, and was not mimicked by dopamine, L-DOPA and NOC-5, a NO generator (Fig. 29). These results indicate that 6R-BH4 activated Ca2+ channels in PC12 cells independent of its cofactor activities, which was compatible to the results of DMNX neurons in the rats. The activating e€ect of 6R-BH4 on Ca2+ channels in PC12 cells was blunted by RpcAMPs, an inhibitor of protein kinase A, and mimicked by 8-bromo cAMP (Fig. 29). The e€ect of 6R-BH4 on Ca2+ uptake was not a€ected by phorbol ester (TPA), an activator of protein kinase C (Fig. 29). These results show that 6R-BH4-induced increase in

Fig. 18. E€ects of sepiapterin and 6R-BH4 on 6R-BH4 content in the striatum (Koshimura et al., 1994).

Fig. 20. Patch clamp method. 4-AP: 4-aminopyridine; TEA: tetraethylammonium; TTX: tetrodotoxin.

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Fig. 21. 6R-BH4-induced ion currents in DMNX neurons of rats measured by cell-attached recording.

Ca2+ uptake was mediated by activation of protein kinase A (Artalejo et al., 1990; Fournier et al., 1993; Sculptoreanu et al., 1993; Wang et al., 1993) but not protein kinase C (Vivaudou et al., 1988; Lacerda et al., 1988; Yang and Tsien, 1993). Dopamine release induced by 6R-BH4 was blunted by Rp-cAMPs and mimicked by 8-bromo cAMP (Fig. 31). These results taken together indicate that 6R-BH4 stimulated dopamine release from PC12 cells by activating protein kinase A (Fig. 32). Recently, by using potential sensitive dye, we observed that 6R-BH4 depolarized membrane potential in PC12 cells (Koshimura et al., 1999b) (Fig. 30B). These data suggest that 6R-BH4 activated Ca2+ channels by inducing membrane deplorization. It remains to be clari®ed whether membrane depolarizing action of 6R-BH4 is mediated by protein kinase A.

Fig. 22. 6R-BH4-induced currents in DMNX neurons of rats measured by whole-cell recording.

Fig. 23. Ca2+ current measurement by whole-cell recording. 4-AP: 4aminopyridine; TEA: tetraethylammonium; TTX: tetrodotoxin.

5. E€ect on survival of PC12 cells Several lines of evidence indicate that high potassium-induced depolarization enhances neuronal survival (Lasher and Zagon, 1972; Gallo et al., 1987; Wakade et al., 1988; Hack et al., 1993; Galli et al., 1995). It is reported that Ca2+ channel activation by high potass-

Fig. 24. Characterization of Ca2+ currents in DMNX neurons of rats (Shiraki et al., 1996). o-CgTx: o-conotoxin.

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Fig. 26. E€ects of sodium nitroprusside (SNP) and L-DOPA on Ca2+ currents in DMNX neurons of rats (Shiraki et al., 1996).

Fig. 25. E€ects of 6R-BH4 and 6S-BH4 on Ca2+ currents in DMNX neurons of rats (Shiraki et al., 1996).

ium enhanced survival of NGF-dependent neurons (Koike et al., 1989; Koike and Tanaka, 1991; Collins et al., 1991). This may be due to an increase in intracellular Ca2+ concentration after activation of Ca2+ channels (Ca2+ set-point hypothesis) (Johnson et al., 1992). At basal concentration of intracellular Ca2+, neuronal cells live dependently on NGF. At high concentration of intracellular Ca2+ induced by depolarization, neuronal cells can live less dependently on NGF. At higher concentration of intracellular Ca2+ induced by ischemia, neurons can no longer live (Fig. 33). According to this hypothesis, it is speculated that endogenous bioactive substances, which activate Ca2+ channels, may stimulate neuronal survival. Recently, trophic e€ect of 6R-BH4 on PC12 cells was reported (Anastasidias et al., 1996, 1997). Thus, we examined the e€ects of 6R-BH4 on survival of PC12 cells di€erentiated by NGF (Koshimura et al., 1999a). When PC12 cells were cultured without serum and NGF,

cells died with DNA fragmentation as reported previously (Mensner et al., 1992; Batistatou and Greene, 1993) (Figs. 34 and 35B). When 25 mM KCl was

Fig. 27. E€ects of various drugs on dopamine release from PC12 cells (Koshimura et al., 1999b). (A): E€ects of 6R-BH4, nicardipine, a-methyl-p-tyrosine (a-MT) and L-nitro-arginine methyl ester (LNAME) on dopamine release from PC12 cells. (B): E€ects of 6RBH4, 6S-BH4 and sepiapterin (SP) on dopamine release from PC12 cells.

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Fig. 30. E€ect of 6R-BH4 on intracellular Ca2+ concentration (A) and membrane potential (B) in PC12 cells (Koshimura et al., 1999b).

2‡

Fig. 28. Timecourse of 45 Ca uptake induced by 6R-BH4 and KCl (Koshimura et al., 1999a). w: control; Q: 6R-BH4 (30 mM); *: KCl (50 mM).

added to the culture medium, viable cell number under depletion of serum and NGF remarkably increased (Fig. 35C). Administration of 6R-BH4 also increased viable cell number under depletion of serum and NGF (Fig. 35D). However, surviving cells showed round shape without neurite extension as observed before di€erentiation. These results show that 6R-BH4 enhanced survival of PC12 cells but that 6R-BH4 had no e€ect on di€erentiation. The cell-surviving e€ect of 6R-BH4 persisted in the presence of a-MT or LNAME (Fig. 36), indicating that the e€ect of 6R-BH4 was independent of its cofactor activity. The e€ect of 6R-BH4 on cell survival was inhibited by nicardipine

(Fig. 36), showing that the e€ect of 6R-BH4 was mediated by Ca2+ channel activation. The cell-surviving e€ect of 6R-BH4 was blunted by Rp-cAMPs and mimicked by 8-bromo cAMP but not TPA (Fig. 36). These results indicate that the e€ect of 6R-BH4 was associated with activation of protein kinase A (Murrell and Tolkovsky, 1993; Kew et al., 1996) but not protein kinase C (Zirrgiebel et al., 1995). The characteristics of the e€ects of 6R-BH4 on cell survival were compatible to those on Ca2+ channel activation. The e€ect of 8bromo-cAMP on cell survival was inhibited by nicardipine, indicating that cell-surviving e€ect of 8-bromocAMP was mediated by Ca2+ channel activation as well as the e€ect of 6R-BH4 (Fig. 36). These results taken together demonstrate that 6R-BH4 enhances survival of PC12 cells under depletion of NGF and serum by activating Ca2+ channels via cAMP-protein kinase A pathway. Compared with 6R-BH4, the e€ects of 6S2‡ uptake and cell survival were less BH4 on 45 Ca potent (Fig. 37). Thus, it is considered that the e€ects of 6R-BH4 on cell survival have stereospeci®city as

2‡

Fig. 29. E€ects of various drugs on 454 Ca uptake into PC12 cells (Koshimura et al., 1999a). 8-br-cAMP, 8-bromo-cAMP; TPA, phorbol 12-myristate 13-acetate.

Fig. 31. E€ects of Rp-cAMPs on 6R-BH4-induced dopamine release from PC12 cells. 8br-cAMP, 8-bromo-cAMP.

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Fig. 34. DNA fragmentation of PC12 cells. (A): 5-Day culture without serum and NGF. (B): Administration of 30 mM 6R-BH4 to culture medium without serum and NGF.

Fig. 32. Action of 6R-BH4 on PC12 cells. A-kinase: protein kinase A; Arg: arginine; DA: dopamine; SP: sepiapterin; TH: tyrosine hydroxylase.

well as those on dopamine release. To further investigate the mechanism of the e€ect of 6R-BH4, we examined the e€ect of 6R-BH4 on mitogen-activated protein (MAP) kinase in PC12 cells. MAP kinase activity was not stimulated by 6R-BH4 (Fig. 38). These results may explain the absence of supporting e€ect of 6R-BH4 on neuronal di€erentiation of PC12 cells. Under depletion of serum and NGF, depolarization-induced Ca2+ channel activation (Fig. 39A) and dopamine release (Fig. 39B) were diminished in PC12 cells even in the presence of 6R-BH4. These results show that 6R-BH4 cannot maintain excitability of PC12 cells under depletion of serum and NGF. Thus, 6R-BH4 had little e€ect on preservation of neuronal function. To further investigate the cell-surviving action of endogenous bioactive substances, we have examined the e€ects of pituitary adenylate cyclase activating polypeptide (PACAP) (Tanaka et al., 1997) or erythropoietin (Koshimura et al., 1999c) on survival of PC12 cells.

Fig. 33. Ca2+ set-point hypothesis (Franklin and Johnson, 1992).

PACAP activated Ca2+ channels (Fig. 40A) and increased intracellular Ca2+ concentrations in PC12 cells (Fig. 40B). PACAP enhanced survival of PC12 cells with neurite extension (Fig. 41). MAP kinase activity was enhanced by PACAP (Fig. 42). Similar e€ects were observed in erythropoietin for PC12 cells. It is reported that activation of Ca2+ channels induces activation of ras-MAP kinase cascade in PC12 cells (Rosen et al., 1994; Rosen and Greenberg, 1996; Rusanescu et al., 1995; Finkbeiner and Greenberg, 1996). Thus, it is speculated that PACAP and erythropoietin enhance survival of PC12 cells with di€erentiated form by activating Ca2+ channel-ras-MAP kinase pathway. Comparing the e€ects of 6R-BH4 on cell survival with those of PACAP and erythropoietin, it is assumed that activation of MAP kinase is critical for neuronal di€erentiation in PC12 cells (Fukuda et al., 1995) but not for survival (Creedom et al., 1996). 6. E€ect on cytotoxicity of NO NO was identi®ed as vasodilator derived from endothels (Palmer et al., 1987). Recently, it has been clari®ed that NO has diverse functions (Snyder and Bredt, 1991; Lowenstein and Snyder, 1992; McCall and Vallance, 1992; Schmidt and Walter, 1994; Reid et al., 1996). Cytotoxic e€ect is one of the major functions of NO which is synthesized in macrophages, lymphocytes or glia cells (Corrazilia et al., 1993; Lewis et al., 1995; BruÈne et al., 1997). In these cells, inducible NOS is present and this enzyme is induced when the cells are activated by interferon-g, interleukin 2 or tumor necrosis factor-a (Evans et al., 1996; Sakai et al., 1993, 1995; Wheeler et al., 1997). Thus, it is considered that NO is involved in cellular immunity. Several lines of evidence indicate that neuronal apoptosis occurs in neurodegenerative disorders (Su et al., 1994;

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Fig. 35. E€ects of high K+ and 6R-BH4 on microscopic view of PC12 cells cultured for ®ve days in the absence of serum and NGF (Koshimura et al., 1999a). (A): control; (B): depletion of serum and NGF; (C): addition of KCl (50 mM) to culture medium without serum and NGF; (D): addition of 6R-BH4 (30 mM) to culture medium without serum and NGF.

Lassmann et al., 1995; Portera-Cailliau et al., 1995; Schreiber and Baudry, 1995; Thompson, 1995). NO was observed to induce neuronal apoptosis (Dawson et al., 1991; Dawson et al., 1994; Chao et al., 1992; Bonfoco et al., 1995; McMillian et al., 1995; Jacobson, 1996; Troy et al., 1996). Thus, cytotoxicity of NO is considered to play a critical role for neurodegenerative disorders. Interestingly, it is reported that neuronal cells containing NOS survive in the brain of neurodegenerative disorders (Ferrante et al., 1985; Kowall and Beal, 1988; Uemura et al., 1990; Lowenstein and Snyder, 1992). These results raise the possibility that selfprotective system for NO is present in NO producing neurons. Free radical scavengers such as glutathione or superoxide dismutase are suggested as protective agents for NO toxicity (Stamler, 1994). However, these substances are not located speci®cally in NO producing neurons. Recently, 6R-BH4 is reported to oxidize

NO into inactive products during its autoxidation (Mayer et al., 1995). These results raise the possibility that 6R-BH4 acts as a self-protective agent for NO toxicity. We investigated the e€ects of 6R-BH4 on cytotoxicity of NO using PC12 cells (Koshimura et al., 1998). Administration of sodium nitroprusside (SNP) or NOC-12, NO generators, at 10 or 30 mM to the incubation medium induced cell death in PC12 cells (Fig. 43B, D, F, H). When cells were cultured with NO generators and 30 mM 6R-BH4, cell death induced by NO generators was considerably inhibited (Fig. 43C, E, G, I). The protective e€ect of 6R-BH4 was maximum at 10 mM (Fig. 44). Cell death induced by NO generators at 100 mM was not prevented by 6RBH4 (Fig. 44). Administration of 6R-BH4 to the culture medium decreased NO metabolite levels, either in the presence or absence of NO generators (Fig. 45). These results taken together indicate that exogenous

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Fig. 36. E€ects of various drugs on survival of PC12 cells (Koshimura et al., 1999a). #: p < 0.05; vs. the control group cultured with serum and NGF. : p < 0.05; vs. the group cultured without serum and NGF (serum (-)/NGF (-)); 8br-cAMP: 8-bromo-cAMP; TPA: phorbol 12-myristate 13-acetate.

2‡

2‡

Fig. 37. E€ects of 6S-BH4 on 45 Ca uptake and cell survival in PC12 cells (Koshimura et al., 1999a). (A): E€ects of 6S-BH4 on 45 Ca uptake. : p < 0.05; vs. the control group. (B): E€ects of 6S-BH4 on the number of viable cells cultured without serum and NGF. #: p < 0.05; vs. the control group cultured with serum and NGF. : p < 0.05; vs. the group cultured without serum and NGF.

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Fig. 38. E€ects of 6R-BH4 on MAP kinase activity in PC12 cells (Koshimura et al., 1999a). : p < 0.05; vs. the control group.

6R-BH4 prevented NO-induced cell death by degrading NO. Then, we investigated the e€ect of endogenous 6R-BH4 on toxicity of NO. When 2,4-diamino-6hydroxypyrimidine (DAHP), an inhibitor for GTP cyclohydrolase I, was added to the culture medium, viable cell number was decreased (Fig. 46A). Cell death induced by DAHP was prevented by 6R-BH4

2‡

Fig. 39. E€ects of 6R-BH4 on cellular functions in PC12 cells cultured without serum and NGF (Koshimura et al., 1999a). (A): 2‡ E€ects of 6R-BH4 on high K+-induced 45 Ca uptake into PC12 cells cultured without serum and NGF. : p < 0.05; vs. the control group cultured with serum and NGF. #: p < 0.05; vs. the control group cultured in the medium supplemented with 6R-BH4 but not serum and NGF. (B): E€ects of 6R-BH4 on high K+-induced dopamine release from PC12 cells cultured without serum and NGF. : p < 0.05; vs. the control group cultured with serum and NGF.

(Fig. 46B). Intracellular concentration of 6R-BH4 was decreased by DAHP (Fig. 46C), showing that DAHP inhibited biosynthesis of 6R-BH4. Administration of 6R-BH4 restored intracellular 6R-BH4 levels (Fig. 46C). These results show that endogeneous 6R-BH4 protects PC12 cells against NO toxicity. Since 6R-BH4 is a cofactor for NOS, reduction of intracellular 6R-

Fig. 40. E€ect of PACAP-38 and its related drugs on 45 Ca uptake into PC12 cells and intracellular Ca2+ concentration (Tanaka et al., 1997). 2‡ (A): E€ects of PACAP and its related drugs on 45 Ca uptake into PC12 cells. : p < 0.05; vs. the control group. 8br-cAMP: 8-bromo-cAMP; TPA: phorbol 12-myristate 13-acetate. (B): E€ects of PACAP on intracellular Ca2+ concentration in PC12 cells.

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431

Fig. 41. E€ects of KCl and PACAP-38 on microscopic view of PC12 cells cultured for three days in the absence of serum and NGF (Tanaka et al., 1997). (A): control; (B): depletion of serum and NGF; (C): addition of 50 mM KCl to culture medium deprived of serum and NGF; (D): addition of 10ÿ8 M PACAP-38 to culture medium deprived of serum and NGF.

Fig. 42. E€ects of PACAP-38 on MAP kinase activity in PC12 cells (Tanaka et al., 1997). : p < 0.05; vs. the control group.

BH4 may cause decrease in NO synthesis. This is not likely because intracellular NO metabolite levels were unchanged in the presence of DAHP (Fig. 46D). These results suggest that NOS was saturated with 6R-BH4 in PC12 cells. It was reported that superoxide was generated during autoxidation of 6R-BH4 (Mayer et al., 1995). To investigate the mechanism of cell protective action of 6R-BH4, we examined the e€ect of superoxide on NO toxicity in PC12 cells. When the cells were cultured with hypoxanthine and xanthine oxidase, superoxide generating system, cell viability was unchanged at 10 mM hypoxanthine. Viable cell number was decreased by hypoxanthine at higher than 100 mM (Fig. 47A). Within non-toxic concentration of hypoxanthine and xanthine oxidase, superoxide generating system prevented NO-induced cell death and degraded NO as observed in the e€ect of 6R-BH4 on NO toxicity (Fig. 47B, C). These results indicate that cell protective e€ect of 6R-BH4 against NO toxicity is mediated by superoxide, which is generated during

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Fig. 43. Photomicrographs of PC12 cells cultured for three days with sodium nitroprusside (SNP), NOC-12 and 6R-BH4 (Koshimura et al., 1998). (A) control; (B) SNP (10 mM); (C) SNP (30 mM) +6R-BH4 (30 mM); (D) SNP (30 mM); (E) SNP (30 mM) +6R-BH4 (30 mM); (F) NOC12 (10 mM); (G) NOC-12 (10 mM) +6R-BH4 (30 mM); (H) NOC-12 (30 mM); (I) NOC-12 (30 mM) +6R-BH4 (30 mM).

autoxidation of 6R-BH4. It is reported that superoxide reacts with NO to generate peroxynitrite (Lipton et al., 1993; EsteÂvez et al., 1995), which is toxic to cells. Peroxynitrite is unstable and rapidly decomposed to nitrate (Pryor and Squadrito, 1995). In our culture system of PC12 cells, administration of SNP and 6R-BH4 decreased nitrate levels (Fig. 45). Thus, it is not likely that peroxynitrite was generated during the culture with NO generators and 6R-BH4 in our system. These data taken together demonstrate that 6R-BH4 protects PC12 cells from NO toxicity by degrading NO during autoxidation. It is reported that NOS is unsaturated with 6R-BH4 under ischemic condition. Under such conditions, 6RBH4 may act as a stimulant for NO synthesis and enhance NO toxicity (Cho et al., 1999). The role of

6R-BH4 for NO toxicity in vivo is assumed to be diverse than expected, and remains to be further investigated. 7. Conclusion In conclusion, exogenous 6R-BH4 stimulates neuronal activities for monoaminergic neurons. The physiological signi®cance of 6R-BH4 in the brain remains to be fully clari®ed. However, since 6R-BH4 is known to be secreted from endothelial cells (Walter et al., 1994), it is possible that endogenous 6R-BH4 plays a role for regulating neuronal activities. Therefore, 6R-BH4 may be a promising drug for neurodegenerative disorders such as Parkinson's disease or Alzheimer's disease. In

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Fig. 44. E€ects of sodium nitroprusside (SNP), NOC-12 and 6R-BH4 on the viable cell number of PC12 cells (Koshimura et al., 1998). (A): E€ects of 6R-BH4 on SNP-induced cell death. : p < 0.05 vs. the control group; #: p < 0.05; vs. the group cultured with 10 mM sodium nitroprusside (SNP); }: p < 0.05 vs. the group cultured with 30 mM SNP. (B) E€ects of 6R-BH4 on NOC-12-induced cell death. : p < 0.05 vs. the control group; #: p < 0.05 vs. the group cultured with 10 mM NOC-12; }: p < 0.05; vs. the group cultured with 30 mM NOC-12.

Fig. 45. E€ects of sodium nitroprusside (SNP) and 6R-BH4 on the concentrations of NO metabolites (nitrite and nitrite) in the culture medium (Koshimura et al., 1998). A: Representative chromatograms of authentic standard (100 pmol of NaNO2 and NaNO3) (A), cell-free DMEM (B), control group (C), the group cultured with 30 mM SNP (D) and the group cultured with 30 mM SNP and 30 mM 6R-BH4 (E). B: Nitrite …NOÿ 2† concentrations in culture medium of PC12 cells. C: Nitrate …NOÿ 3 † concentrations in culture medium of PC12 cells. : p < 0.05 vs. control group; #: p < 0.05 vs. the group cultured with 10 mM SNP; }: p < 0.05; vs. the group cultured with 30 mM SNP.

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Fig. 46. E€ect of 2,4-diamino-6-hydroxypyrimidine (DAHP) on PC12 cells (Koshimura et al., 1998). (A) E€ect of DAHP on viable cell number. : p < 0.05 vs. control group. (B) E€ect of 6R-tetrahydrobiopterin (6R-BH4) on number of viable cells cultured with DAHP. : p < 0.05; vs. control group. (C) E€ect of DAHP and 6R-BH4 on cellular 6R-BH4 concentrations. : p < 0.05; vs. control group. (D) E€ect of DAHP on nitrite concentration in culture medium of PC12 cells.

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435

Fig. 47. E€ect of hypoxanthine (HX) and xanthine oxidase (XO) on viability of PC12 cells (Koshimura et al., 1998). (A) E€ect of HX and XO on viable cell number. : p < 0.05 vs. control group. (B) E€ect of HX and XO on sodium nitroprusside (SNP)-induced decrease in viable cell number. : p < 0.05 vs. control group. #: p < 0.05 vs. the group cultured with 30 mM SNP. (C) E€ect of HX and XO on SNP-induced increase in nitrite concentration in the culture medium. : p < 0.05; vs. control group. #: p < 0.05; vs. the group cultured with 30 mM SNP.

the brain, 6R-BH4 enters neurons independent of glucose and extracellular sodium (Anastasidias et al., 1994). However, 6R-BH4 poorly entered into the brain when it was peripherally administered (Levine et al., 1987). Although several derivatives of 6R-BH4 showed good entrance into the brain (Levine et al., 1987), the e€ects of 6R-BH4 have stereospeci®city. Therefore, more e€ective system of drug delivery to the brain is critically required for the strategy of the therapy with 6R-BH4.

References Anastasidias, P.Z., Kuhn, D.M., Levine, R.A., 1994. Tetrahydrobiopterin uptake into rat brain synaptosomes, cultured PC12 cells, and rat striatum. Brain Res 665, 77±84. Anastasidias, P.Z., States, J.C., Imerman, B.A., Louie, M.C., Kuhn, D.M., Levine, R.A., 1996. Mitogenic e€ects of tetrahydrobiopterin in PC12 cells. Mol Pharmacol 49, 149±155. Anastasidias, P.Z., Bezin, L., Imerman, B.A., Kuhn, D.M., Louie, M.C., Levine, R.A., 1997. Tetrahydrobiopterin as a mediator of PC12 cell proliferation induced by EGF and NGF. Eur. J. Neurosci 9, 1831±1837. Artalejo, C.R., Ariano, M.A., Perlman, R.L., Fox, A.P., 1990. Activation of facilitation calcium channels in chroman cells by D1 dopamine receptors through a cAMP/protein kinase A-dependent mechanism. Nature 348, 239±242. Bailey, S.W., Dillard, S.B., Ayling, J.E., 1991. Role of C6 chirality of tetrahydrobiopterin cofactor in catalysis and regulation of tyrosine and phenylalanine hydroxylases. Biochemistry 30, 10226± 10235.

Batistatou, A., Greene, L.A., 1993. Internucleosomal DNA cleavage and neuronal cell survival/death. J. Cell Biol 122, 523±532. Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P., Lipton, S.A., 1995. Apoptosis and necrosis: Two distinct events induced respectively by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. USA 92, 7162±7166. Brenneman, A.R., Kaufman, S., 1964. The role of tetrahydropteridines in the enzymatic conversion of tyrosine to 3,4-dihydroxyphenylalanine. Biochem. Biophys. Res. Commun 17, 177±183. BruÈne, B., GoÈtz, C., Meûmer, U.K., Sandau, K., Hirvonen, M.-R., Lapetina, E.G., 1997. Superoxide formation and macrophage resistance to nitric oxide-mediated apoptosis. J. Biol. Chem 272, 7253±7258. Bullard, W.P., Guthrie, P.B., Russo, P.V., Mandell, A.J., 1978. Regional and subcellular distribution and some factors in the regulation of reduced pterins in the brain. J. Pharmacol. Exp. Ther 205, 4±20. Chao, C.C., Hu, S., Molitor, T.W., Shaskan, E.G., Peterson, P.K., 1992. Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J. Immunol 149, 2736±2741. Cho, S., Volpe, B.T., Bae, Y., Hwang, O., Choi, H.J., Gal, J., Park, L.C.H., Chu, C.K., Du, J., Joh, T.H., 1999. Blockade of tetrahydrobiopterin synthesis protects neurons after transient forebrain ischemia in rat: a novel role for the cofactor. J. Neurosci 19, 878± 889. Collins, F., Schmidt, M.F., Guthrie, P.B., Kater, S.B., 1991. Sustained increase in intracellular calcium promotes neuronal survival. J. Neurosci 11, 2582±2587. Corrazilia, I.M., Campo, M.L., Fuentes, J.M., Campos-Portuguez, S., Soler, G., 1993. Parallel induction of nitric oxide and glucose6-phosphate dehydrogenase in activated bone marrow derived macrophages. Biochem. Biophys. Res. Commun 196, 342±347. Creedom, D.J., Johnson Jr, E.M., Lawrence Jr, J.C., 1996. Mitogenactivated protein kinase-independent pathways medicate the

436

K. Koshimura et al. / Progress in Neurobiology 61 (2000) 415±438

e€ects of nerve growth factor and cAMP on neuronal survival. J. Biol. Chem 271, 20713±20718. Curtius, H.-C., Niederwieser, A., Levine, R.A., Lovenberg, W., Woggon, B., Angst, J., 1983. Successful treatment of depression with tetrahydrobiopterin, Lancet I 657±658. Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S., Snyder, S.H., 1991. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. Natl. Acad. Sci. USA 88, 6368± 6371. Dawson, T.M., Dawson, V.L., Snyder, S.H., 1994. Molecular mechanisms of nitric oxide actions in the brain. Ann. NY Acad. Sci 738, 76±85. EsteÂvez, A.G., Radi, R., Barbeito, L., Shin, J.T., Thompson, J.A., Beckman, J.S., 1995. Peroxynitrite-induced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism di€erentially modulated by neurotrophic factors. J. Neurochem 65, 1543±1550. Evans, T.J., Buttery, L.D.K., Carpenter, A., Springall, D.R., Polak, J.M., Cohen, J., 1996. Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingested bacteria. Proc. Natl. Acad. Sci. USA 93, 9553±9558. Ferrante, R.J., Kowall, N.W., Beal, M.F., Richardson Jr, E.P., Bird, E.D., Martin, J.B., 1985. Selective sparing of a class of striatal neurons in Huntington's disease. Science 230, 561±563. Finkbeiner, S., Greenberg, M.E., 1996. Ca2+-dependent routes to ras: mechanisms for neuronal survival di€erentiation and plasticity? Neuron 16, 233±236. Fournier, F., Bourinet, E., Nargeot, J., Charnet, P., 1993. Cyclic AMP-dependent regulation of P-type calcium channels expressed in Xenopus oocytes. P¯uÈger Arch. Eur. J. Physiol 423, 173±180. Franklin, J.L., Johnson Jr, E.M., 1992. Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium. Trends Neurosci. Carcia-Cardena, G 15, 501±508. Fujishiro, K.-I., Hagihara, M., Takahashi, A., Nagatsu, T., 1990. Concentrations of neopterin and biopterin in the cerebrospinal ¯uid of patients with Parkinson's disease. Biochem. Med. Metabol. Biol 44, 97±100. Fukuda, M., Gotoh, Y., Tachibana, T., Dell, K., Hattori, S., Yoneda, Y., Nishida, E., 1995. Induction of neurite outgrowth by MAP kinase in PC12 cells. Oncogene 11, 239±244. Fukushima, T., Nixon, J.C., 1980. Analysis of reduced forms of biopterin in biological tissues and ¯uids. Anal. Biochem 102, 176±188. Furukawa, Y., Kondo, T., Nishi, K., Yokochi, F., Narabayashi, H., 1991. Total biopterin levels in the ventricular CSF of patients with Parkinson's disease: a comparison between akineto-rigid and tremor types. J. Neurol. Sci 103, 232±237. Galli, C., Meucci, O., Scorziello, A., Werge, T.M., Calissano, P., Schettini, G., 1995. Apoptosis in cerebellar granule cells is blocked by high KCl forskoline and IGF-1 through distinct mechanisms of action: the involvement of intracellular calcium and RNA synthesis. J. Neurosci 15, 1172±1179. Gallo, V., Kingsbury, A., BalaÂzs, R., Jùrgensen, O.S., 1987. The role of depolarization in the survival and di€erentiation of cerebellar granule cells in culture. J. Neurosci 7, 2203±2213. Hasegawa, H., Imaizumi, S., Ichiyama, A., Sugimoto, T., Matsuura, S., Oka, K., Kato, T., Nagatsu, T., Akino, M., 1979. Cofactor activity of diastereoisomers of tetrahydrobiopterin. In: Kisliuk, R.L., Brown, G.M. (Eds.), Chemistry and Biology of Pteridines. Elsevier±North Holland, New York, pp. 183±188. Hack, N., Hidaka, H., Eake®eld, M.J., BalaÂzs, R., 1993. Promotion of granule cell survival by high K+ or excitatory amino acid treatment and Ca2+/calmodulin-dependent protein kinase activity. Neuroscience 57, 9±20. Jacobson, M.D., 1996. Reactive oxygen species and programmed cell death. Trends Biochem. Sci 21, 83±86. Johnson Jr, E.M., Koike, T., Franklin, J., 1992. A ``calcium set-

point hypothesis'' of neuronal dependence on neurotrophic factor. Exp. Neurol 115, 163±166. Kaufman, S., 1959. Studies on the mechanism of the enzymatic conversion of phenylalanine to tyrosine. J. Biol. Chem 234, 2677± 2682. Kay, A.D., Milstien, S., Kaufman, S., Creasey, H., Hazby, J.U., Cutler, N.R., Rapoport, S.I., 1986. Cerebrospinal ¯uid biopterin is decreased in Alzheimer's disease. Arch. Neurol 43, 996±999. Kew, J.N.C., Smith, D.W., Sofroniew, M.V., 1996. Nerve growth factor withdrawal induces the apoptotic death of developing septal cholinergic neurons in vitro protection by cyclic AMP analogue and high potassium. Neuroscience 70, 329±339. Klatt, P., Schmid, M., Leopold, E., Schmidt, K., Werner, E.R., Mayer, B., 1994. The pteridine binding site of brain nitric oxide synthase Ð tetrahydrobiopterin binding kinetics speci®city and allosteric interaction with the substrate domain. J. Biol. Chem. 269, 13861±13866. Koike, T., Martin, D.P., Johnson Jr, E.M., 1989. Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation: evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells. Proc. Natl. Acad. Sci. USA 86, 6421±6425. Koike, T., Tanaka, S., 1991. Evidence that nerve growth factor dependence of sympathetic neurons for survival in vitro may be determined by levels of cytoplasmic free Ca2+. Proc. Natl. Acad. Sci. USA 88, 3892±3896. Koshimura, K., Miwa, S., Lee, K., Fujiwara, M., Watanabe, Y., 1990. Enhancement of dopamine release in vivo from the rat striatum by dialytic perfusion of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin. J. Neurochem 54, 1391±1397. Koshimura, K., Miwa, S., Ohue, T., Lee, K., Fujiwara, M., Watanabe, Y., 1991. A novel action of 6R-BH4: direct stimulation of dopamine release in the brain. In: Blau, N., Curtius, H.C., Levine, R.A., Cotton, R.G.H. (Eds.), Pteridines and Biogenic Amines in Neurology, Pediatrics, and Immunology. Lakeshore Publishing Company, Grosse Pointe, pp. 299±312. Koshimura, K., Miwa, S., Watanabe, Y., 1994. Dopamine releasing action of 6R-L-erythro-tetrahydrobiopterin: analysis of its action site using sepiapterin. J. Neurochem 63, 649±654. Koshimura, K., Takagi, Y., Miwa, S., Kido, T., Watanabe, Y., Murakami, Y., Kato, Y., Masaki, T., 1995. Characterization of a dopamine releasing action of 6R-L-erythro-tetrahydrobiopterin: comparison with a 6S-form. J. Neurochem 65, 827±830. Koshimura, K., Murakami, Y., Tanaka, J., Kato, Y., 1998. Self-protection of PC12 cells by 6R-tetrahydrobiopterin from nitric oxide toxicity. J. Neurosci. Res 54, 664±672. Koshimura, K., Tanaka, J., Murakami, Y., Kato, Y., 1999a. Enhancement of neuronal survival by 6R-tetrahydrobiopterin. Neuroscience 88, 561±569. Koshimura, K., Tanaka, J., Murakami, Y., Kato, Y., 1999b. E€ect of tetrahydrobiopterin on dopamine release from PC12 cells. Biomed. Res 20, 141±144. Koshimura, K., Murakami, Y., Sohmiya, M., Tanaka, J., Kato, Y., 1999c. E€ects of erythropoietin on neuronal activity. J. Neurochem 72, 2565±2572. Kowall, N.W., Beal, M.F., 1988. Cortical somatostatin, neuropeptide Y, and NADPH diaphorse neurons: Normal anatomy and alterations in Alzheimer's disease. Ann. Neurol 23, 105±114. Kwon, N.S., Nathan, C.F., Stuehr, D.J., 1989. Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J. Biol. Chem. 264, 20496±20501. Lacerda, A.E., Rampe, D., Brown, A.M., 1988. E€ects of protein kinase C activators on cardiac Ca2+ channels. Nature 335, 249± 251. Lasher, R.S., Zagon, L.S., 1972. The e€ect of potassium on neuronal

K. Koshimura et al. / Progress in Neurobiology 61 (2000) 415±438 di€erentiation in cultures of dissociated newborn rat cerebellum. Brain Res 41, 482±488. Lassmann, H., Bancher, C., Breitschopf, H., Wegiel, J., Bobinski, M., Jellinger, K., Wisniewski, H.M., 1995. Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ. Acta Neuropathologica 89, 35±41. Levine, R.A., Miller, L.P., Lovenberg, W., 1981. Tetrahydrobiopterin in striatum localization in dopamine nerve terminals and role in catecholamine synthesis. Science 214, 919± 921. Levine, R.A., 1988. Tetrahydrobiopterin and biogenic amine metabolism in neuropsychiatry immunology and aging. Ann. NY Acad. Sci 521, 129±139. Levine, R.A., Zoephel, G.P., Niederwieser, A., Curtius, H.C., 1987. Entrance of tetrahydrobiopterin derivatives in brain after peripheral administration e€ect on biogenic metabolism. J. Pharmacol. Exp. Ther 242, 514±522. Lewis, R.S., Tamir, S., Tannenbaum, S.R., Deen, W.M., 1995. Kinetic analysis of the fate of nitric oxide synthesized by macrophages in vitro. J. Biol. Chem 270, 29350±29355. LeWitt, P.A., Miller, L.P., Levine, R.A., Lovenberg, W., Newman, R.P., Papavasiliou, A., Rayes, A., Eldridge, R., Burns, R.S., 1988. Pterin abnormalities in dystonia: A metabolic marker with therapeutic implications. Adv. Neurol 50, 193±201. Liang, L.P., Kaufman, S., 1998. The regulation of dopamine release from striatum slices by tetrahydrobiopterin and L-arginine-derived nitric oxide. Brain Res 800, 181±186. Lipton, S.A., Choi, Y.B., Pan, Z.H., Lei, S.Z., Chen, H.S.V., Sucher, N.J., Lascalzo, J., Singel, D.J., Stamler, J.S., 1993. A redox-based mechanism for the neuroprotective and neurodestructive e€ects of nitric oxide and related nitroso-compounds. Nature 364, 626±631. Lovenberg, W., Jequier, E., Sjoerdsma, A., 1967. Tryptophan hydroxylation: measurement in pineal gland brainstem and carcinoid tumor. Science 155, 217±219. Lovenberg, W., Levine, R.A., Robinson, D.S., Ebert, M., Kalne, A.C., Kalne, D.B., 1979. Hydroxylase cofactor activity in cerebrospinal ¯uid of normal subjects and patients with Parkinson's disease. Science 204, 624±626. Lowenstein, C.J., Snyder, S.H., 1992. Nitric oxide, a novel biogenic messenger. Cell 70, 705±707. Mataga, N., Imamura, K., Watanabe, Y., 1991. 6R-tetrahydrobiopterin perfusion enhances dopamine, serotonin, and glutamate outputs in dialysate from rat striatum and frontal cortex. Brain Res 551, 64±71. Mayer, B., John, M., BoÈhme, E., 1990. Puri®cation of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin. FEBS Lett 227, 215±219. Mayer, B., John, M., Heinzel, B., Werner, E.R., Wachter, H., Schltz, G., BoÈhme, E., 1991. Brain nitric oxide synthase is a biopterinand ¯avin-containing multi-functional oxide-reductase. FEBS Lett 288, 187±191. Mayer, B., Klatt, P., Werner, E.R., Schmidt, K., 1995. Kinetics and mechanism of tetrahydrobiopterin-induced oxidation of nitric oxide. J. Biol. Chem 270, 665±669. McCall, T., Vallance, P., 1992. Nitric oxide takes centre-stage with newly de®ned roles. Trends Pharmacol Sci 13, 1±6. McMillian, M., Kong, L.-Y., Sawin, S.-M., Wilson, B., Das, K., Hudson, P., Hong, J.-S., Bing, G., 1995. Selective killing of cholinergic neurons by microglial activation in basal forebrain mixed neuronal/glial cultures. Biochem. Biophys. Res. Commun 215, 572±577. Mensner, P.W., Winters, T.R., Greeen, S.H., 1992. Nerve Growth factor withdrawal-induced cell death in neuronal PC12 cells resembles that in sympathetic neurons. J. Cell Biol 119, 1669±1680. Minami, M., Takahashi, T., Maruyama, W., Takahashi, A., Nagatsu, T., Naoi, M., 1992. Allosteric e€ect of tetrahydrobiop-

437

terin cofactors on tyrosine hydroxylase activity. Life Sci 50, 15± 20. Miwa, S., Watanabe, Y., Hayaishi, O., 1985. 6R-L-erythroTetrahydrobiopterin as a regulator of dopamine and serotonin biosynthesis in the rat brain. Arch. Biochem. Biophys 239, 234± 241. Murrell, R.D., Tolkovsky, A.M., 1993. Role of voltage-gated Ca2+ channels and intracellular Ca2+ in rat sympathetic neuron survival and function promoted by high K+ and cyclic AMP in the presence or absence of NGF. Eur. J. Neurosci 5, 1261±1272. Nagatsu, T., Levitt, M., Udenfriend, S., 1964. Tyrosine hydroxylase. The initial step in norepinephrine biosynthesis. J. Biol. Chem 239, 2910±2917. Nagatsu, Y., Yamaguchi, T., Katyo, T., Sugimoto, T., Matsuura, S., Akino, M., Nagatsu, I., Iizuka, R., Narabayashi, H., 1981. Biopterin in human brain and urine from control and Parkinsonism patients: application of a new radioimmunoassay. Clin. Chim. Acta 661, 45±53. Nagatsu, T., Yamaguchi, T., Rahman, M.K., Trocewicz, J., Oka, K., Hirota, Y., Nagatsu, I., Narabayashi, H., Kondo, T., Iizuka, R., 1984. Catecholamine-related enzymes and the biopterin cofactor in Parkinson's disease and related extrapyramidal diseases. In: Hassler, R.G., Christ, J.F. (Eds.), Advanced in Neurology. Raven Press, New York, pp. 467±473. Naruse, H., Hayashi, T., Takesada, M., Nakane, A., Yamazaki, K., Noguchi, T., Watanabe, Y., Hayaishi, O., 1987. Therapeutic e€ect of tetrahydrobiopterin in infantile autism. Proc. Japan Acad 63 (B), 231±233. Niederwieser, A., Blau, N., Wang, M., Joller, P., Atares, M., Cardesa-Garcia, J., 1984. GTP cyclohydrolase I de®ciency a new enzyme defect causing hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin de®ciencies and muscular hypotonia. Eur. J. Pediatr 141, 208±214. Niederwieser, A., Leimbacher, W., Curtius, H.-C., Pronzone, A., Rey, F., Leupold, D., 1985. Atypical phenylketonuria with ``dihydrobiopterin synthase'' de®ciency absence of phosphate-eliminating enzyme activity demonstrated in liver. Eur. J. Pediatr 144, 13±16. Numata, T., Kato, T., Nagatsu, T., Sugimoto, T., Matsuura, S., 1977. E€ects of stereochemical structures of tetrahydrobiopterin on tyrosine hydroxylase. Biochim. Biophys. Acta 480, 104±112. Ohue, T., Lee, K., Koshimura, K., Miwa, S., 1991a. Characterization of a novel, hydrophilic dihydropyridine, NKY722, as a Ca2+ antagonist in bovine cultured adrenal chroman cells. Br. J. Pharmacol 103, 1889±1892. Ohue, T., Koshimura, K., Lee, K., Watanabe, Y., Miwa, S., 1991b. A novel action of 6R-L-erythro-tetrahydrobiopterin a cofactor for hydroxylases of phenylalanine tyrosine and tryptophan: enhancement of acetylcholine release in vivo in the rat hippocampus. Neurosci. Lett 128, 93±96. Ohue, T., Koshimura, K., Akiyama, Y., Ito, A., Kido, T., Takagi, Y., Miwa, S., 1992a. Regulation of acetylcholine release in vivo from rat hippocampus by monoamines as revealed by novel column-switching HPLC with electrochemical detection. Brain Res 572, 340±344. Ohue, T., Koshimura, K., Akiyama, Y., Watanabe, Y., Miwa, S., 1992b. Monoamine-mediated enhancement of acetylcholine release in rat hippocampus by 6R-L-erythro-tetrahydrobiopterin. Brain Res 570, 173±179. Oka, K., Kato, T., Sugimoto, T., Matsuura, S., Nagatsu, T., 1981. Kinetic properties of tyrosine hydroxylase with natural tetrahydropterin as a cofactor. Biochem. Biophys. Acta 661, 45±53. Palmer, R.M.J., Ferrige, A.G., Moncada, S., 1987. Nitric oxide release acounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524±526. Portera-Cailliau, C., Hedreen, J.C., Price, D.L., Koliatsos, V.E.,

438

K. Koshimura et al. / Progress in Neurobiology 61 (2000) 415±438

1995. Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. J. Neurosci 15, 3775±3787. Pryor, W.A., Squadrito, G.L., 1995. The chemistry of peroxynitrite: A product from the reaction of nitric oxide and superoxide. Am. J. Physiol 268, L699±L722. Reid, J.J., Beart, P.M., Marley, P.D., 1996. NO problems downunder. Trends Pharmacol. Sci 17, 205±208. Rosen, L.B., Ginty, D.D., Weber, M.J., Greenberg, M.E., 1994. Membrane depolarization and calcium in¯ux stimulate MEK and MAP kinase via activation of Ras. Neuron 12, 1207±1221. Rosen, L.B., Greenberg, M.E., 1996. Stimulation of growth factor receptor signal transduction by activation of voltage-sensitive calcium channels. Proc. Natl. Acad. Sci. USA 93, 1113±1118. Rusanescu, G., Qi, H., Thomas, S.M., Brugge, J.S., Halegoua, S., 1995. Calcium in¯ux induces neurite growth through a Src-Ras signaling cassette. Neuron 15, 1415±1425. Sakai, N., Kaufman, S., Milstein, S., 1993. Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264). Mol. Pharmacol 43, 6±10. Sakai, N., Kaufman, S., Milstein, S., 1995. Parallel induction of nitric oxide and tetrahydrobiopterin synthesis by cytokines in rat glial cells. J. Neurochem 65, 895±902. Schmidt, H.H.H.W., Walter, U., 1994. NO at work. Cell 78, 919± 925. Schreiber, S.S., Baudry, M., 1995. Selective neuronal vulnerability in the hippocampus Ð a role for gene expression. Trends Neurosci 18, 446±451. Sculptoreanu, A., Scheuer, T., Catterall, W.A., 1993. Voltage-dependent potentiation of L-type Ca2+ channels due to phosphorylation by cAMP-dependent protein kinase. Nature 364, 240±243. Shiraki, T., Koshimura, K., Kobayashi, S., Miwa, S., Masaki, T., Watanabe, Y., Murakami, Y., Kato, Y., 1996. Stimulating e€ect of 6R-tetrahydrobiopterin on Ca2+ channels in neurons of rat dorsal motor nucleus of the vagus. Biochem. Biophys. Res. Comm 221, 181±185. Smith, I., Clayton, B.E., Wol€, O.H., 1975. New variant of phenylketonuria with progressive neurological illness unresponsive to phenylalanine restriction, Lancet I 1108±1111. Snyder, S.H., Bredt, D.S., 1991. Nitric oxide as a neuronal messenger. Trends Pharmacol. Sci 12, 125±128. Stamler, J.S., 1994. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78, 931±936. Su, J.H., Anderson, A.J., Cummings, B.J., Cotman, C.W., 1994. Immunohistochemical evidence for apoptosis in Alzheimer's disease. Neuroreport 5, 2529±2533. Tanaka, J., Koshimura, K., Murakami, Y., Sohmiya, M., Yanaihara, N., Kato, Y., 1997. Neuronal protection from apoptosis by pituitary adenylate cyclase activating polypeptide. Regulat. Pept 72, 1±8.

Thompson, C.B., 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456±1462. Troy, C.M., Derossi, D., Prochiantz, A., Greene, L.A., Shelanski, M.L., 1996. Down regulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J. Neurosci 16, 253±261. Uemura, Y., Kowall, N.W., Beal, M.F., 1990. Selective sparing of NADPH-diaphorase-somatostatin-neuropeptide Y neurons in ischemic gerbil striatum. Ann. Neurol 27, 620±625. Vivaudou, M.B., Clapp, L.H., Walsh Jr, J.V., Singer, J.J., 1988. Regulation of one type of Ca2+ current in smooth muscle cells by diacylglycerol and acetylcholine. FASEB J 2, 2497±2504. Wakade, A.R., Wakade, T.D., Malhotra, R.K., Bhave, S.V., 1988. Excess K+ and phorbol ester activate protein kinase C and support the survival of chick sympathetic neurons in culture. J. Neurochem 51, 975±983. Walter, R., Sch€ner, A., Blau, N., Kierat, L., Schoedon, G., 1994. Tetrahydrobiopterin is a secretary product of murine vascular endothelial cells. Biochem. Biophys. Res. Commun 203, 1522±1526. Wang, Y., Townsend, C., Rosenberg, R.L., 1993. Regulation of cardiac L-type Ca channels in planar lipid bilayers by G proteins and protein phosphorylation. Am. J. Physiol 264, C1473±C1479. Wheeler, M.A., Smith, S.D., Garcia-Cardena, Nathan, C.F., Weiss, R.M., Sessa, W.C., 1997. Bacterial infection induces nitric oxide synthase in human neutrophils, J. Clin. Invest. 99, 110±116. Williams, A.C., Eldridge, R., Levine, R.A., Lovenberg, W., Paulson, G., 1979. Low CSF hydroxylase cofactor (tetrahydrobiopterin) levels in inherited dystonia. Lancet II 410±411. Williams, A.C., Levine, R.A., Calne, D.B., Chase, T.N., Lovenberg, W., 1980. CSF hydroxylase cofactor levels in some neurological diseases. J. Neurol. Neurosurg. Psychiatry 43, 735±738. Wolf, W.A., Ziaja, E., Arthur Jr, R.A., Anastasidias, P.Z., Levine, R.A., Kuhn, D.M., 1991. E€ect of tetrahydrobiopterin on serotonin synthesis release and metabolism in superfused hippocampal slices. J. Neurochem 57, 1191±1197. Yang, J., Tsien, R.W., 1993. Enhancement of N- and L-type calcium currents by protein kinase C in frog sympathetic neurons. Neuron 10, 127±136. Zhu, X.-Z., Luo, L.-G., 1992. E€ect of nitroprusside (nitric oxide) on endogenous dopamine release from rat striatal slices. J. Neurochem 59, 932±935. Zirrgiebel, U., Ohga, Y., Carter, B., Berninger, B., Inagaki, N., Thonene, H., Lindholm, D., 1995. Characterization of Trk Breceptor-mediated signaling pathways in rat cerebellar granule neurons: involvement of protein kinase C in neuronal survival. J. Neurochem 65, 2241±2250.