A novel action of 6R-l-erythro-5,6,7,8-tetrahydrobiopterin , a cofactor for hydroxylases of phenylalanine, tyrosine and tryptophan: Enhancement of acetylcholine release in vivo in the rat hippocampus

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Neuroscience Letters, 128 (1991) 93-96 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100335F NSL 07853

A novel action of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin, a cofactor for hydroxylases of phenylalanine, tyrosine and tryptophan: enhancement of acetylcholine release in vivo in the rat hippocampus Tetsuya Ohue 1, K u n i o K o s h i m u r a 1, Ken Lee 1, Yasuyoshi W a t a n a b e 2 and Soichi Miwa 1 1Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto (Japan) and ZDepartmentof Neuroscience, Osaka Bioscience Institute, Suita (Japan) (Received 18 February 1991; Revised version received 28 March 1991; Accepted 2 April 1991)

Key words: Acetylcholine release; 6R-BH4; Hippocampus; Brain microdialysis; Intracerebroventricular injection Recently, we have reported that 6R-L-erythro-tetrahydrobiopterin (6R-BH4), a natural cofactor for hydroxylases of phenylalanine, tyrosine and tryptophan, has a dopamine releasing action in the rat striatum independent of its cofactor role. Here we studied the effects of 6R-BH4 on acetylcholine (ACh) release in the rat hippocampus using brain microdialysis. Intracerebroventricular injection of 6R-BH4 dose-dependently increased extracellular ACh levels monitored by brain microdialysis. Biopterin, an oxidized form of 6R-BH4, and 6S-BH4, an unnatural diastereoisomer of 6R-BH4, had little effect on the ACh levels. The 6R-BH4-induced increase in ACh levels was eliminated after inhibition of voltage-dependent Na ÷ channels by tetrodotoxin, but not after depletion of catecholamines by reserpine. These results show that 6R-BH4 has direct ACh releasing action in vivo.

6R-L-erythro-5,6,7,8-Tetrahydrobiopterin (6R-BH4) is a natural cofactor for hydroxylases of phenylalanine [5], tyrosine [20] and tryptophan [18], and is considered to play an important role in the biogenic amine neurotransmitter synthesis. Recently, using brain microdialysis technique, we have shown that 6R-BH4 has dopamine releasing action in the rat brain, which is clearly distinguished from its cofactor role [14]. In the present study, to clarify whether this neurotransmitter releasing action of 6R-BH4 is specific for neuron systems in which 6R-BH4 serves as a cofactor for neurotransmitter synthesis, we investigate the effects of 6R-BH4 on in vivo release of acetylcholine (ACh), the biosynthesis of which does not require 6R-BH4 as a cofactor, using the brain microdialysis technique. Under ether anesthesia, a U-shaped probe for microdialysis (BDP 21-03, EICOM, Kyoto, Japan) was stereotactically implanted into the right hippocampus of male Wistar rats (250-300g) (coordinates taken from the bregma with the skull fiat: A, - 3.0; L, 2.5; V, - 3.5 mm, from the atlas of Pellegrino et al. [21]). After rats had recovered from the anesthesia, brain microdialysis was carried out under a free-moving condition [14, 15]. The

Correspondence." S. Miwa, Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606, Japan.

dialysis probe was continuously perfused at a flow rate of 4.8 pl/min with Ringer solution (147 mM NaCI, 4 mM CaC12 and 2.3 mM KC1, pH 6.1) containing 30/tM physostigmine sulfate, an inhibitor of acetylcholinesterase, and the dialysates were collected every 20 min. At the time of implantation of a microdialysis probe, a stainless-steel cannula (external diameter, 0.5 mm) for intracerebroventricular (i.c.v.) injection was stereotactically implanted into the lateral cerebral ventricle (A, 0.0; L, 1.7; V, -3.5) ipsilateral to brain microdialysis. Immediately before use, 6R-BH4 dihydrochloride or 6S-Lerythro-5,6,7,8-tetrahydrobiopterin (6S-BH4) dihydrochloride was initially dissolved in 100 mM sodium phosphate buffer (pH 7.4) and L-erythro-biopterin (biopterin) was dissolved in 1 M NaOH. The pH of these solutions was adjusted to 7.4 and 5 /tl of these solutions was injected into the cerebral ventricle using a microsyringe connected to the ventricular cannula. The final concentrations of 6R-BH4 or 6S-BH4 in the solution used for i.c.v, administration of 0.5 mg, 1.0 mg and 2.0 mg were 0.360 M, 0.720 M and 1.440 M, respectively, and that of biopterin for i.c.v, administration of 1.0 mg was 0.844 M. Doses of 6R-BH4 or 6S-BH4 are given in terms of the weights of the respective salts. For control rats, vehicle alone was injected. As described recently [7, 15], ACh and choline in hippocampal dialysates were determined using HPLC with an enzyme reactor, in which acetylcholinesterase and

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Fig. 1. Effect of various doses of 6R-BH4 injected intracerebroventricularly on levels of ACh (A) and choline (B) in hippocampal dialysates. One hundred min after the beginning of the brain microdialysis, 5/11 of either vehicle solution (©) or test solution containing various doses of 6R-BH4 (0.5 mg, A; 1.0 mg, • ; 2.0 mg, 0 ; in a salt form) was injected intracerebroventricularly as indicated by an arrow. Levels of ACh and choline collected in each fraction were expressed as percentages of the control value in the third fraction (ACh, 3.1 +0.2 pmol/20 min; choline, 85.3+6.5 pmol/20 min). Each value is the mean + S.E.M. of 4 determinations. *P<0.05; **P<0.01; significantly different from a control group which was injected with vehicle alone.

choline oxidase were immobilized, and with an electrochemical detector. The recoveries of ACh and choline through the dialysis membrane (concentration in dialysate/concentration in surrounding fluid x 100%) at the used flow rate were constant at the concentration range examined: 14.7 + 1.0 and 15.4+1.0% at 1 /IM, 15.1___0.9 and 15.8+0.8% at 10/~M, 14.9 __+1.2 and 14.8 + 0.7 % at 100/zM, respectively (n = 4). Therefore, the data in the present study were presented without correction for recovery. Fig. 1 shows the effects of various doses (0.5, 1.0 and 2.0 mg) of 6R-BH4 administered intracerebroventricularly on levels of ACh and choline in hippocampal dialysates. In control rats, which were intracerebroventricularly injected with vehicle alone, ACh levels in dialysates were constant from the beginning of the brain microdialysis up to 240 min. After i.c.v, injection of various doses of 6R-BH4, ACh levels in dialysates increased dosedependently and reached the maximum level in the fraction collected between 20 min and 40 min after the injection: the maximum levels were 150, 200 and 300% of the control value, respectively, following injection of 0.5, 1.0 and 2.0 mg of 6R-BHa (Fig. 1A). In contrast, choline levels in dialysates were virtually unchanged (Fig. 1B). Because 6R-BH4 is rapidly oxidized to biopterin in the neutral solution [12, 14], there is the possibility that the increase in ACh levels in dialysates is induced by biopterin but not by 6R-BH4. To test this assumption, we examined the effects of biopterin on ACh levels in dialy-

sates. Administration of 1 mg of biopterin had little effect on ACh levels in dialysates (Fig. 2). These results suggest that the increase in ACh levels in dialysates following administration of 6R-BH4 is due to the action of 6R-BH4 itself. Intracerebroventricular administration of 1 mg of 6SBH4, an unnatural diastereoisomer of 6R-BH4, could not increase ACh levels in dialysates (Fig. 2). This result suggests that stereospecificity is required for enhancement of ACh levels in dialysates. Thus it is likely that the effects of 6R-BH4 are not the result of nonspecific changes such as those in osmotic pressure, ionic strength, reducing power, etc. Generally, an increase in extracellular ACh levels is considered to be an increase in exocytotic release of ACh, which depends on neuronal activity. Alternatively, the increase could be induced by an increase in nonexocytotic leakage of ACh, which does not depend on neuronal activity. To distinguish between release and leakage, we examined the effects of 6R-BH4 on ACh levels in dialysates after pretreatment with tetrodotoxin (TTX), which inhibits neuronal activity by selectively inhibiting voltage-dependent sodium channels [1, 22] (Fig. 3A). After addition of TTX to the perfusion fluid

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Time (minutes) Fig. 2. Comparison of the effects of 6R-BH4, 6S-BH4 and biopterin on ACh levels in hippocampal dialysates. One hundred min after the beginning of brain microdialysis, 5/11 of vehicle solution (©) or test solution containing 1 mg of either 6R-BH4 (O), 6S-BH4 ( • ) or biopterin (A) was injected intracerebroventricularly as indicated by an arrow. ACh levels in each fraction were expressed as percentages of the control value in the third fraction (3.3 + 0.2 pmol/20 min). Each value is the mean + S.E.M. of 4 determinations. *P<0.05; **P<0.01; significantly different from a control group which was injected with vehicle alone.

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at a final concentration of 40 ~tM, the concentration which completely inhibits neuronal activity-dependent (i.e. exocytotic) neurotransmitter release [14, 23], ACh levels rapidly decreased to 30 % of the control value and remained low. When 1 mg of 6R-BH4 was injected intracerebroventricularly after addition of TTX, no increase in ACh levels in dialysates was observed. These results suggest that the 6R-BHa-induced increase in ACh levels in dialysates is the result of an increase in exocytotic release of ACh but not an increase in nonexocytotic leakage. As described recently [14], 6R-BH4 has dopamine releasing action. On the other hand, it is reported that ACh release in the brain is modified by catecholamines [2-4, 6, 9, 10]. Thus there is the possibility that the 6RBH4-induced increase in ACh release is mediated by an increase in catecholamine release. Therefore, to test this possibility, we examined the effects of 6R-BH4 on ACh release after pretreatment with reserpine, a chemical depleter of catecholamines [17, 19]. After pretreatment with reserpine (intraperitoneal injection of 5 mg/kg and 3 mg/kg, 48 h and 24 h before experiments, respectively),

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the content of dopamine and noradrenaline decreased to 0.1+_0.1% and 1.3___1.2%, respectively, of the control value in the hippocampus, and to 8.3+1.9% and 3.3 + 1.0%, respectively, of the control value in the whole brain (n = 6). Using these catecholamine-depleted rats, we examined the effects of 6R-BH4 on ACh release (Fig. 3B). Even after depletion of catecholamines by pretreatment with reserpine, 6R-BH4 could induce an increase in ACh release, which was as large as the value without reserpine pretreatment (see results in Fig. 1). These results rule out the possibility that 6R-BHa-induced increase in ACh release is mediated by an increase in catecholamine release. In conclusion, we showed that 6R-BH4, a cofactor for hydroxylases of phenylalanine, tyrosine and tryptophan, possesses direct ACh releasing action, in addition to dopamine releasing action [14]. From clinical view points, 6R-BH4 is expected to be a therapeutic drug for Alzheimer's disease, in which the activity of cholinergic neurons in the brain is decreased [8, 13, 16, 24]. Furthermore, considering that 6R-BH4 is present in the brain [11] and that only the natural form (6R-form) is effective for enhancement of release of ACh as well as dopamine, 6R-BH4 might play at~ important role in physiological regulation of release of these neurotransmitters in the brain. The effects of 6R-BH4 on release of neurotransmitters other than dopamine and ACh, and the mechanism of neurotransmitter releasing action are now under investigation in our laboratory.

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Fig. 3. Effects of pretreatment with tetrodotoxin (TTX) (A) or reserpine (B) on 6R-BH4-induced increases in ACh levels in hippocampal dialysates. A: 100 rain after the beginning of brain microdialysis, TTX was added to the perfusion fluid at a final concentration of 40/~M and continued to be present up to the end of the experiment (indicated by a bar). Sixty min after addition of TTX, either vehicle solution (A) or test solution containing 1 mg of 6R-BH4 ( • ) was injected intracerebroo ventricularly as indicated by an arrow. For comparison, control experiments without TTX were carried out in the same manner as described in Fig. 1, except that the injection of vehicle solution (©) or test solution containing I mg of 6R-BH4 (O) was done 160 min instead of I00 min after the beginning of the brain microdialysis. B: after pretreatment with reserpine, rats were subjected to brain microdialysis. One hundred min after the beginning of brain microdialysis, vehicle solution (©) or test solution containing 1 mg of 6R-BH4 (O) was injected intracerebroventricularly as indicated by an arrow. ACh levels in each fraction were expressed as percentages of the control value in the fifth (A: 3.3 + 0.2 pmol/20 min) or third (B: 3.0 + 0.2 pmol/20 min) fraction. Each value is the mean _ S.E.M. of 4 determinations. *P<0.05; **P<0.01; significantly different from a control group which was injected with vehicle alone.

This work was supported by a Grant-in-Aid for New Drug Development from the Ministry of Health and Welfare of Japan and by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science and Culture, Japan. We thank Suntory Institute for Biomedical Research, Osaka, Japan, for a kind donation of 6R-BHa dihydrochloride, 6S-BH4 dihydrochloride and biopterin. 1 Agnew, W.S., Tomilo, S.A., Rosenberg, P.L., Emerick, M.C. and Cooper, E.C,, The structure and function of the voltage-sensitive Na channel, Ann. NY Acad, Sci., 479 (1986) 238-256. 2 Ajima, A, Yamaguchi, T. and Kato, T., Modulation of acetylcholine release by Dr, D2 dopamine recepors in rat striatum under free moving conditions, Brain Res., 518 (1990) 193-198. 3 Beani, L. and Bianchi, C., Effect of amantadine on cerebral acetylcholine release and content in the guinea pig, Neuropharmacology, 12 (1973) 283-289. 4 Beani, L., Bianchi, C. and Castellucci, A., Correlation of brain catecholamines with cortical acetylcholine outflow, behaviour and electrocorticogram, Eur. J. Pharmacol., 26 (1974) 63-72. 5 Brenneman, A.R. and Kaufman, S., The role of tetrahydropteridines in the enzymatic conversion of tyrosine to 3,4-dihydroxyphenylalanine, Biochem. Biophys. Res. Commun., 17 (1964) 177-183.

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