Neuroscience Letters, 140 (1992) 153-156 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
153
NSL 08665
Evidence for L-DOPA systems responsible for cardiovascular control in the nucleus tractus solitarii of the rat T a k a o K u b o 1, J i n - L i a n g Yue, Yoshio G o s h i m a , Shinichi N a k a m u r a a n d Yoshimi M i s u Department of Pharmacology, Yokohama City University School of Medieine, Yokohama (Japan) (Received 5 February 1992; Accepted 9 March 1992)
Key words: L-DOPA; D-DOPA; L-DOPA methyl ester, competitive L-DOPA antagonist; Dopamine; Noradrenaline; Nucleus tractus solitarii; Blood pressure; Release of endogenous DOPA; Rat Microinjections of L-DOPA (10-100 ng) into the medial area of the nucleus tractus solitarii (NTS) led to dose~lependent decreases in arterial blood pressure and heart rate in rats treated with i.p. 3-hydroxybenzylhydrazine, a central inhibitor of DOPA decarboxylase, or similarly with intraventricular 6-hydroxydopamine. D-DOPA, dopamine or noradrenaline (100 ng) produced no effect. L-DOPA methyl ester (1/tg), a competitive antagonist for L-DOPA, microinjected into NTS, blocked the depressor and bradycardic responses to L-DOPA. High K ÷ (40 mM) released endogenous DOPA in a Ca2+-dependent manner from slices of the rat dorsomedial medulla including NTS. These results support the hypothesis that there exist systems of L-DOPA itself responsible for cardiovascular regulation in NTS of rats. This regulatory action of L-DOPA seems to be postsynaptic in nature.
DOPA is believed to be an inert amino acid which exerts its actions via its conversion to dopamine (DA) by L-aromatic amino acid decarboxylase (AADC). Conversely, we have proposed that DOPA itself is an endogenous neuroactive substance in the central nervous system (CNS). DOPA is released by depolarizing stimuli from rat striatal slices in a transmitter-like manner [5, 14]. The transmitter-like basal release of DOPA was also seen under physiological conditions in striata of freely moving rats [16]. On the other hand, in rat striatal and hypothalamic slices, the nanomolar concentrations of exogenously applied DOPA stereoselectively facilitated the evoked release of DA and noradrenaline (NA) via stereoselectively propranolol-sensitive presynaptic fladrenoceptors, even in the presence of an AADC inhibitor [4, 6, 13]. However, this facilitation of the NA release was found to be antagonized competitively by L-DOPA methyl ester, a prodrug for L-DOPA [2], whereas it was non-competitively antagonized by propranolol [6]. Furthermore, the picomolar concentrations of DOPA stereoselectively potentiated the isoproterenol-induced facilitation of the NA release [7]. This potentiation was selec~Present address: Department of Pharmacology, Showa College of Pharmaceutical Sciences, Tokyo 194, Japan. Correspondence: Y. Misu, Department of Pharmacology, Yokohama City University School of Medicine, Yokohama 236, Japan. Fax: (81) (45) 785 3645.
tively antagonized by L-DOPA methyl ester, whereas propranolol antagonized both the facilitation by isoproterenol alone and its potentiation by L-DOPA. Thus, L-DOPA may act on a recognition site for itself, which differs from presynaptic fl-adrenoceptors. In addition, there exist DOPA-immunoreactive neurons and nerve fibers, which display no DA immunoreactivity, in the nucleus tractus solitarii (NTS) of rats [17], the principal site of termination of the primary afferent fibers from peripheral baroreceptors [3, 15]. Thus, we have attempted to clarify whether or not microinjections of L- and DDOPA or DA into the medial area of NTS in rats modulate blood pressure and, further, whether or not endogenous DOPA is released in a transmitter-like manner from superfused slices of the caudal dorsomedial medulla including NTS. Male Wistar rats (240-320 g) were anaesthetized with urethane (1.2 g/kg, i.p.). The femoral artery was cannulated. Rats were paralyzed with D-tubocurarine (1 mg/kg, i.m.) and artificially ventilated with a respirator [10]. In some rats, 3-hydroxybenzylhydrazine (NSD-1015 100 mg/kg, Aldrich) was injected i.p. 30 min before microinjections to inhibit brain AADC. Rats were placed in a stereotaxic apparatus with the head fixed at 45 °. The dorsal surface of the lower brainstem was exposed by a limited occipital craniotomy. A glass micropipette pulled to an outside tip diameter of 50-100/am was introduced into NTS (0.6 mm rostral and 0.6 mm lateral to the cau-
154
A
min i
i
,.,.z,2oI"r360L ~ L-DOPA 10 ng
L-DOPA 30 ng
L-DOPA 100 ng
B
C 0_ tl~
t2. m
42°r 7-36o'
~.
420r 36°L t
t
L-DOPA 100ng
D-DOPA L-DOPA 100 ng 100 ng
DA 100 rN
L-DOPA 100ng
Fig. 1. Effects of unilateral microinjections of L-DOPA (A), D-DOPA (B) and DA (C) into NTS, on blood pressure (BP, mmHg) and heart rate (HR, beats/min) in rats treated with NSD-1015 (100 mg/kg, i.p.)
dal tip of the area postrema and 0.6 mm beneath the dorsal surface of the brainstem) [12]. Drugs dissolved in saline (0.9% NaC1 in 10 mM phosphate buffer, pH 7.4) were injected unilaterally into NTS in a volume of 50 nl in 2 s. At the end of some experiments, the injection site was marked by injecting 50 nl of Evans blue dye solution. The brain was removed, frozen sections were cut (50/lm) and the injection site was identified. In some rats to destruct brainstem NA fibers, 6-hydroxydopamine (6-OHDA 250 /lg, Sigma) or vehicle (0.5% ascorbic acid in saline) was injected twice into the lateral brain ventricle at 3 day intervals 1 week before experiments, using a stereotaxic technique [11]. After the end of experiments, NA contents in the medulla oblongata were estimated. For superfusion experiments, rats were decapitated and the brain was rapidly removed and placed in ice-cold Krebs-Henseleit medium, containing (in mM): NaC1 113, NaHCO3 24, KCI 4.75, KHzPO4 1.18, CaC12 2.52, M g S O 4 1.19, glucose 11.1, disodium EDTA 0.029 and ascorbic acid 0.29. The caudal dorsomedial medulla region was dissected out and cut 0.3 mm in thickness [8]. The slices were superfused with the Krebs medium containing cocaine 20/IM at a rate of 0.45 ml/min [5]. After 41 min, perfusate was successively collected every 4 min. High K ÷ stimulation (40 mM) was applied for 4 min
aj --'0t
A
B - 40
40-
-
-30o.. ,~ 20-
~-20
-
-10-
-10]
0--
-i
I--'1 Before DOPAester ~ After DOPA ester lpg
i
0
~
~
~
_
10r~l 30ng lO0nc] L-IX)PA
-
-
-1 L-DOPA 100ng
Glutamate 30 ng
Fig. 2. A: dose-effect columns for depressor (BE mmHg) and bradycardic (HR, beats/min) responses to microinjections of L-DOPA into NTS in rats treated with NSD-1015 (100 mg/kg, i.p.). B: effects of L-DOPA methyl ester (DOPA ester) on depressor and bradycardic responses to microinjections of L-DOPA and L-glutamate. The effects were determined 1 min after microinjection of DOPA ester. Values are mean + S.E.M. from 5-6 experiments. *P
153
NSL 08665
Evidence for L-DOPA systems responsible for cardiovascular control in the nucleus tractus solitarii of the rat T a k a o K u b o 1, J i n - L i a n g Yue, Yoshio G o s h i m a , Shinichi N a k a m u r a a n d Yoshimi M i s u Department of Pharmacology, Yokohama City University School of Medieine, Yokohama (Japan) (Received 5 February 1992; Accepted 9 March 1992)
Key words: L-DOPA; D-DOPA; L-DOPA methyl ester, competitive L-DOPA antagonist; Dopamine; Noradrenaline; Nucleus tractus solitarii; Blood pressure; Release of endogenous DOPA; Rat Microinjections of L-DOPA (10-100 ng) into the medial area of the nucleus tractus solitarii (NTS) led to dose~lependent decreases in arterial blood pressure and heart rate in rats treated with i.p. 3-hydroxybenzylhydrazine, a central inhibitor of DOPA decarboxylase, or similarly with intraventricular 6-hydroxydopamine. D-DOPA, dopamine or noradrenaline (100 ng) produced no effect. L-DOPA methyl ester (1/tg), a competitive antagonist for L-DOPA, microinjected into NTS, blocked the depressor and bradycardic responses to L-DOPA. High K ÷ (40 mM) released endogenous DOPA in a Ca2+-dependent manner from slices of the rat dorsomedial medulla including NTS. These results support the hypothesis that there exist systems of L-DOPA itself responsible for cardiovascular regulation in NTS of rats. This regulatory action of L-DOPA seems to be postsynaptic in nature.
DOPA is believed to be an inert amino acid which exerts its actions via its conversion to dopamine (DA) by L-aromatic amino acid decarboxylase (AADC). Conversely, we have proposed that DOPA itself is an endogenous neuroactive substance in the central nervous system (CNS). DOPA is released by depolarizing stimuli from rat striatal slices in a transmitter-like manner [5, 14]. The transmitter-like basal release of DOPA was also seen under physiological conditions in striata of freely moving rats [16]. On the other hand, in rat striatal and hypothalamic slices, the nanomolar concentrations of exogenously applied DOPA stereoselectively facilitated the evoked release of DA and noradrenaline (NA) via stereoselectively propranolol-sensitive presynaptic fladrenoceptors, even in the presence of an AADC inhibitor [4, 6, 13]. However, this facilitation of the NA release was found to be antagonized competitively by L-DOPA methyl ester, a prodrug for L-DOPA [2], whereas it was non-competitively antagonized by propranolol [6]. Furthermore, the picomolar concentrations of DOPA stereoselectively potentiated the isoproterenol-induced facilitation of the NA release [7]. This potentiation was selec~Present address: Department of Pharmacology, Showa College of Pharmaceutical Sciences, Tokyo 194, Japan. Correspondence: Y. Misu, Department of Pharmacology, Yokohama City University School of Medicine, Yokohama 236, Japan. Fax: (81) (45) 785 3645.
tively antagonized by L-DOPA methyl ester, whereas propranolol antagonized both the facilitation by isoproterenol alone and its potentiation by L-DOPA. Thus, L-DOPA may act on a recognition site for itself, which differs from presynaptic fl-adrenoceptors. In addition, there exist DOPA-immunoreactive neurons and nerve fibers, which display no DA immunoreactivity, in the nucleus tractus solitarii (NTS) of rats [17], the principal site of termination of the primary afferent fibers from peripheral baroreceptors [3, 15]. Thus, we have attempted to clarify whether or not microinjections of L- and DDOPA or DA into the medial area of NTS in rats modulate blood pressure and, further, whether or not endogenous DOPA is released in a transmitter-like manner from superfused slices of the caudal dorsomedial medulla including NTS. Male Wistar rats (240-320 g) were anaesthetized with urethane (1.2 g/kg, i.p.). The femoral artery was cannulated. Rats were paralyzed with D-tubocurarine (1 mg/kg, i.m.) and artificially ventilated with a respirator [10]. In some rats, 3-hydroxybenzylhydrazine (NSD-1015 100 mg/kg, Aldrich) was injected i.p. 30 min before microinjections to inhibit brain AADC. Rats were placed in a stereotaxic apparatus with the head fixed at 45 °. The dorsal surface of the lower brainstem was exposed by a limited occipital craniotomy. A glass micropipette pulled to an outside tip diameter of 50-100/am was introduced into NTS (0.6 mm rostral and 0.6 mm lateral to the cau-
154
A
min i
i
,.,.z,2oI"r360L ~ L-DOPA 10 ng
L-DOPA 30 ng
L-DOPA 100 ng
B
C 0_ tl~
t2. m
42°r 7-36o'
~.
420r 36°L t
t
L-DOPA 100ng
D-DOPA L-DOPA 100 ng 100 ng
DA 100 rN
L-DOPA 100ng
Fig. 1. Effects of unilateral microinjections of L-DOPA (A), D-DOPA (B) and DA (C) into NTS, on blood pressure (BP, mmHg) and heart rate (HR, beats/min) in rats treated with NSD-1015 (100 mg/kg, i.p.)
dal tip of the area postrema and 0.6 mm beneath the dorsal surface of the brainstem) [12]. Drugs dissolved in saline (0.9% NaC1 in 10 mM phosphate buffer, pH 7.4) were injected unilaterally into NTS in a volume of 50 nl in 2 s. At the end of some experiments, the injection site was marked by injecting 50 nl of Evans blue dye solution. The brain was removed, frozen sections were cut (50/lm) and the injection site was identified. In some rats to destruct brainstem NA fibers, 6-hydroxydopamine (6-OHDA 250 /lg, Sigma) or vehicle (0.5% ascorbic acid in saline) was injected twice into the lateral brain ventricle at 3 day intervals 1 week before experiments, using a stereotaxic technique [11]. After the end of experiments, NA contents in the medulla oblongata were estimated. For superfusion experiments, rats were decapitated and the brain was rapidly removed and placed in ice-cold Krebs-Henseleit medium, containing (in mM): NaC1 113, NaHCO3 24, KCI 4.75, KHzPO4 1.18, CaC12 2.52, M g S O 4 1.19, glucose 11.1, disodium EDTA 0.029 and ascorbic acid 0.29. The caudal dorsomedial medulla region was dissected out and cut 0.3 mm in thickness [8]. The slices were superfused with the Krebs medium containing cocaine 20/IM at a rate of 0.45 ml/min [5]. After 41 min, perfusate was successively collected every 4 min. High K ÷ stimulation (40 mM) was applied for 4 min
aj --'0t
A
B - 40
40-
-
-30o.. ,~ 20-
~-20
-
-10-
-10]
0--
-i
I--'1 Before DOPAester ~ After DOPA ester lpg
i
0
~
~
~
_
10r~l 30ng lO0nc] L-IX)PA
-
-
-1 L-DOPA 100ng
Glutamate 30 ng
Fig. 2. A: dose-effect columns for depressor (BE mmHg) and bradycardic (HR, beats/min) responses to microinjections of L-DOPA into NTS in rats treated with NSD-1015 (100 mg/kg, i.p.). B: effects of L-DOPA methyl ester (DOPA ester) on depressor and bradycardic responses to microinjections of L-DOPA and L-glutamate. The effects were determined 1 min after microinjection of DOPA ester. Values are mean + S.E.M. from 5-6 experiments. *P
