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Involvement of nitric oxide production via kynurenic acid-sensitive glutamate receptors in DOPA-induced depressor responses in the nucleus tractus solitarii of anesthetized rats
Neuroscience Research 43 (2002) 231 /238 www.elsevier.com/locate/neures
Involvement of nitric oxide production via kynurenic acid-sensitive glutamate receptors in DOPA-induced depressor responses in the nucleus tractus solitarii of anesthetized rats Kaori Yamanashi a,1, Takeaki Miyamae a, Yukio Sasaki a, Masanobu Maeda b, Hideyasu Hirano c, Yoshimi Misu a,2, Yoshio Goshima a,* a
Department of Pharmacology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan b Department of Physiology II, Wakayama Medical College, Wakayama 641-0012, Japan c Department of Biochemistry, School of Medicine, University of Occupational and Environmental Health, Kitakyusyu 807-8555, Japan Received 6 February 2002; accepted 12 March 2002
Abstract We have proposed the hypothesis that L-3,4-dihydroxyphenylalanine (DOPA) plays a role of neurotransmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii (NTS). In the present study, we tried to clarify whether glutamate receptors and/or nitric oxide (NO), important modulators for central cardiovascular regulation, are involved in the DOPA-induced cardiovascular responses in the nucleus. Male Wistar rats were anesthetized with urethane and artificially ventilated. Compounds or antisense oligos (17-mer) for neuronal NO synthase were microinjected into depressor sites of the unilateral nucleus. DOPA 30 /300 pmol microinjected into the nucleus dose-dependently induced depressor and bradycardic responses. Prior injection of kynurenic acid (600 pmol) suppressed DOPA (300 pmol)-induced responses by :/80%. Prior injection of N G-monomethyl-L-arginine 100 nmol, a potent NO synthase inhibitor, reversibly attenuated by :/90% DOPA-induced responses, while the D-isomer 100 nmol produced no effect. Furthermore, prior injection of neuronal NO synthase antisense oligos (20 pmol) reversibly reduced by :/70% responses to DOPA. Sense or scrambled oligos produced no effect. A NO precursor L-arginine (30 nmol) induced depressor and bradycardic responses, but these responses were not affected by kynurenic acid. These results suggest important roles for glutamate receptors and NO in DOPA induced-depressor and bradycardic responses in the NTS. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: DOPA; Glutamate receptors; Nitric oxide; Depressor response; Nucleus tractus solitarii; Kynurenic acid
1. Introduction L-3,4-Dihydroxyphenylalanine (DOPA) has been believed to be only a precursor that exerts its actions via conversion to dopamine (DA) by aromatic L-amino acid decarboxylase (AADC). Since 1986, we have proposed that DOPA is a neurotransmitter and/or neuromodulator as well as a DA precursor in the CNS (Goshima et
* Corresponding author. Tel.: /81-45-787-2593; fax: /81-45-7853645 E-mail address: [email protected] (Y. Goshima). 1 Present address: Department of Pharmacy, Yokohama City University Medical Center, 4-57, Urafune-cho, Minami-ku, Yokohama 232-0024, Japan. 2 Present address: Shinobu Hospital, Fukushima 960-1101, Japan.
al., 1986, 1988; Misu and Goshima, 1993; Misu et al., 1996). Immunohistochemical studies have revealed some neuronal cells that may contain DOPA as an end product (Okamura et al., 1988; Tison et al., 1989). Transmitter-like release of DOPA is seen under in vitro and in vivo conditions (Goshima et al., 1988; Nakamura et al., 1992; Yue et al., 1994; Miyamae et al., 1999b; Nishihama et al., 1999). Exogenously applied DOPA itself produces pre- and postsynaptic responses. All the effects are stereoselective and most are antagonized by competitive antagonists for DOPA, such as DOPA methyl ester (DOPA ME) or DOPA cyclohexyl ester (DOPA CHE) (Goshima et al., 1991; Misu et al., 1997; Furukawa et al., 2000, 2001; Arai et al., 2001). These findings provide evidence that there exist recognition sites for DOPA. In addition, we recently found a
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transporter unit candidate molecule for Na -dependent uptake of DOPA (Ishii et al., 2000) and that neuronal and/or glial cells, which take up DOPA in Na dependent manner, exist in the CNS (Sugaya et al., 2001). The lower brain stem plays a vital role in baroreflex and regulation of cardiovascular function. We have provided evidence for a neurotransmitter role of DOPA in the lower brain stem nuclei (Yue et al., 1994; Miyamae et al., 1999b; Nishihama et al., 1999). DOPA is a probable neurotransmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii (NTS) (Kubo et al., 1992; Yue et al., 1994; Misu et al., 1996). There exist strong DOPA immunoreactive neurons that are tyrosine hydroxylase (TH)positive and DOPA-positive, but AADC-negative and DA-negative in the NTS (Tison et al., 1989; Misu et al., 1996). Denervation peripheral to the ganglion nodosum, in which cell bodies are located, decreases TH- and DOPA-immunoreactivity, but not DA- and DA-bhydroxylase-immunoreactivity, in the medial and intermediate NTS in the ipsilateral lesioned side (Yue et al., 1994). The decreased area of TH-immunoreactivity is roughly compatible with the projection area of the aortic depressor nerve (ADN). The release of endogenous DOPA is evoked in a tetrodotoxin-sensitive manner by electrical ADN stimulation. DOPA microinjected into depressor sites of the NTS produces hypotension and bradycardia (Kubo et al., 1992). These responses are dose-dependent, stereoselective, seen under inhibition of central AADC and antagonized by DOPA ME or DOPA CHE (Kubo et al., 1992; Misu et al., 1997; Furukawa et al., 2000). DOPA ME microinjected into the NTS antagonizes depressor and bradycardic responses to ADN stimulation (Yue et al., 1994) in accord with those to exogenous DOPA microinjected (Kubo et al., 1992). Furthermore, bilateral injection of DOPA ME alone dose-dependently produces pressor and tachycardic responses. These responses are abolished by pretreatment with an inhibitor for TH, a-methyl-p tyrosine that potently inhibits endogenous DOPA release (Yue et al., 1994; Miyamae et al., 1999b). These findings provide evidence that endogenously released DOPA tonically functions to activate depressor neurons in the NTS. On the other hand, much evidence suggests that glutamate is a highly probable transmitter candidate of the primary baroreceptor afferents terminating in the NTS (Talman et al., 1980; Reis et al., 1981; Leone and Gordon, 1989; Kubo and Kihara, 1991; Van Giersbergen et al., 1992; Foley et al., 1999). In addition, DOPA induces the release of glutamate in the central nervous system (Goshima et al., 1993; Maeda et al., 1997; Furukawa et al., 2001). It is therefore important to clarify possible interactions between DOPA and glutamate.
Nitric oxide (NO), synthesized from L-arginine through the NO synthase (NOS), is also an important modulator for central cardiovascular regulation (Tagawa et al., 1994; Ma et al., 1995; Tseng et al., 1996; Chan and Sawchenko, 1998; Lin et al., 1998; Maeda et al., 1999). Microinjection of L-arginine, a NO donor, into the NTS produces hypotension and bradycardia (Tseng et al., 1996). Microinjection of N G-monomethylL-arginine (L-NMMA), a NOS inhibitor, into the NTS inhibits the L-arginine-induced depressor responses (Wu and Morris, 1998). Neuronal NOS (nNOS) is present in pre- and post-synaptic structures in the NTS and the majority of NOS immunoreactivity or NOS mRNA in the NTS is found in intrinsic structures in the nucleus (Chan and Sawchenko, 1998; Lin et al., 1998). Some evidence for possible interaction between nNOS and glutamate receptors in the NTS has been presented. LNMMA reduces the depressor response evoked by glutamate in the NTS (Di Paola et al., 1991; Lo et al., 1997). There have been some discrepancies between the effects of some pharmacological agents against hypotensive actions induced by ADN stimulation and by exogenous glutamate applied into the NTS (Talman et al., 1980; Leone and Gordon, 1989). Kynurenic acid, a non-selective ionotropic glutamate receptor antagonist, antagonizes these responses to ADN stimulation, but not those to glutamate microinjected (Talman et al., 1980; Leone and Gordon, 1989; Misu et al., 1996). Although the reasons are not clear at present, there might be several explanations for little antagonism by kynurenic acid against glutamate. Microinjection of a metabotropic glutamate receptor (mGluR) agonist, into the NTS, produced kynurenic acid-insensitive depressor responses in a manner similar to those to exogenous glutamate (Pawloski-Dahm and Gordon, 1992). If mGluRs are located at extrasynaptic locations, as observed in the hippocampus and the cerebellum (Baude et al., 1993), glutamate, when exogenously applied into the NTS, could preferentially activate mGluRs and then induce kynurenic acid-insensitive depressor responses. Alternatively, an unknown excitatory amino acid, the responses to which are antagonized by kynurenic acid, might be a primary transmitter of ADN (Leone and Gordon, 1989). In the present study, we have thus attempted to clarify whether glutamate receptors and/or NO are involved in the DOPA-induced cardiovascular responses in the NTS and whether NO production is an upstream or downstream event of activation of glutamate receptors in the NTS. We here propose a third pathway that DOPA is a neurotransmitter and/or neuromodulator of primary ADN neurons and glutamate is a neurotransmitter of primary afferent and/or secondary neurons in the microcircuits of the NTS.
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2. Materials and methods 2.1. General procedures All procedures were carried out according to the institutional guidelines outlined in the Institutional Animal Care and Use Committee of the Yokohama City University School of Medicine and to the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023). Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their suffering. The experimental procedures in this study were described previously (Yue et al., 1994; Miyamae et al., 1999b; Nishihama et al., 1999). In brief, male Wistar rats (Charles River), weighing 250/350 g, were anesthetized with urethane (1.2 g/kg, i.p.). After the trachea was cannulated, the animal was paralyzed with D-tubocurarine (1 mg/kg, i.m.) and artificially ventilated at a rate of 70/75 breaths/min and a volume of 2.5 /3.5 ml with a respirator (Shinano, SN-480) to avoid cardiovascular effects secondary to blood gas changes. The femoral artery was cannulated for recording systolic/diastolic arterial blood pressure (BP) and heart rate (HR). BP was measured with a pressure transducer (NEC Sanei, P23XL). HR was determined using a tachograph (NEC Sanei, 180) triggered by BP waves. BP and HR were recorded on a polygraph (NEC Sanei, 8S). Body temperature was maintained at 37.0 /37.5 8C with a temperature controller (BAS, CMA/150). The animal was placed in a stereotaxic apparatus (Narishige, SR-6) with the head fixed at 458. The dorsal surface of the lower brainstem was exposed by a limited occipital craniotomy (Yue et al., 1994). 2.2. Microinjection A glass micropipette pulled to an outside diameter of :/50 mm was inserted into the unilateral NTS. The coordinate for the depressor sites of the NTS was 0.6 mm rostral and 0.6 mm lateral to the caudal tip of the area postrema and 0.6 mm beneath the dorsal surface of the brainstem. Glutamate (Nacalai Tesque), DOPA (Nacalai Tesque), D-NMMA (Wako), L-NMMA (Wako) or L-arginine (Wako) was dissolved in saline (0.9% NaCl in 10 mM phosphate buffer, pH 7.4). Kynurenic acid (Sigma) was dissolved in 1 N NaOH, diluted in saline (0.9% NaCl in 10 mM phosphate buffer, pH 7.4) and adjusted pH to 7.4 using 1 N HCl. Each compound was given in a volume of 50 nl in 2 s. Antisense, sense and scrambled oligothionucleotides for the rat nNOS mRNA were synthesized as described previously (Maeda et al., 1999). Each of these oligos dissolved in artificial cerebrospinal fluid (132 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 0.65 mM MgCl2, 25 mM
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NaHCO3, pH 7.3) mixed with cationic lipids (LipofectAMINE PLUS reagent and LipofectAMINE reagent, Life Technologies) was given in a volume of 50 nl in 10 s as described (Maeda et al., 1999). Analysis was carried out with data only from the experiments, in which glutamate (160 pmol) microinjected into the NTS produced hypotension of more than /20 mmHg and in which saline microinjected into the NTS produced no effect. 2.3. Statistics Data shown are means9/S.E.M. Statistical significance was calculated using Dunn’s multiple comparison test following repeated measures ANOVA, Student’s ttest or Welch’s t-test. A P value of B/0.05 was considered statistically significant.
3. Results 3.1. Effect of prior injection of kynurenic acid on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats In this series, respective resting mean BP was 859/6 mmHg and HR was 3789/14 beats/min (n / 6). DOPA at 300 pmol microinjected into the NTS produced depressor and bradycardic responses (Fig. 1A). We confirmed that DOPA at 30, 100 and 300 pmol dosedependently produced depressor and bradycardic responses (Kubo et al., 1992). Kynurenic acid at 600 pmol microinjected, which blocks the depressor and bradycardic responses to ADN stimulation (Kubo and Kihara, 1991), produced no effect on resting BP and HR. Injection of kynurenic acid 2 min prior to DOPA reduced depressor and bradycardic responses to DOPA by :/80% (Table 1). Reduced depressor responses required :/30 min to recover to 90% of control. 3.2. Effect of prior injection of L-NMMA on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats In this series, respective resting mean BP was 859/5 mmHg in L-NMMA (n/6) and 859/5 mmHg in DNMMA (n/ 5). Respective resting mean HR was 3799/ 9 beats/min in L-NMMA (n / 6) and 3589/3 beats/min in D-NMMA (n / 5). L-NMMA at 100 nmol microinjected into the NTS produced a slight pressor response: the maximal change in BP was 119/6 mmHg. After recovery to the resting level, DOPA (300 pmol) was microinjected into the NTS. Injection of LNMMA 10 min prior to DOPA reduced depressor and bradycardic responses to DOPA by :/90% (Fig. 1A,
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Fig. 1. Typical traces for reversible effects of prior injection of L-NMMA (100 nmol) (A) and nNOS antisense oligos (20 pmol) (B) on depressor and bradycardic responses to DOPA (300 pmol) microinjected into depressor sites of the unilateral NTS in anesthetized rats. Table 1 Effects of prior injection of kynurenic acid and L- or D-NMMA on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats Pretreatments
n DBP (mmHg) Before
Kynurenic acid L-NMMA D-NMMA
6 /399/4 6 /359/5 5 /399/4
DHR (beats/min)
After *
/99/5 /59/3* /349/4
Before
After
/639/14 /559/12 /649/9
/129/5* /59/4$ /429/4
Kynurenic acid (600 pmol), L- or D-NMMA (100 nmol) was microinjected into the NTS prior to DOPA (300 pmol). Data are mean9/S.E.M. * P B/0.01 (Student’s t -test) versus before. $ P B/0.01 (Welch’s t -test) versus before.
Table 1). We observed dose-dependent inhibition by LNMMA (10, 30 and 100 nmol) against DOPA-induced depressor and bradycardic responses (data not shown). Reduced depressor responses required :/20/95 min to recover 90% of control. Prior injection of D-NMMA
(100 nmol) produced no effect on responses to DOPA microinjected. 3.3. Effect of prior injection of neuronal NO synthase antisense oligos on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats In this series, respective resting mean BP was 909/3 mmHg in antisense oligos (n / 9), 909/7 mmHg in sense oligos (n/5) and 919/8 mmHg in scrambled oligos (n / 4). Respective resting mean HR was 3859/15 beats/ min in antisense oligos (n / 9), 3949/17 beats/min in sense oligos (n / 5) and 4139/8 beats/min in scrambled oligos (n/ 4). Antisense, sense or scrambled nNOS oligos each 20 pmol microinjected into the unilateral NTS produced no effect on the resting BP and HR. After microinjection of nNOS oligos (45 min), DOPA (300 pmol) was microinjected into the NTS. nNOS antisense oligos reduced the depressor and bradycardic responses to DOPA by :/70% (Fig. 1B, Table 2). Reduced depressor responses required :/75 /175 min
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Table 2 Effects of prior injection of neuronal NO synthase antisense oligos on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats Pretreatments
n
DBP (mmHg) Before
Antisense oligos Sense oligos Scrambled oligos
9 5 4
/449/4 /399/6 /419/5
DHR (beats/min) After *,$
/159/7 /399/5 /429/6
Before
After
/1169/20 /679/21 /979/26
/399/20* /649/21 /1139/36
After depressor responses to DOPA (300 pmol) microinjected into the unilateral NTS, nNOS oligos (20 pmol) were microinjected into the ipsilateral NTS. The second DOPA into the NTS was challenged 45 min after nNOS oligos. Data are mean9/S.E.M. * P B/0.01 (Student’s t -test) versus before. $ P B/0.05 (Dunn’s multiple comparison test following repeated measures ANOVA) versus after injection of sense oligos, or scrambled oligos.
to recover 90% of control. Sense or scrambled nNOS oligos produced no effect on the responses to DOPA microinjected. 3.4. Effect of prior injection of kynurenic acid on depressor and bradycardic responses to L-arginine microinjected into depressor sites of the unilateral NTS in anesthetized rats
Table 3 L-Arginine microinjected into depressor sites of the unilateral NTS in anesthetized rats Pretreatments
n
DBP (mmHg)
DHR (beats/min)
Saline Kynurenic acid
9 8
/129/2 /109/2
/169/6 /109/2
Kynurenic acid (600 pmol) was microinjected into the NTS prior to Data are mean9/S.E.M.
L-arginine.
In this series, respective resting mean BP and HR was 859/4 mmHg and 4159/9 beats/min in saline (n/ 9) and 869/3 mmHg and 4049/14 beats/min in L-arginine (n / 8). L-Arginine at 30 nmol microinjected into the NTS produced depressor and bradycardic responses. Injection of kynurenic acid (600 pmol) 2 min prior to Larginine failed to reduce the responses to L-arginine (Fig. 2, Table 3).
4. Discussion We demonstrated that exogenously microinjected DOPA into the NTS produced depressor and bradycardic responses via activation of kynurenic acid-sensitive ionotropic glutamate receptors and nNOS. Kynurenic acid is a broad-spectrum antagonist for ionotropic glutamate receptors. Kynurenic acid has been shown to abolish the depressor response to ADN
Fig. 2. Typical traces for effects of prior injection of kynurenic acid (600 pmol) on depressor and bradycardic responses to L-arginine (30 nmol) microinjected into depressor sites of the unilateral NTS in anesthetized rats.
stimulation (Talman et al., 1980; Leone and Gordon, 1989; Kubo and Kihara, 1991; Misu et al., 1996). In the present study, kynurenic acid reduced by :/80% depressor responses to DOPA, thereby suggesting that a major pathway for DOPA is mediated by kynurenic acid-sensitive ionotropic glutamate receptors. We previously tested possible interactions of DOPA-related compounds with ionotropic glutamate receptors (Miyamae et al., 1999a). Among binding sites labeled with available glutamatergic [3H]-ligands in rat brain membrane, DOPA acts only on a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with a low affinity (IC50 /260 mM), whereas competitive DOPA antagonists, DOPA esters, act only on N -methyl-Daspartate (NMDA) ion channel domain with a low affinity (mM IC50s). DOPA ester and DOPA do not displace the selective binding of [3H]D1, D2, a2 and bligands in brain membranes (Goshima et al., 1991; Furukawa et al., 2000). DOPA antagonists do not displace kynurenic acid-sensitive specific binding of the [3H]-ligands for NMDA glycine site and NMDA binding site. In addition, we confirm that DOPA CHE does not inhibit the uptake of [14C]levodopa into oocytes (Ishii et al., 2000). Thus, DOPAergic agonist and competitive antagonist should act on the same sites, which may differ from the transport sites, catecholamine receptors and ionotropic glutamate receptors. How does DOPA induce kynurenic acid-sensitive depressor responses? Our previous findings indicate that DOPA evokes release of endogenous glutamate in striatum in vitro and in vivo (Goshima et al., 1993; Maeda et al., 1997; Furukawa et al., 2001). It is
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therefore likely that DOPA microinjected into the NTS elicits glutamate release via DOPA recognition sites, and glutamate, thus released, activates kynurenic acid-sensitive glutamate receptors, leading to depressor and bradycardic responses. In the present study, we attempted to clarify whether DOPA microinjected into the NTS elicits the release of endogenous glutamate during microdialysis of the NTS. However, DOPA apparently did not evoke glutamate release over the basal release (data not shown). Although the exact reasons remain unknown, this is probably due to a minor component of the amount of glutamate released from ADN in the perfusate samples from the NTS region. This negative result is consistent with previous findings that evoked release of endogenous glutamate over the basal release is undetectable upon ADN and baroreceptor stimulation (Sved and Curtis, 1993; Misu et al., 1994). Stereoselective and reversible antagonism by NMMA against the cardiovascular responses to DOPA suggests that microinjected DOPA produces cardiovascular effect via NOS activation. Several lines of evidence support the idea that some interaction exists between glutamate receptors and NOS in the NTS. There is an anatomical link between glutamate and NO systems on immunohistochemical studies in the NTS (Aoki et al., 1997; Lin et al., 1998). L-NMMA reduces the depressor responses to glutamate in the NTS (Di Paola et al., 1991; Lo et al., 1997). In the rat brain, activation of NMDA glutamate receptors leads to synthesis and release of NO (Garthwaite et al., 1989). Our present findings argue for the idea that the interaction between glutamate and NO also exists in the cardiovascular responses to DOPA. NOS has three isozymes: nNOS, endothelial NOS (eNOS) and inducible NOS (iNOS) and L-NMMA is a competitive inhibitor of all NOSs with low specificity (Reif and McCreedy, 1995). It is unlikely that iNOS is involved in DOPA-induced responses because L-
NMMA inhibits nNOS and eNOS reversibly, but iNOS irreversibly. Furthermore, nNOS antisense oligos (Maeda et al., 1999) selectively reduced the depressor responses to DOPA. This nNOS antisense oligo markedly suppresses the nNOS immunoreactivity in the NTS only 45 min after injection (Maeda et al., 1999), the time course which was coincident with the blocking effect of the antisense on DOPA-induced hypotensive and bradycardic responses in the present study (Fig. 1). Our finding therefore indicates that nNOS is involved in DOPA-induced depressor responses in the NTS. Accordingly, NO has been shown to have an excitatory action on some neurons in the NTS (Tagawa et al., 1994; Ma et al., 1995). It is also possible that DOPA-induced NO production further facilitates DOPA release like glutamate (Matsuo et al., 2001). NO production is probably a downstream event of activation of the glutamate receptors involved in depressor responses to DOPA, since kynurenic acid at the dose which reduced the cardiovascular response to DOPA, failed to affect the responses to L-arginine. L-Arginine is considered to induce depressor responses by accelerating NO synthesis because many cells utilize L-arginine to generate NO (Wu and Morris, 1998) and L-NMMA reduces the cardiovascular responses to L-arginine (Tseng et al., 1996). It is noteworthy that bilateral injection of LNMMA or antisense oligo to nNOS into the NTS alone evokes hypertension (Harada et al., 1993; Maeda et al., 1999). These findings suggest that nNOS and NO synthesized tonically functions to activate depressor neurons for regulation of blood pressure in the NTS. Our preliminary experiments indicate that D(/)-2amino-5-phosphonopentanoate, a selective NMDA antagonist, partially blocks depressor and bradycardic responses to DOPA microinjected into the NTS (unpublished observation). These findings suggest that NMDA glutamate receptors are at least in part involved
Fig. 3. A model for neuronal microcircuits in depressor sites of NTS.
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in DOPA-induced depressor responses. Additional possibility is that responses to DOPA remained after kynurenic acid may be antagonized by metabotropic glutamate antagonist (Foley et al., 1999) or DOPA activates directly the depressor sites (Fig. 3). In conclusion, DOPA produces depressor and bradycardic responses via activation of kynurenic acid-sensitive ionotropic glutamate receptors and nNOS. DOPA may release undetectable but functioning endogenous glutamate via recognition sites for DOPA. We herein propose a new pathway that DOPA is a neurotransmitter and/or neuromodulator of the primary ADN and glutamate is a neurotransmitter of primary and/or secondary neurons in neuronal microcircuits of the NTS (Fig. 3).
Acknowledgements This work was partially supported by Japan SRF Grants for Biomedical Research.
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Reis, D.J., Granata, A.R., Perrone, M.H., Talman, W.T. 1981. Evidence that glutamic acid is the neurotransmitter of baroreceptor afferent terminating in the nucleus tractus solitarius (NTS). J. Auton. Nerv. Syst. 3, 321 /334. Sugaya, Y., Sasaki, Y., Goshima, Y., Kitahama, K., Kusakabe, T., Miyamae, T., Kato, T., Misu, Y. 2001. Autoradiographic studies using L-[14C] and [3H]DOPA reveal regional Na -dependent uptake of the neurotransmitter candidate L-DOPA in the central nervous system. Neuroscience 104, 1 /14. Sved, A.F., Curtis, J.T. 1993. Amino acid neurotransmitters in nucleus tractus solitarius: an in vivo microdialysis study. J. Neurochem. 61, 2089 /2098. Tagawa, T., Imaizumi, T., Harada, S., Endo, T., Shiramoto, M., Hirooka, Y., Takeshita, A. 1994. Nitric oxide influences neuronal activity in the nucleus tractus solitarius of rat brainstem slices. Circ. Res. 75, 70 /76. Talman, W.T., Perrone, M.H., Reis, D.J. 1980. Evidence for Lglutamate as the neurotransmitter of baroreceptor afferent nerve fibers. Science 209, 813 /815. Tison, F., Mons, N., Rouet-Karama, S., Geffard, M., Henry, P. 1989. Endogenous L-DOPA in the rat dorsal vagal complex: an immunocytochemical study by light and electron microscopy. Brain Res. 497, 260 /270. Tseng, C.-J., Liu, H.-Y., Lin, H.-C., Ger, L.-P., Tung, C.-S., Yen, M.H. 1996. Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 27, 36 /42. Van Giersbergen, P.L., Palkovits, M., De Jong, W. 1992. Involvement of neurotransmitters in the nucleus tractus solitarii in cardiovascular regulation. Physiol. Rev. 72, 789 /824. Wu, G., Morris, S.M., Jr 1998. Arginine metabolism: nitric oxide and beyond. Biochem. J. 336, 1 /17. Yue, J.-L., Okamura, H., Goshima, Y., Nakamura, S., Geffard, M., Misu, Y. 1994. Baroreceptor-aortic nerve-mediated release of endogenous L-3,4-dihydroxyphenylalanine and its tonic depressor function in the nucleus tractus solitarii of rats. Neuroscience 62, 145 /161.
Involvement of nitric oxide production via kynurenic acid-sensitive glutamate receptors in DOPA-induced depressor responses in the nucleus tractus solitarii of anesthetized rats Kaori Yamanashi a,1, Takeaki Miyamae a, Yukio Sasaki a, Masanobu Maeda b, Hideyasu Hirano c, Yoshimi Misu a,2, Yoshio Goshima a,* a
Department of Pharmacology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan b Department of Physiology II, Wakayama Medical College, Wakayama 641-0012, Japan c Department of Biochemistry, School of Medicine, University of Occupational and Environmental Health, Kitakyusyu 807-8555, Japan Received 6 February 2002; accepted 12 March 2002
Abstract We have proposed the hypothesis that L-3,4-dihydroxyphenylalanine (DOPA) plays a role of neurotransmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii (NTS). In the present study, we tried to clarify whether glutamate receptors and/or nitric oxide (NO), important modulators for central cardiovascular regulation, are involved in the DOPA-induced cardiovascular responses in the nucleus. Male Wistar rats were anesthetized with urethane and artificially ventilated. Compounds or antisense oligos (17-mer) for neuronal NO synthase were microinjected into depressor sites of the unilateral nucleus. DOPA 30 /300 pmol microinjected into the nucleus dose-dependently induced depressor and bradycardic responses. Prior injection of kynurenic acid (600 pmol) suppressed DOPA (300 pmol)-induced responses by :/80%. Prior injection of N G-monomethyl-L-arginine 100 nmol, a potent NO synthase inhibitor, reversibly attenuated by :/90% DOPA-induced responses, while the D-isomer 100 nmol produced no effect. Furthermore, prior injection of neuronal NO synthase antisense oligos (20 pmol) reversibly reduced by :/70% responses to DOPA. Sense or scrambled oligos produced no effect. A NO precursor L-arginine (30 nmol) induced depressor and bradycardic responses, but these responses were not affected by kynurenic acid. These results suggest important roles for glutamate receptors and NO in DOPA induced-depressor and bradycardic responses in the NTS. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: DOPA; Glutamate receptors; Nitric oxide; Depressor response; Nucleus tractus solitarii; Kynurenic acid
1. Introduction L-3,4-Dihydroxyphenylalanine (DOPA) has been believed to be only a precursor that exerts its actions via conversion to dopamine (DA) by aromatic L-amino acid decarboxylase (AADC). Since 1986, we have proposed that DOPA is a neurotransmitter and/or neuromodulator as well as a DA precursor in the CNS (Goshima et
* Corresponding author. Tel.: /81-45-787-2593; fax: /81-45-7853645 E-mail address: [email protected] (Y. Goshima). 1 Present address: Department of Pharmacy, Yokohama City University Medical Center, 4-57, Urafune-cho, Minami-ku, Yokohama 232-0024, Japan. 2 Present address: Shinobu Hospital, Fukushima 960-1101, Japan.
al., 1986, 1988; Misu and Goshima, 1993; Misu et al., 1996). Immunohistochemical studies have revealed some neuronal cells that may contain DOPA as an end product (Okamura et al., 1988; Tison et al., 1989). Transmitter-like release of DOPA is seen under in vitro and in vivo conditions (Goshima et al., 1988; Nakamura et al., 1992; Yue et al., 1994; Miyamae et al., 1999b; Nishihama et al., 1999). Exogenously applied DOPA itself produces pre- and postsynaptic responses. All the effects are stereoselective and most are antagonized by competitive antagonists for DOPA, such as DOPA methyl ester (DOPA ME) or DOPA cyclohexyl ester (DOPA CHE) (Goshima et al., 1991; Misu et al., 1997; Furukawa et al., 2000, 2001; Arai et al., 2001). These findings provide evidence that there exist recognition sites for DOPA. In addition, we recently found a
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 0 3 7 - 8
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transporter unit candidate molecule for Na -dependent uptake of DOPA (Ishii et al., 2000) and that neuronal and/or glial cells, which take up DOPA in Na dependent manner, exist in the CNS (Sugaya et al., 2001). The lower brain stem plays a vital role in baroreflex and regulation of cardiovascular function. We have provided evidence for a neurotransmitter role of DOPA in the lower brain stem nuclei (Yue et al., 1994; Miyamae et al., 1999b; Nishihama et al., 1999). DOPA is a probable neurotransmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii (NTS) (Kubo et al., 1992; Yue et al., 1994; Misu et al., 1996). There exist strong DOPA immunoreactive neurons that are tyrosine hydroxylase (TH)positive and DOPA-positive, but AADC-negative and DA-negative in the NTS (Tison et al., 1989; Misu et al., 1996). Denervation peripheral to the ganglion nodosum, in which cell bodies are located, decreases TH- and DOPA-immunoreactivity, but not DA- and DA-bhydroxylase-immunoreactivity, in the medial and intermediate NTS in the ipsilateral lesioned side (Yue et al., 1994). The decreased area of TH-immunoreactivity is roughly compatible with the projection area of the aortic depressor nerve (ADN). The release of endogenous DOPA is evoked in a tetrodotoxin-sensitive manner by electrical ADN stimulation. DOPA microinjected into depressor sites of the NTS produces hypotension and bradycardia (Kubo et al., 1992). These responses are dose-dependent, stereoselective, seen under inhibition of central AADC and antagonized by DOPA ME or DOPA CHE (Kubo et al., 1992; Misu et al., 1997; Furukawa et al., 2000). DOPA ME microinjected into the NTS antagonizes depressor and bradycardic responses to ADN stimulation (Yue et al., 1994) in accord with those to exogenous DOPA microinjected (Kubo et al., 1992). Furthermore, bilateral injection of DOPA ME alone dose-dependently produces pressor and tachycardic responses. These responses are abolished by pretreatment with an inhibitor for TH, a-methyl-p tyrosine that potently inhibits endogenous DOPA release (Yue et al., 1994; Miyamae et al., 1999b). These findings provide evidence that endogenously released DOPA tonically functions to activate depressor neurons in the NTS. On the other hand, much evidence suggests that glutamate is a highly probable transmitter candidate of the primary baroreceptor afferents terminating in the NTS (Talman et al., 1980; Reis et al., 1981; Leone and Gordon, 1989; Kubo and Kihara, 1991; Van Giersbergen et al., 1992; Foley et al., 1999). In addition, DOPA induces the release of glutamate in the central nervous system (Goshima et al., 1993; Maeda et al., 1997; Furukawa et al., 2001). It is therefore important to clarify possible interactions between DOPA and glutamate.
Nitric oxide (NO), synthesized from L-arginine through the NO synthase (NOS), is also an important modulator for central cardiovascular regulation (Tagawa et al., 1994; Ma et al., 1995; Tseng et al., 1996; Chan and Sawchenko, 1998; Lin et al., 1998; Maeda et al., 1999). Microinjection of L-arginine, a NO donor, into the NTS produces hypotension and bradycardia (Tseng et al., 1996). Microinjection of N G-monomethylL-arginine (L-NMMA), a NOS inhibitor, into the NTS inhibits the L-arginine-induced depressor responses (Wu and Morris, 1998). Neuronal NOS (nNOS) is present in pre- and post-synaptic structures in the NTS and the majority of NOS immunoreactivity or NOS mRNA in the NTS is found in intrinsic structures in the nucleus (Chan and Sawchenko, 1998; Lin et al., 1998). Some evidence for possible interaction between nNOS and glutamate receptors in the NTS has been presented. LNMMA reduces the depressor response evoked by glutamate in the NTS (Di Paola et al., 1991; Lo et al., 1997). There have been some discrepancies between the effects of some pharmacological agents against hypotensive actions induced by ADN stimulation and by exogenous glutamate applied into the NTS (Talman et al., 1980; Leone and Gordon, 1989). Kynurenic acid, a non-selective ionotropic glutamate receptor antagonist, antagonizes these responses to ADN stimulation, but not those to glutamate microinjected (Talman et al., 1980; Leone and Gordon, 1989; Misu et al., 1996). Although the reasons are not clear at present, there might be several explanations for little antagonism by kynurenic acid against glutamate. Microinjection of a metabotropic glutamate receptor (mGluR) agonist, into the NTS, produced kynurenic acid-insensitive depressor responses in a manner similar to those to exogenous glutamate (Pawloski-Dahm and Gordon, 1992). If mGluRs are located at extrasynaptic locations, as observed in the hippocampus and the cerebellum (Baude et al., 1993), glutamate, when exogenously applied into the NTS, could preferentially activate mGluRs and then induce kynurenic acid-insensitive depressor responses. Alternatively, an unknown excitatory amino acid, the responses to which are antagonized by kynurenic acid, might be a primary transmitter of ADN (Leone and Gordon, 1989). In the present study, we have thus attempted to clarify whether glutamate receptors and/or NO are involved in the DOPA-induced cardiovascular responses in the NTS and whether NO production is an upstream or downstream event of activation of glutamate receptors in the NTS. We here propose a third pathway that DOPA is a neurotransmitter and/or neuromodulator of primary ADN neurons and glutamate is a neurotransmitter of primary afferent and/or secondary neurons in the microcircuits of the NTS.
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2. Materials and methods 2.1. General procedures All procedures were carried out according to the institutional guidelines outlined in the Institutional Animal Care and Use Committee of the Yokohama City University School of Medicine and to the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023). Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their suffering. The experimental procedures in this study were described previously (Yue et al., 1994; Miyamae et al., 1999b; Nishihama et al., 1999). In brief, male Wistar rats (Charles River), weighing 250/350 g, were anesthetized with urethane (1.2 g/kg, i.p.). After the trachea was cannulated, the animal was paralyzed with D-tubocurarine (1 mg/kg, i.m.) and artificially ventilated at a rate of 70/75 breaths/min and a volume of 2.5 /3.5 ml with a respirator (Shinano, SN-480) to avoid cardiovascular effects secondary to blood gas changes. The femoral artery was cannulated for recording systolic/diastolic arterial blood pressure (BP) and heart rate (HR). BP was measured with a pressure transducer (NEC Sanei, P23XL). HR was determined using a tachograph (NEC Sanei, 180) triggered by BP waves. BP and HR were recorded on a polygraph (NEC Sanei, 8S). Body temperature was maintained at 37.0 /37.5 8C with a temperature controller (BAS, CMA/150). The animal was placed in a stereotaxic apparatus (Narishige, SR-6) with the head fixed at 458. The dorsal surface of the lower brainstem was exposed by a limited occipital craniotomy (Yue et al., 1994). 2.2. Microinjection A glass micropipette pulled to an outside diameter of :/50 mm was inserted into the unilateral NTS. The coordinate for the depressor sites of the NTS was 0.6 mm rostral and 0.6 mm lateral to the caudal tip of the area postrema and 0.6 mm beneath the dorsal surface of the brainstem. Glutamate (Nacalai Tesque), DOPA (Nacalai Tesque), D-NMMA (Wako), L-NMMA (Wako) or L-arginine (Wako) was dissolved in saline (0.9% NaCl in 10 mM phosphate buffer, pH 7.4). Kynurenic acid (Sigma) was dissolved in 1 N NaOH, diluted in saline (0.9% NaCl in 10 mM phosphate buffer, pH 7.4) and adjusted pH to 7.4 using 1 N HCl. Each compound was given in a volume of 50 nl in 2 s. Antisense, sense and scrambled oligothionucleotides for the rat nNOS mRNA were synthesized as described previously (Maeda et al., 1999). Each of these oligos dissolved in artificial cerebrospinal fluid (132 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 0.65 mM MgCl2, 25 mM
233
NaHCO3, pH 7.3) mixed with cationic lipids (LipofectAMINE PLUS reagent and LipofectAMINE reagent, Life Technologies) was given in a volume of 50 nl in 10 s as described (Maeda et al., 1999). Analysis was carried out with data only from the experiments, in which glutamate (160 pmol) microinjected into the NTS produced hypotension of more than /20 mmHg and in which saline microinjected into the NTS produced no effect. 2.3. Statistics Data shown are means9/S.E.M. Statistical significance was calculated using Dunn’s multiple comparison test following repeated measures ANOVA, Student’s ttest or Welch’s t-test. A P value of B/0.05 was considered statistically significant.
3. Results 3.1. Effect of prior injection of kynurenic acid on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats In this series, respective resting mean BP was 859/6 mmHg and HR was 3789/14 beats/min (n / 6). DOPA at 300 pmol microinjected into the NTS produced depressor and bradycardic responses (Fig. 1A). We confirmed that DOPA at 30, 100 and 300 pmol dosedependently produced depressor and bradycardic responses (Kubo et al., 1992). Kynurenic acid at 600 pmol microinjected, which blocks the depressor and bradycardic responses to ADN stimulation (Kubo and Kihara, 1991), produced no effect on resting BP and HR. Injection of kynurenic acid 2 min prior to DOPA reduced depressor and bradycardic responses to DOPA by :/80% (Table 1). Reduced depressor responses required :/30 min to recover to 90% of control. 3.2. Effect of prior injection of L-NMMA on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats In this series, respective resting mean BP was 859/5 mmHg in L-NMMA (n/6) and 859/5 mmHg in DNMMA (n/ 5). Respective resting mean HR was 3799/ 9 beats/min in L-NMMA (n / 6) and 3589/3 beats/min in D-NMMA (n / 5). L-NMMA at 100 nmol microinjected into the NTS produced a slight pressor response: the maximal change in BP was 119/6 mmHg. After recovery to the resting level, DOPA (300 pmol) was microinjected into the NTS. Injection of LNMMA 10 min prior to DOPA reduced depressor and bradycardic responses to DOPA by :/90% (Fig. 1A,
K. Yamanashi et al. / Neuroscience Research 43 (2002) 231 /238
234
Fig. 1. Typical traces for reversible effects of prior injection of L-NMMA (100 nmol) (A) and nNOS antisense oligos (20 pmol) (B) on depressor and bradycardic responses to DOPA (300 pmol) microinjected into depressor sites of the unilateral NTS in anesthetized rats. Table 1 Effects of prior injection of kynurenic acid and L- or D-NMMA on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats Pretreatments
n DBP (mmHg) Before
Kynurenic acid L-NMMA D-NMMA
6 /399/4 6 /359/5 5 /399/4
DHR (beats/min)
After *
/99/5 /59/3* /349/4
Before
After
/639/14 /559/12 /649/9
/129/5* /59/4$ /429/4
Kynurenic acid (600 pmol), L- or D-NMMA (100 nmol) was microinjected into the NTS prior to DOPA (300 pmol). Data are mean9/S.E.M. * P B/0.01 (Student’s t -test) versus before. $ P B/0.01 (Welch’s t -test) versus before.
Table 1). We observed dose-dependent inhibition by LNMMA (10, 30 and 100 nmol) against DOPA-induced depressor and bradycardic responses (data not shown). Reduced depressor responses required :/20/95 min to recover 90% of control. Prior injection of D-NMMA
(100 nmol) produced no effect on responses to DOPA microinjected. 3.3. Effect of prior injection of neuronal NO synthase antisense oligos on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats In this series, respective resting mean BP was 909/3 mmHg in antisense oligos (n / 9), 909/7 mmHg in sense oligos (n/5) and 919/8 mmHg in scrambled oligos (n / 4). Respective resting mean HR was 3859/15 beats/ min in antisense oligos (n / 9), 3949/17 beats/min in sense oligos (n / 5) and 4139/8 beats/min in scrambled oligos (n/ 4). Antisense, sense or scrambled nNOS oligos each 20 pmol microinjected into the unilateral NTS produced no effect on the resting BP and HR. After microinjection of nNOS oligos (45 min), DOPA (300 pmol) was microinjected into the NTS. nNOS antisense oligos reduced the depressor and bradycardic responses to DOPA by :/70% (Fig. 1B, Table 2). Reduced depressor responses required :/75 /175 min
K. Yamanashi et al. / Neuroscience Research 43 (2002) 231 /238
235
Table 2 Effects of prior injection of neuronal NO synthase antisense oligos on depressor and bradycardic responses to DOPA microinjected into depressor sites of the unilateral NTS in anesthetized rats Pretreatments
n
DBP (mmHg) Before
Antisense oligos Sense oligos Scrambled oligos
9 5 4
/449/4 /399/6 /419/5
DHR (beats/min) After *,$
/159/7 /399/5 /429/6
Before
After
/1169/20 /679/21 /979/26
/399/20* /649/21 /1139/36
After depressor responses to DOPA (300 pmol) microinjected into the unilateral NTS, nNOS oligos (20 pmol) were microinjected into the ipsilateral NTS. The second DOPA into the NTS was challenged 45 min after nNOS oligos. Data are mean9/S.E.M. * P B/0.01 (Student’s t -test) versus before. $ P B/0.05 (Dunn’s multiple comparison test following repeated measures ANOVA) versus after injection of sense oligos, or scrambled oligos.
to recover 90% of control. Sense or scrambled nNOS oligos produced no effect on the responses to DOPA microinjected. 3.4. Effect of prior injection of kynurenic acid on depressor and bradycardic responses to L-arginine microinjected into depressor sites of the unilateral NTS in anesthetized rats
Table 3 L-Arginine microinjected into depressor sites of the unilateral NTS in anesthetized rats Pretreatments
n
DBP (mmHg)
DHR (beats/min)
Saline Kynurenic acid
9 8
/129/2 /109/2
/169/6 /109/2
Kynurenic acid (600 pmol) was microinjected into the NTS prior to Data are mean9/S.E.M.
L-arginine.
In this series, respective resting mean BP and HR was 859/4 mmHg and 4159/9 beats/min in saline (n/ 9) and 869/3 mmHg and 4049/14 beats/min in L-arginine (n / 8). L-Arginine at 30 nmol microinjected into the NTS produced depressor and bradycardic responses. Injection of kynurenic acid (600 pmol) 2 min prior to Larginine failed to reduce the responses to L-arginine (Fig. 2, Table 3).
4. Discussion We demonstrated that exogenously microinjected DOPA into the NTS produced depressor and bradycardic responses via activation of kynurenic acid-sensitive ionotropic glutamate receptors and nNOS. Kynurenic acid is a broad-spectrum antagonist for ionotropic glutamate receptors. Kynurenic acid has been shown to abolish the depressor response to ADN
Fig. 2. Typical traces for effects of prior injection of kynurenic acid (600 pmol) on depressor and bradycardic responses to L-arginine (30 nmol) microinjected into depressor sites of the unilateral NTS in anesthetized rats.
stimulation (Talman et al., 1980; Leone and Gordon, 1989; Kubo and Kihara, 1991; Misu et al., 1996). In the present study, kynurenic acid reduced by :/80% depressor responses to DOPA, thereby suggesting that a major pathway for DOPA is mediated by kynurenic acid-sensitive ionotropic glutamate receptors. We previously tested possible interactions of DOPA-related compounds with ionotropic glutamate receptors (Miyamae et al., 1999a). Among binding sites labeled with available glutamatergic [3H]-ligands in rat brain membrane, DOPA acts only on a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with a low affinity (IC50 /260 mM), whereas competitive DOPA antagonists, DOPA esters, act only on N -methyl-Daspartate (NMDA) ion channel domain with a low affinity (mM IC50s). DOPA ester and DOPA do not displace the selective binding of [3H]D1, D2, a2 and bligands in brain membranes (Goshima et al., 1991; Furukawa et al., 2000). DOPA antagonists do not displace kynurenic acid-sensitive specific binding of the [3H]-ligands for NMDA glycine site and NMDA binding site. In addition, we confirm that DOPA CHE does not inhibit the uptake of [14C]levodopa into oocytes (Ishii et al., 2000). Thus, DOPAergic agonist and competitive antagonist should act on the same sites, which may differ from the transport sites, catecholamine receptors and ionotropic glutamate receptors. How does DOPA induce kynurenic acid-sensitive depressor responses? Our previous findings indicate that DOPA evokes release of endogenous glutamate in striatum in vitro and in vivo (Goshima et al., 1993; Maeda et al., 1997; Furukawa et al., 2001). It is
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therefore likely that DOPA microinjected into the NTS elicits glutamate release via DOPA recognition sites, and glutamate, thus released, activates kynurenic acid-sensitive glutamate receptors, leading to depressor and bradycardic responses. In the present study, we attempted to clarify whether DOPA microinjected into the NTS elicits the release of endogenous glutamate during microdialysis of the NTS. However, DOPA apparently did not evoke glutamate release over the basal release (data not shown). Although the exact reasons remain unknown, this is probably due to a minor component of the amount of glutamate released from ADN in the perfusate samples from the NTS region. This negative result is consistent with previous findings that evoked release of endogenous glutamate over the basal release is undetectable upon ADN and baroreceptor stimulation (Sved and Curtis, 1993; Misu et al., 1994). Stereoselective and reversible antagonism by NMMA against the cardiovascular responses to DOPA suggests that microinjected DOPA produces cardiovascular effect via NOS activation. Several lines of evidence support the idea that some interaction exists between glutamate receptors and NOS in the NTS. There is an anatomical link between glutamate and NO systems on immunohistochemical studies in the NTS (Aoki et al., 1997; Lin et al., 1998). L-NMMA reduces the depressor responses to glutamate in the NTS (Di Paola et al., 1991; Lo et al., 1997). In the rat brain, activation of NMDA glutamate receptors leads to synthesis and release of NO (Garthwaite et al., 1989). Our present findings argue for the idea that the interaction between glutamate and NO also exists in the cardiovascular responses to DOPA. NOS has three isozymes: nNOS, endothelial NOS (eNOS) and inducible NOS (iNOS) and L-NMMA is a competitive inhibitor of all NOSs with low specificity (Reif and McCreedy, 1995). It is unlikely that iNOS is involved in DOPA-induced responses because L-
NMMA inhibits nNOS and eNOS reversibly, but iNOS irreversibly. Furthermore, nNOS antisense oligos (Maeda et al., 1999) selectively reduced the depressor responses to DOPA. This nNOS antisense oligo markedly suppresses the nNOS immunoreactivity in the NTS only 45 min after injection (Maeda et al., 1999), the time course which was coincident with the blocking effect of the antisense on DOPA-induced hypotensive and bradycardic responses in the present study (Fig. 1). Our finding therefore indicates that nNOS is involved in DOPA-induced depressor responses in the NTS. Accordingly, NO has been shown to have an excitatory action on some neurons in the NTS (Tagawa et al., 1994; Ma et al., 1995). It is also possible that DOPA-induced NO production further facilitates DOPA release like glutamate (Matsuo et al., 2001). NO production is probably a downstream event of activation of the glutamate receptors involved in depressor responses to DOPA, since kynurenic acid at the dose which reduced the cardiovascular response to DOPA, failed to affect the responses to L-arginine. L-Arginine is considered to induce depressor responses by accelerating NO synthesis because many cells utilize L-arginine to generate NO (Wu and Morris, 1998) and L-NMMA reduces the cardiovascular responses to L-arginine (Tseng et al., 1996). It is noteworthy that bilateral injection of LNMMA or antisense oligo to nNOS into the NTS alone evokes hypertension (Harada et al., 1993; Maeda et al., 1999). These findings suggest that nNOS and NO synthesized tonically functions to activate depressor neurons for regulation of blood pressure in the NTS. Our preliminary experiments indicate that D(/)-2amino-5-phosphonopentanoate, a selective NMDA antagonist, partially blocks depressor and bradycardic responses to DOPA microinjected into the NTS (unpublished observation). These findings suggest that NMDA glutamate receptors are at least in part involved
Fig. 3. A model for neuronal microcircuits in depressor sites of NTS.
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in DOPA-induced depressor responses. Additional possibility is that responses to DOPA remained after kynurenic acid may be antagonized by metabotropic glutamate antagonist (Foley et al., 1999) or DOPA activates directly the depressor sites (Fig. 3). In conclusion, DOPA produces depressor and bradycardic responses via activation of kynurenic acid-sensitive ionotropic glutamate receptors and nNOS. DOPA may release undetectable but functioning endogenous glutamate via recognition sites for DOPA. We herein propose a new pathway that DOPA is a neurotransmitter and/or neuromodulator of the primary ADN and glutamate is a neurotransmitter of primary and/or secondary neurons in neuronal microcircuits of the NTS (Fig. 3).
Acknowledgements This work was partially supported by Japan SRF Grants for Biomedical Research.
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