Evidence for l-DOPA systems responsible for cardiovascular control in the nucleus tractus solitarii of the rat

Microinjections of l-DOPA (10–100 ng) into the medial area of the nucleus tractus solitarii (NTS) led to dose-dependent 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 μg), 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.     

Involvement of nitric oxide production via kynurenic acid-sensitive glutamate receptors in DOPA-induced depressor responses in the nucleus tractus solitarii of anesthetized rats

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 approximately 80%. Prior injection of N(G)-monomethyl-L-arginine 100 nmol, a potent NO synthase inhibitor, reversibly attenuated by approximately 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 approximately 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.     

GABA may function tonically via GABA(A) receptors to inhibit hypotension and bradycardia by L-DOPA microinjected into depressor sites of the nucleus tractus solitarii in anesthetized rats

We have proposed that DOPA is a transmitter of the primary baroreceptor afferents terminating in the rat nucleus tractus solitarii (NTS). GABA is a putative inhibitory neuromodulator for baroreflex inputs in the NTS. GABA may inhibit DOPAergic transmission. Drugs were microinjected into depressor sites of the NTS in anesthetized rats. DOPA (10-60 ng) elicited dose-dependent depressor responses. GABA (3-300 ng) elicited dose-dependent pressor responses. Nipecotic acid (100 ng) elicited pressor responses. Bicuculline (10 ng) elicited depressor responses. Responses to DOPA (30 ng) were inhibited by pretreatment with GABA and nipecotic acid, but potentiated by bicuculline, when vascular responses to pretreated drugs returned to basal levels. DOPA ME, a competitive DOPA antagonist, did not displace specific [3H]GABA binding. Prior DOPA ME (1 microg) inhibited by one-half pressor responses to 300 ng GABA. GABA seems to inhibit tonically via GABA(A) receptors depressor responses to DOPA and to elicit pressor responses partially by inhibition of tonic function of endogenous DOPA to activate depressor sites in the NTS. These findings further support the above proposal.     

NMDA receptor antagonism blocks the cardiovascular responses to microinjection of trans-ACPD into the NTS of awake rats

The possible interaction of glutamatergic metabotropic agonists and N-methyl- d -aspartate (NMDA) receptors was investigated in the nucleus tractus solitarii (NTS) of awake rats. The cardiovascular responses to unilateral microinjection of trans -1-amino-1,3-cyclopentanediocarboxylic acid ( trans -ACPD; 250 pmol/50 nL) into the NTS ( n = 8) produced hypotension (−64 ± 4 mmHg) and bradycardic (−206 ± 11 bpm) responses, which were blocked by previous microinjection of 2-amino-5-phosphonovaleric acid (AP-5; 10 nmol/50 nL), a selective antagonist of NMDA ionotropic receptors, into the same site. Intravenous injection of methyl-atropine blocked both the bradycardic and hypotensive responses to microinjection of trans -ACPD into the NTS, indicating that the hypotension was secondary to the intense bradycardic response. The data also showed that the bradycardic and hypotensive responses to microinjection of an NMDA agonist (10 pmol/50 nL) into the NTS were not affected by previous microinjection of α-methyl-4-carboxyphenylglycine (MCPG; 5 nmol/50 nL), a non-selective antagonist of metabotropic receptors. The results showing that the cardiovascular responses to microinjection of trans -ACPD into the NTS were blocked by AP-5 indicate that the responses to metabotropic agonists in the NTS involves NMDA receptors.     

Depressor and bradycardic actions of L-proline injected into the nucleus tractus solitarii of anesthetized rats

L-Glutamate has been considered to be a neurotransmitter in the nucleus tractus solitarius (NTS) of the afferent baroreflex pathway, though this has not yet been decisively shown. A bolus injection of a neurotransmitter candidate amino acid L-proline into the cisterna magna and that of L-glutamate shows the same pressor action in the freely moving rat, but the actual nuclei responding L-proline remain undetermined. Besides L-glutamate, L-proline might be another candidate amino acid in the NTS. The present study was therefore performed to characterize the circulatory action of L-proline injected into the NTS where responses to glutamate in the anesthetized rat had already been shown. The NTS was first determined as a site on the dorsal surface of the medulla where a microinjection of L-glutamate decreased arterial pressure and heart rate. Microinjected L-proline (1.65 to 13.2 nmol, 33 nl) into the NTS decreased arterial pressure and heart rate in a dose-dependent manner. The injection of a mixed solution (66 nl) of kynurenate, an ionotropic excitatory amino acid receptors antagonist (1.32 nmol), and L-proline (6.6 nmol) into the NTS abolished the depressor and bradycardic actions with L-proline alone (6.6 nmol, 66 nl). However, a mixture of an increased concentration of kynurenate (6.6 nmol) with glutamate augmented the actions seen with glutamate alone (0.66 nmol, 66 nl). D-Proline (13.2 nmol, 66 nl), the optic isomer of L-proline, produced no change in arterial pressure or heart rate, suggesting that the actions of L-proline in the NTS were optically specific. The results indicate that L-proline but not D-proline induces its depressor and bradycardic actions through ionotropic excitatory amino acid receptors in the NTS of the anesthetized rat. L-Proline may become a candidate transmitter of baroreceptor information in the NTS.     

Cardiovascular responses to microinjection of nociceptin and endomorphin-1 into the nucleus tractus solitarii in conscious rats

Increasing evidence suggests an active participation of nociceptinergic transmission in the central control of cardiovascular activity and reflex. In this study, the role of the classic opioid mu receptor and the nociceptin/orphanin FQ receptor, a novel opioid receptor, in the nucleus tractus solitarii (NTS) in the regulation of cardiovascular activity was investigated and compared in chronically cannulated and freely moving conscious rats. Microinjections of nociceptin, an endogenous ligand for the nociceptin receptor, into the relatively rostral NTS produced dose-related (0.04, 0.2, and 1 nmol) increases in blood pressure and heart rate. Intra-NTS injection of the selective nociceptin receptor antagonist [Nphe(1)]Nociceptin(1-13)NH(2) (NOR-AN) at 1 nmol blocked the increases in blood pressure and heart rate induced by nociceptin. In contrast, pretreatment with the nonselective opioid receptor antagonist naloxone (5 nmol) had no effects on the cardiovascular responses to nociceptin. Like nociceptin, microinjection of endomorphin-1 (EM-1), an endogenous ligand for the opioid mu receptor, into the rostral NTS increased blood pressure and heart rate in a dose-dependent manner (0.04, 0.2, and 1 nmol). Pretreatment with naloxone (5 nmol), but not NOR-AN, blocked cardiovascular responses elicited by EM-1. Neither NOR-AN nor naloxone alone had significant effects on the baseline blood pressure and heart rate. Injection of excitatory amino acid l-glutamate (1 nmol) into the same sites caused the typical depressor and bradycardic responses. In the caudal NTS areas, nociceptin and EM-1 seemed to induce opposite responses: hypotension and bradycardia. These results suggest that the novel nociceptin receptors and traditional opioid receptors in the NTS may be independently involved in the regulation of cardiovascular activity.     

Cardiovascular actions of adrenocorticotropin microinjections into the nucleus tractus solitarius of the rat

The presence of adrenocorticotropin (ACTH) containing cells and melanocortin (MC) receptors has been reported in the nucleus tractus solitarius (NTS) of the rat. The importance of the NTS in the regulation of cardiovascular function is also well established. Based on these reports, it was hypothesized that ACTH acting within the NTS may modulate the central regulation of cardiovascular function. To test this hypothesis, cardiovascular effects of ACTH in the NTS were investigated in intact urethane-anesthetized and unanesthetized decerebrate, artificially ventilated, adult male Wistar rats. Microinjections of ACTH (0, 0.5, 1, 2, and 4 mM) into the medial subnucleus of NTS (mNTS) elicited decreases in mean arterial pressure (MAP; 0+/-0, 24.4+/-3.5, 35.7+/-4.3, 44.5+/-5.8 and 53.7+/-5.6 mm Hg, respectively) and heart rate (HR; 0+/-0, 25.7+/-5.3, 35.5+/-6.4, 47.5+/-12.1 and 55.0+/-5.6 beats/min, respectively). The onset and duration of the responses to microinjections of ACTH (0.5-4 mM) were 5-10 s and 45-120 s, respectively. Control microinjections of artificial cerebrospinal fluid (aCSF) did not elicit any response. The volume of all microinjections was 100 nl. The concentrations of ACTH that elicited depressor and bradycardic responses when microinjected into the mNTS (e.g. 1 or 2 mM, 100 nl), did not elicit a response when injected i.v. (n=5) or i.c.v. (n=2) indicating that there was no leakage of the drug from the injection site in the mNTS. Microinjections of MC3/4 receptor antagonists (acetyl-[Nle(4), Asp(5), d-2-Nal(7), Lys(10)]-cyclo-alpha-MSH amide, fragments 4-10 (SHU9119) and agouti-related protein (83-132) amide) into the mNTS blocked the responses to ACTH. Microinjections of ACTH (2 mM) into the mNTS decreased efferent greater splanchnic nerve activity. Bilateral vagotomy significantly attenuated ACTH-induced bradycardia. These results indicated that: 1) microinjections of ACTH into the mNTS elicited depressor and bradycardic responses, 2) these responses were mediated via MC3/4 receptors, 3) the depressor effects were mediated via a decrease in the activity of the sympathetic nervous system, and 4) the bradycardic responses were vagally mediated.     

DOPA cyclohexyl ester, a competitive DOPA antagonist, protects glutamate release and resultant delayed neuron death by transient ischemia in hippocampus CA1 of conscious rats

In rat striata, DOPA released is a causal factor for glutamate release and resultant delayed neuron death by four-vessel occlusion. Nanomolar DOPA cyclohexyl ester (CHE), a potent and relatively stable competitive DOPA antagonist, protects these events. We tried to clarify whether DOPA CHE protects these events in hippocampal CA1 pyramidal cell layers most vulnerable against ischemia. Five to 10 min ischemia caused slight to mild glutamate release in 10 min samples during microdialysis and mild to severe neuron death 96 h after reperfusion. DOPA and dopamine were under assay limit in this design, but were basally detected by 20 min sampling and released by 20 min ischemia. In 10 min samples, intrahippocampal perfusion of 100 nM DOPA CHE 10 min before ischemia for 70 min did not inhibit glutamate release by 10 min ischemia, while it abolished glutamate release and protected delayed neuron death by 5 min ischemia. DOPA CHE is neuroprotective under a mild ischemic condition in rat hippocampus CA1.     

DOPA cyclohexyl ester potently inhibits aglycemia-induced release of glutamate in rat striatal slices

Brain ischemic insult causes glutamate release and resultant neuronal cell death. We here show that L-3,4-dihydroxyphenylalanine (DOPA) is a positive regulatory factor for glutamate release elicited by a mild brain insult using in vitro superfused rat striatal slices as a model system. Glucose deprivation for 18 min elicited release of glutamate, DOPA and dopamine (DA). Either tetrodotoxin (TTX) (1 microM) or alpha-methyl-p-tyrosine (alpha-MPT) (1 mM), a tyrosine hydroxylase inhibitor reduced markedly each of these releases. NSD-1015 (20 microM), an aromatic L-amino acid decarboxylase inhibitor restored the inhibition by alpha-MPT of glutamate and DOPA but not DA release. DOPA cyclohexyl ester (DOPA CHE) (0.3-1 microM), a competitive DOPA antagonist, concentration-dependently suppressed aglycemia-induced glutamate release, the effect which was mimicked neither by S-sulpiride nor SCH23390, a DA D(1) or D(2) receptor antagonist, respectively. Zonisamide (1-1000 microM), an anticonvulsant or YM872 (1 microM), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) a receptor antagonist produced no effect on aglycemia-induced glutamate release. DOPA CHE thus showed a relatively potent inhibitory action on aglycemia-induced glutamate release among several neuroprotective agents tested.     

The evidence for tonic GABAergic regulation of basal L-DOPA release via activation of inhibitory GABA(A) receptors in the nucleus tractus solitarii of anesthetized rats

We have proposed that DOPA is a neurotransmitter of the primary baroreceptor afferents terminating in the rat nucleus tractus solitarii (NTS). GABA is a putative inhibitory neuromodulator for baroreflex inputs in the NTS. Thus, GABA may inhibit DOPAergic transmission in the NTS. We tried to clarify whether basal DOPA release is inhibited by muscimol, a GABA(A) agonist, and facilitated by bicuculline, a GABA(A) antagonist, during microdialysis of the NTS in anesthetized rats. DOPA release was consistently detectable. Muscimol 10-100 microM perfused via probes gradually inhibited concentration-dependently DOPA release. Peak 30% inhibition occurred 2 h after perfusion. Muscimol (30 microM)-induced inhibition was antagonized by non-effective 10 microM bicuculline. Bicuculline (30 microM) elicited peak 30% facilitation of DOPA release 2 h after perfusion. Endogenous GABA seems to regulate tonically basal DOPA release via activation of inhibitory GABA(A) receptors in the rat NTS. These findings further support the above proposal.     

Nicotine suppression of gustatory responses of neurons in the nucleus of the solitary tract

This study investigated effects of nicotine applied to the tongue surface on responses of gustatory neurons in the nucleus of the solitary tract (NTS) in rats. In pentobarbital-anesthetized rats, single-unit recordings were made from NTS units responsive to one or more tastants (sucrose, NaCl, citric acid, monosodium glutamate, quinine). Application of nicotine (0.87, 8.7, or 600 mM) excited gustatory NTS units and significantly attenuated NTS unit responses to their preferred tastant in a dose-dependent manner. The depressant effect of nicotine was equivalent regardless of which tastant best excited the NTS unit. Nicotinic excitation of NTS units and depression of their tastant-evoked responses were both significantly attenuated by the nicotinic antagonist mecamylamine, which itself did not excite NTS units. In rats with bilateral trigeminal ganglionectomy, nicotine still excited nearly all NTS units but no longer depressed tastant-evoked responses. Nicotine did not elicit plasma extravasation when applied to the tongue. The results indicate that nicotine directly excites NTS units by gustatory nerves and inhibits their tastant-evoked responses by a nicotinic acetylcholine receptor-mediated excitation of trigeminal afferents that inhibit NTS units centrally.     

L-dopaergic components in the caudal ventrolateral medulla in baroreflex neurotransmission.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is probably a transmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii; L-DOPA functions tonically to activate depressor sites of the caudal ventrolateral medulla, which receives input from the nucleus tractus solitarii [Misu Y. et al. (1996) Prog. Neurobiol. 49, 415-454]. We have attempted to clarify whether or not L-DOPAergic components within the caudal ventrolateral medulla are involved in baroreflex neurotransmission in anesthetized rats. Electrolytic lesions of the right nucleus tractus solitarii (1 mA d.c. for 10 s, 10 days before measurement) selectively decreased by 45% the tissue content of L-DOPA in the dissected ipsilateral caudal ventrolateral medulla. Electrolytic lesions did not decrease dopamine, norepinephrine and epinephrine levels. During microdialysis of the right caudal ventrolateral medulla, extracellular levels of L-DOPA, norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid were consistently detectable using high-performance liquid chromatography with electrochemical detection. However, extracellular dopamine levels were lower than the assay limit. Baroreceptor activation by i.v. phenylephrine selectively evoked L-DOPA without increasing the levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. This L-DOPA release was suppressed by acute lesion in the ipsilateral nucleus tractus solitarii. Intermittent stimulation of the right aortic depressor nerve (20 Hz, 3 V, 0.3 ms duration, for 30 min) repetitively and constantly caused L-DOPA release, hypotension and bradycardia, without increases in levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. Local inhibition of L-DOPA synthesis with alpha-methyl-p-tyrosine (30 microM) infused into the ipsilateral caudal ventrolateral medulla gradually decreased basal levels of L-DOPA and 3,4-dihydroxyphenylacetic acid without decreasing norepinephrine and epinephrine. The inhibition of L-DOPA synthesis interrupted L-DOPA release and decreased by 65% depressor responses elicited by aortic nerve stimulation; however, it produced no effect on bradycardic responses. CoCl2 (119 ng), a mainly presynaptic inhibitory transmission marker, and L-DOPA methyl ester (1 microg), a competitive L-DOPA antagonist, when microinjected into depressor sites of the right caudal ventrolateral medulla, reduced by 60% depressor responses to transient ipsilateral stimulation of the aortic nerve (20 Hz, 3 V, 0.1 ms duration, for 10 s). No changes in bradycardic responses were observed. There may exist an L-DOPAergic relay from the nucleus tractus solitarii to the caudal ventrolateral medulla. L-DOPAergic components in the caudal ventrolateral medulla are involved in baroreflex neurotransmission via a baroreceptor-aortic depressor nerve-nucleus tractus solitarii-caudal ventrolateral medulla relay in the rat.     

Evidence of N-methyl-D-aspartate receptor-mediated modulation of the aortic baroreceptor reflex in the rat nucleus tractus solitarii

Microinjections of N-methyl-D-aspartate (NMDA) into the medial area of the nucleus tractus solitarii (NTS) of the rat led to a decrease in arterial pressure and heart rate. The NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP5) reduced the cardiovascular responses to NMDA. Depressor and bradycardic responses to aortic nerve stimulation were reduced by AP5 but not by a substance P antagonist, injected into the NTS. High K+ stimulation caused a calcium-dependent release of glutamate and aspartate from tissues in the area of the NTS. These results provide evidence of NMDA receptor-mediated modulation of the aortic baroreceptor reflex in the rat NTS.     

Intravenous morphine-induced activation of vagal afferents: peripheral, spinal, and CNS substrates mediating inhibition of spinal nociception and cardiovascular responses.

1. Intravenous administration of 1.0 mg/kg of morphine produces inhibition of the nociceptive tail-flick (TF) reflex, hypotension, and bradycardia in the pentobarbital-anesthetized rat. The present experiments examined peripheral, spinal, and supraspinal relays for inhibition of the TF reflex and cardiovascular responses produced by morphine (1.0 mg/kg iv) in the pentobarbital-anesthetized rat using 1) bilateral cervical vagotomy, 2) spinal cold block or mechanical lesions of the dorsolateral funiculi (DLFs), or 3) nonselective local anesthesia or soma-selective lesions of specific CNS regions. Intravenous morphine-induced inhibition of responses of unidentified, ascending, and spinothalamic tract (STT) lumbosacral spinal dorsal horn neurons to noxious heating of the hindpaw were also examined in intact and bilateral cervical vagotomized rats. 2. Bilateral cervical vagotomy significantly attenuated inhibition of the TF reflex and bradycardia produced by intravenous administration of morphine. Bilateral cervical vagogtomy changed the normal depressor response produced by morphine into a sustained pressor response. Inhibition of the TF reflex in intact rats was not due to changes in tail temperature. 3. Spinal cold block significantly attenuated inhibition of the TF reflex, the depressor response, and the bradycardia produced by intravenous administration of morphine. However, bilateral mechanical transections of the DLFs failed to significantly affect either inhibition of the TF reflex or cardiovascular responses produced by this dose of intravenous morphine. 4. Microinjection of either lidocaine or ibotenic acid into the nuclei tracti solitarii (NTS), rostromedial medulla (RMM), or ventrolateral pontine tegmentum (VLPT) attenuated morphine-induced inhibition of the TF reflex. Similar microinjections into either the periaqueductal gray (PAG) or the dorsolateral pons (DLP) failed to affect morphine-induced inhibition of the TF reflex. 5. Microinjection of either lidocaine or ibotenic acid into the NTS, RMM, VLPT, DLP, or rostral ventrolateral medulla (RVLM) attenuated the depressor response produced by morphine, although baseline arterial blood pressure (ABP) was affected by ibotenic acid microinjections in the DLP. In all these cases, the microinjections failed to reveal a sustained pressor response as was observed with bilateral cervical vagotomy. Similar microinjections into the PAG failed to affect the depressor response produced by morphine. 6. The lidocaine and ibotenic acid microinjection treatments also showed that the bradycardic response produced by morphine depends on the integrity of the NTS, RMM, RVLM, and possibly the DLP, but not the PAG or VLPT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nitrergic stimulation of the locus coeruleus modulates blood pressure and heart rate in the anaesthetized rat.

To investigate whether nitric oxide is involved in the cardiovascular responses mediated via the locus coeruleus, the effects of microinjections of L-arginine and L-glutamate into the locus coeruleus on blood pressure and heart rate were investigated in sodium pentobarbitone-anaesthetized rats. Unilateral microinjection of L-arginine (25, 50 nmol) elicited dose-related depressor (-17 +/- 4, -25 +/- 4 mmHg) and bradycardic (13 +/- 3, 24 +/- 6 b.p.m.) effects. Furthermore, these effects were attenuated by prior local microinjection of N(G)-nitro-L-arginine (40 nmol). Peripheral muscarinic receptor blockade with atropine methyl nitrate (1 mg/kg, i.v.) attenuated the bradycardic but not the depressor responses to L-arginine. L-Glutamate (2 nmol) microinjections also mediated depressor (-27 +/- 6 mmHg) and bradycardic (53 +/- 23 b.p.m.) effects that were attenuated by microinjections of dizocilpine maleate (1 nmol) into the locus coeruleus. In addition, pretreatment with N(G)-nitro-L-arginine (40 nmol) also significantly attenuated the depressor response elicited by L-glutamate. These results suggest that nitrergic and glutamatergic pathways are operative within the locus coeruleus to modulate cardiovascular function, and also that a functional interaction may exist between the nitrergic and glutamatergic systems within the rat locus coeruleus.     

Role of nucleus tractus solitarius 5-HT3 receptors in the defense reaction-induced inhibition of the aortic baroreflex in rats

Different stressful conditions elicit a typical behavior called the defense reaction. Our aim was to determine whether 5-HT3 receptors in the nucleus tractus solitarius (NTS) are involved in 1) the inhibition of the baroreflex bradycardia and 2) the rise in blood pressure, which are known to occur during the defense reaction. In urethane-anesthetized rats, the defense reaction was elicited by electrical stimulation of the dorsomedial nucleus of the hypothalamus (DMH) or the dorsal part of the periaqueductal gray (dPAG). Direct electrical stimulation of the aortic depressor nerve was used to trigger the typical baroreflex responses. Aortic stimulation at high (100-150 microA) and low (50-90 microA) intensity produced a decrease in heart rate of -39 to -44% (relative to baseline, Group 1 responses, n = 113) and -19 to -24% (Group 2 responses, n = 43), respectively. In spontaneously breathing rats, Group 1 and Group 2 bradycardiac responses were inhibited during DMH (-75 +/- 4% and -96 +/- 4%, n = 38 and n = 11, respectively), as well as dPAG (-81 +/- 3% and -95 +/- 4%, n = 36 and n = 10, respectively) stimulation. The aortic baroreflex bradycardia was hardly affected by DMH or dPAG stimulation when bicuculline (5 pmol), a specific GABAA receptor antagonist, had previously been microinjected into the NTS. Likewise, NTS microinjections of granisetron, a specific 5-HT3 receptor antagonist, prevented, in a dose-dependent manner, the baroreflex bradycardia inhibition. In addition, intra-NTS granisetron did not affect the rise in blood pressure induced by either site stimulation. These data show that 5-HT3 receptors in the NTS are involved in the GABAergic inhibition of the aortic baroreflex bradycardia, but not in the rise in blood pressure, occurring during the defense reaction elicited by DMH or dPAG stimulation.     

Glutamatergic systems in the bed nucleus of the stria terminalis,effects on cardiovascular system

The bed nucleus of the stria terminalis (BST) is a part of the limbic system. Two studies have shown that microinjection of l-glutamate in the BST elicited cardiovascular depressive and bradycardic responses, but in one study, both pressor and depressor responses were observed in the chemical stimulation of BST by glutamate in the urethane-anesthetized rats. Also, the roles of glutamate receptor subtypes have not been investigated yet. The aim of this study was to find the effects of glutamate and its receptors on the blood pressure and heart rate in the BST of urethane-anesthetized rats. The drugs (50 nl) were microinjected into the BST of anaesthetized rats. The blood pressure and heart rate were recorded throughout each experiment. The average changes in the mean arterial pressure and heart rate at different intervals were compared both within each case group and between the case and the control groups, using repeated measures ANOVA. Microinjection of l-glutamate (0.25 M) into the BST resulted in the decrease of the mean arterial pressure (−18.85 ± 3.84 mmHg) and heart rate (−18 ± 4 beats/min). Injection of AP5, antagonist of glutamate NMDA receptor (2.5 , 5 mM) and CNQX, antagonist of glutamate AMPA receptor (0.5, 1 mM) had no significant effect on the mean arterial pressure and heart rate. Either Ap5 or CNQX, when co-injected with glutamate, abolished the depressor and bradycardic effects of glutamate, suggesting that simultaneous activation of both glutamate receptors is necessary for the effect of glutamate system to emerge.     

Involvement of the purinergic system in central cardiovascular modulation at the level of the nucleus ambiguus of anaesthetized rats

Anatomical studies have demonstrated the existence of purinergic P2 receptors in the nucleus ambiguus (NA), a site containing cardiac vagal motoneurons. However, very little is known about the functional role of these receptors in central cardiac vagal regulation. The aims of our study were to evaluate the following: (1) the blood pressure and heart rate responses following purinoceptor activation within the NA; (2) the role of purinoceptors and excitatory amino acid (EAA) receptors in mediating the cardiovascular responses evoked by ATP and L-glutamate stimulation of NA; and (3) the role of NA purinoceptors in mediating the cardiovascular responses of the Bezold-Jarisch reflex. In anaesthetized rats, microinjection of L-glutamate (5.0 nmol/50 nl) into the NA induced a marked and immediate onset bradycardia with minimal change in arterial pressure. Microinjection of ATP into the NA induced a dose-dependent (0.31-6.0 nmol/50 nl) bradycardia and pressor responses. It is noteworthy that the bradycardia occurred either before or simultaneously with a pressor response (when present), indicating that it was not a baroreceptor reflex mediated response due to the rise in arterial pressure. The pressor response was prevented by α(1)-adrenergic blockade with prazosin, whereas muscarinic blockade with methyl-atropine abolished the evoked bradycardia. Ipsilateral microinjection of PPADS (a P2 receptor antagonist; 500 pmol/100 nl) into the NA significantly attenuated the ATP-induced bradycardia but spared the pressor response. In contrast, PPADS in the NA had no effect on the L-glutamate-evoked bradycardic response. Ipsilateral injection of kynurenic acid (a non-selective EAA receptor antagonist; 10 nmol/50 nl) into the NA totally blocked the bradycardia induced by l-glutamate and partly attenuated the ATP induced bradycardia. Finally, both the depressor and the bradycardic responses of the Bezold-Jarisch reflex were attenuated significantly (P < 0.01 and P < 0.05, respectively) following bilateral microinjection of PPADS into the NA. These results identify ATP and purinergic P2 receptors within the ventrolateral medulla as excitatory to cardiovagal neurons. Additionally, our data show that P2 receptors within the ventrolateral medulla are integral to the cardiovascular responses of the Bezold-Jarisch reflex.     

Neuronal Nitric Oxide Synthase Activation Is Involved in Insulin-Mediated Cardiovascular Effects in the Nucleus Tractus Solitarii of Rats

Neuronal nitric oxide synthases (nNOS) is distributed throughout the central nervous system (CNS) and has been proposed to modulate neuronal activity in the nucleus tractus solitarii (NTS). Here, we investigated whether the activation of nNOS is involved in insulin-induced cardiovascular responses in the NTS. Insulin (100 IU/ml) was unilaterally microinjected into the NTS, and the cardiovascular effects were evaluated before and after microinjection of the nNOS inhibitors 7-nitroindazole (7-NI) (5 pmol) and N(5)-(1-imino-3-butenyl)-l-ornithine (vinyl-L-NIO) (600 pmol). Western blot and immunohistochemical analyses were performed to determine nNOS phosphorylation levels after insulin or phosphoinositide 3-kinase (PI3K) inhibitor LY294002 microinjection into the NTS. Unilateral microinjection of insulin into the NTS produced prominent depressor and bradycardic effects in WKY rats. Pretreatment with the nNOS inhibitors 7-NI and Vinyl-L-NIO attenuated the cardiovascular response evoked by insulin in Wistar-Kyoto (WKY) rats. Moreover, Western blot analysis showed a significant increase in nNOS (16.5±0.4-fold; P<0.05; n=4) phosphorylation after insulin injection, whereas the PI3K inhibitor LY294002 abolished the insulin-induced effects. In situ nNOS phosphorylation was found to be increased in the NTS after insulin injection. Furthermore, co-immunoprecipitation assay showed Akt and nNOS can bind to each other as detected by phospho-Akt^S473 and phospho-nNOS^S1416 antibodies. In vitro kinase assay showed insulin activated Akt can directly phosphorylate nNOS^S1416. These results demonstrated that nNOS may couple with the activation of the insulin receptor, via the liberation of NO, in order to participate in central cardiovascular regulation of WKY rats.     

The involvement of nitric oxide on the adenosine A(2) receptor-induced cardiovascular inhibitory responses in the posterior hypothalamus of rats

This study was performed to investigate the putative relationship between nitric oxide (NO) and adenosine A(2) receptors on central cardiovascular regulation in the posterior hypothalamus of rats. Posterior hypothalamic injection of drugs was performed in anesthetized, artificially ventilated male Sprague-Dawley rats. Injection of adenosine A(2) receptor agonist 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA; 1, 2 and 5 nmol) produced a dose-dependent decrease of blood pressure and heart rate. Pretreatment with adenosine A(2) receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 nmol) blocked the depressor and bradycardiac effects of CPCA (5 nmol). Pretreatment with soluble guanylate cyclase inhibitor LY-83,583 (5 nmol) attenuated the depressor and bradycardiac effects of CPCA (5 nmol). In addition, pretreatment with NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (40 nmol) attenuated the depressor and bradycardiac responses of CPCA (5 nmol). These results suggest that adenosine A(2) receptor in the posterior hypothalamus plays an inhibitory role in central cardiovascular regulation and that NO participates in the inhibitory response induced by adenosine A(2) receptor stimulation in the posterior hypothalamus.