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Nitric oxide reduces blood pressure in the nucleus tractus solitarius: a real time electrochemical study
Brain Research Bulletin, Vol. 57, No. 2, pp. 171–177, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/02/$–see front matter
PII S0361-9230(01)00737-7
Nitric oxide reduces blood pressure in the nucleus tractus solitarius: A real time electrochemical study W. C. Wu,1 Y. Wang,2 L. S. Kao,3 F. I. Tang3 and C. Y. Chai1* 1
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 2National Institute on Drug Abuse, Baltimore, MD, USA; and 3National Yang-Ming University, Taipei, Taiwan [Received 2 May 2001; Accepted 26 September 2001]
ABSTRACT: Increasing evidence has demonstrated that nitric oxide (NO) is involved in central cardiovascular regulation. In this study, we directly measured extracellular NO levels, in real-time, in the nucleus tractus solitarius (NTS) of anesthetized cats using Nafion/Porphyrine/o-Phenylenediamine-coated NO sensors. We found that local application of L-arginine (L-Arg) induced NO overflow in NTS and hypotension. These responses were potentiated in the vagotomized animals. Pretreatment with NO synthase (NOS)/guanylate cyclase inhibitor methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or NO scavenger hemoglobin attenuated L-Arg-induced hypotension, suggesting that exogenous supplement of NO suppressed cardiac functions through the NOS/cyclic guanosine monophosphate mechanism. The role of endogenous NO was examined after local application of NG-nitro-L-arginine methyl ester (L-NAME). We found that L-NAME suppressed endogenous NO levels in NTS and elicited hypertension and tachycardia. Taken together, our data suggest that NO is tonically released in the NTS to inhibit blood pressure. © 2002 Elsevier Science Inc.
Our data indicate that NO is tonically released in the NTS and that increasing NO level in NTS results in hypotension. MATERIALS AND METHODS Animals Adult cats of either sex, weighing 2.5–3.7 kg, were anesthetized intraperitoneally with urethane (400 mg/kg) and ␣-chloralose (40 mg/kg). The procedures of experimentation have been described previously [29]. These include cannulation of the right femoral artery for monitoring SAP, mean SAP (MSAP), and heart rate (HR), cannulation of the right femoral vein for drug injections, and tracheal intubation for artificial ventilation to maintain the end-tide CO2 concentration at 4%. The body temperature was maintained at 37.5°C. A subgroup of animals received vagotomy. Bilateral cervical vagal and carotid sinus nerves were cut in these animals. Drug Administration
KEY WORDS: Nucleus tractus solitarius, Nitric oxide, L-Arginine, L-NAME, Methylene blue, ODQ, Hemoglobin.
The head of each animal was fixed in a David-Kopf stereotaxic apparatus. The dorsal surface of the brain stem was exposed and the obex was used as the reference point. A three barrel-glass micropipette (outside tip diameter of 30 – 40 m) was glued together with a NO electrode. The distance between the tips of pipette and electrode was 150 –250 m. This electrode/pipette, inclined at 34° from the stereotaxic frame, was inserted to the NTS (0.0 –2.0 mm anterior to the obex, 1.0 –2.0 mm lateral to the midline and 0.5–1.5 mm ventral to the dorsal surface of the medulla). Each barrel containing different a chemical was connected to separate pneumatic pressure pump (PPS-2, PPM-2, Medical Systems Corp., Great Neck, NY, USA) for microinjection. All drugs, except 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), were dissolved in artificial cerebrospinal fluid (aCSF). ODQ was first dissolved in dimethyl sulfoxide (1% DMSO) and then diluted in aCSF. The injection volume was measured by monitoring the movement of the fluid meniscus in the micropipette through a stereomicroscope. At the end of each experiment, animals were euthanized by an overdose of pentobarbital. Brains were removed and immersed in 10% formalin saline for 8 h. After fixation, frozen transverse sections (50 m) were stained with cresyl violet to identify the electrode tracks.
INTRODUCTION Nitric oxide (NO) is an important neurotransmitter in central cardiovascular regulation [3,24,26]. Local application of L-arginine (L-Arg), a NO donor, to the nucleus tractus solitarius (NTS) induced hypotension [25]. Administration of NO synthase (NOS) inhibitors; NG-nitro-L-arginine methyl ester (L-NAME) or NGmonomethyl-L-arginine, produced hypertension [12,27,30] and attenuated the excitatory amino acid -induced hypotension in NTS [15,22]. These data suggest that systemic blood pressure is regulated by the activity of NOS in NTS. Because these studies did not measure the NO level in the NTS, the roles of NO in regulating systemic arterial pressure (SAP) in NTS is still not clearly identified. We, and others, have previously reported that extracellular NO levels can be monitored real-time in rats through Porphryinecoated NO sensors [14,28]. The selectivity to NO is greatly enhanced by Nafion and o-Phenylenediamine [9]. In the present study, we measured NO levels in NTS using these NO sensors.
* Address for correspondence: Dr. Chok-Yung Chai, Institute of Biomedical Science, Academia Sinica, Taipei 11529, Taiwan. Fax: ⫹886-2-2782-9224; E-mail: [email protected]
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NO Measurement Voltammetric measurements of extracellular NO concentration in vivo were performed with microcomputer-controlled apparatus (IVEC-10, Medical systems Co., Greenvale, NY, USA) as described previously [14,28]. Remote from the NTS, a miniature Ag/AgCl reference electrode was inserted into the brain and cemented in place with dental acrylic. The working electrode, a double carbon fiber filaments (each 30 m in diameter; Textron, Lowell, MA, USA), was placed into the NTS. The sensor was first coated with Nafion (5% solution; Aldrich Chemical Co., Milwaukee, WI, USA) at 65°C to decrease any interference from extracellular ascorbic acid (AA) [11]. The electrodes were then electropolymerized with 2 mM Ni-meso-tetra (N-methyl-4-pyridyl) porphyrine-tetratosylate in 0.1 M NaOH at ⫹0.9 V for 25–50 min [16] and then with 5 mM o-Phenylenediamine solution in 0.1 M phosphate buffer solution (PBS, pH 7.4). Each electrode was tested for selectivity and sensitivity to NO in vitro. Calibration of NO was made using 1.0 to 3.0 M S-nitroso-N-acetyl-DL-penicillamine (SNAP) in 0.1 mM PBS. One M of SNAP can generate 1 nM NO in vitro [7]. Only electrodes showing selectivity for NO, compared with AA greater than 100,000:1 in vitro were used in the in vivo recordings. Data Analysis All values were presented as mean ⫾ SEM. Student’s t-test was used to determine the effect of drugs induced into the NTS or intravenously on cardiovascular responses and changes in NO levels. A one-way analysis of variance was used to evaluate the significance (p ⬍ 0.05) of the mean differences between groups of different animals. RESULTS Selectivity, Sensitivity, and Reproducibility of NO Sensors In Vitro and In Vivo We found that our NO electrodes were sensitive to SNAP (2 M), but not to other chemicals, i.e., norepinephrine (2 M), dopamine (2 M), serotonin (2 M), tyrosine (2 M), nitrite (2 M), glycine (2 M), glutamate (2 M), L-NAME (185 M), L-Arg (287 M), methylene blue (MB; 89 M), ODQ (116 M), hemoglobin (Hb; 15 nM), and AA (2 M) in vitro. Moreover, microinjection of AA (100 M ⫻ 100 nl, n ⫽ 5) or 1% DMSO (100 nl, n ⫽ 5) to the NTS did not induce changes in NO level. The sensitivity of NO sensors was examined in vitro before and after 3-h in vivo recording; NO signal generated by exogenous application of SNAP in vitro was not reduced (85 ⫾ 14% of control, n ⫽ 6). The reproducibility of NO sensor was also examined in vivo. Thirty minutes after the first injection of L-Arg in NTS, the ⌬MSAP and peak NO signals induced by the second injection of same dose of L-Arg were not altered (87 ⫾ 5% and 92 ⫾ 6% of the control, n ⫽ 7). These data suggest that our NO sensors have high selectivity and sensitivity to NO. L-Arg and L-NAME Interactions A total of 45 NTS sites recorded from 27 cats were used to study NO release in vivo (Fig. 1). Drug-evoked NO release (⌬NO) was measured by comparing the peak NO value before and after each injection. Microinjection of L-Arg (9 –10, 25–30, 45–50 nmol in 30 –175 nl) produced dose-dependent decreases in ⌬MSAP (12.3 ⫾ 4.5, 24.5 ⫾ 5.4, 33.7 ⫾ 6.5 mmHg; p ⬍0.05, n ⫽ 5) and ⌬HR (23.2 ⫾ 4.6, 42.3 ⫾ 5.2, 49.6 ⫾ 5.4 beats per minute [bpm]; p ⬍0.05, n ⫽ 5) and increases in ⌬NO (1.1 ⫾ 0.3, 2.1 ⫾ 0.4, 3.0 ⫾ 0.4 nM; p ⬍0.05, n ⫽ 5), respectively (Fig. 2). Microinjection of
FIG. 1. The reactive sites of nucleus tractus solitarius (NTS) showing the changes of systemic arterial pressure, heart rate, and nitric oxide level following microinjection of L-arginine (L-Arg; 25–30 nmol) and of NGnitro-L-arginine methyl ester (L-NAME; 25–30 nmol). Dot (●) in the brain drawing shows the point of stimulation. The drawing slices are coronal sections of medulla, 1 mm and 2 mm anterior to the obex. Scale bar: 2 mm. Abbreviations: AN, nucleus of ambiguus; CX, external cuneate nucleus; ION, inferior olivary nucleus; NTS, nucleus tractus solitarius; P, pyramidal tract; VIN, inferior vestibular nucleus; 5sp, spinal trigeminal nucleus; 5st, spinal trigeminal tract; 12N, hypoglossal nucleus; 12n, hypoglossal nerve.
L-NAME (9 –10, 25–30, 45–50 nmol in 50 –270 nl), on the other hand, increased ⌬MSAP (13.3 ⫾ 4.8, 31.4 ⫾ 6.4, 39.3 ⫾ 7.5 mmHg; p ⬍0.05, n ⫽ 5) and ⌬HR (11.3 ⫾ 3.8, 15.2 ⫾ 4.3, 21.6 ⫾ 5.4 bpm; p ⬍0.05, n ⫽ 5) and decreases in ⌬NO levels (0.3 ⫾ 0.2, 0.9 ⫾ 0.3, 1.5 ⫾ 0.5 nM; p ⬍0.05, n ⫽ 5, Fig. 3). Based on the dose response relationship, we selected doses of 25–30 nmol as the microinjected dose of L-Arg and L-NAME in the following study. The interaction between L-Arg and L-NAME was further studied in nine cats. Pretreatment with L-NAME (20 mg/kg, intravenous [i.v.]) significantly antagonized the L-Arg-induced electrochemical (⌬NO levels, 2.0 ⫾ 0.4 nM vs. 0.8 ⫾ 0.3 nM; p ⬍0.05, n ⫽ 5, Fig. 4) and hemodynamic responses (⌬MSAP: ⫺23.2 ⫾ 8.5 vs. ⫺12.4 ⫾ 5.6 mmHg; ⌬HR: ⫺26.4 ⫾ 6.5 vs. ⫺15.8 ⫾ 7.6 bpm; p ⬍0.05) in NTS. In another 4 cats, pretreatment with L-Arg (20 mg/kg, i.v.) reduced the responses evoked by L-NAME in NTS (⌬MSAP: 38.3 ⫾ 7.5 to 16.4 ⫾ 5.8 mmHg; ⌬HR: 28.2 ⫾ 5.9 to 10.9 ⫾ 4.3 bpm; ⌬NO: ⫺0.8 ⫾ 0.3 to ⫺0.4 ⫾ 0.2 nM; p ⬍0.05, Fig. 5). Effects of Baroreceptors To further examine if the changes in ⌬NO in NTS were affected by the activation of baroreceptor reflex, L-Arg and LNAME were injected systemically to nine vagotomized and nine control cats. No significant difference in SAP and HR was found between control and vagotomized animals before injection. In five control animals studied, intravenous administration of L-Arg (20 mg/kg) induced an increase in ⌬NO (0.3 ⫾ 0.1 nM) in NTS, a
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FIG. 2. An example of dose-dependent changes in cardiovascular responses and nitric oxide (NO) release elicited by microinjection of L-arginine (L-Arg) at the same site in nucleus tractus solitarius. Microinjection of 10 (A), 30 (B), and 50 nmole (C) L-Arg elicited decreases in mean systemic arterial pressure (MSAP; 15, 25, 35 mmHg) and heart rate (HR; 30, 45, 50 bpm) associated with increase in NO (1.2, 2.0, 2.8 nM). Please note that the decreases in MSAP and HR, and increase in NO were proportional to the dose of L-Arg. Arrowhead (1) indicates the time of microinjection of chemicals. Bar (—) indicates the time scale of SAP, MSAP, and HR tracings. Please note that the time scale of NO is two times of that of cardiovascular responses (SAP, MSAP, HR).
21.9% decrease in MSAP (from 129.6 ⫾ 16.7 to 101.2 ⫾ 12.5 mmHg; p ⬍0.05), and a 18.3% decrease in HR (from 194.5 ⫾ 25.8 to 159 ⫾ 19.6 bpm; p ⬍0.05, Fig. 6A). L-NAME (20 mg/kg, i.v.) produced a 35.5% increases in MSAP (p ⬍0.05), a 9.1% increase in HR (p ⬍0.05), and a decrease in ⌬NO (⫺0.2 ⫾ 0.1 nM, Fig. 6B) in another four control animals. These L-Arg and L-NAMEinduced responses were all potentiated by vagotomy. In five va-
gotomized cats, L-Arg (20 mg/kg) increased ⌬NO (0.6 ⫾ 0.2 nM) and reduced blood pressure from 138.5 ⫾ 19.7 to 94.9 ⫾ 15.7 mmHg (31.5%, p ⬍0.05). A much greater increase in MSAP (53.8%, p ⬍0.05) and decrease in ⌬NO (⫺0.4 ⫾ 0.1 nM), induced by L-NAME, were also noted in four vagotomized cats. These data suggest that NOS activity in NTS and NOS-mediated depressor responses can be modulated by baroreceptor reflex.
FIG. 3. An example of dose-related changes in cardiovascular response and nitric oxide (NO) concentration elicited by of NG-nitro-L-arginine methyl ester (L-NAME) at the same site in nucleus tractus solitarius. Microinjection of 10 (A), 30 (B), and 50 nmol (C) L-NAME elicited a dose-dependent increases in mean systemic arterial pressure (MSAP) (13, 35, 45 mmHg) and heart rate (HR) (10, 12, 15 bpm) associated with decreases in NO (0.4, 0.9, 1.4 nM). Please note that the increases in MSAP and HR, and decrease in NO were proportional to the dose of L-NAME.
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FIG. 4. Prior intravenous administration of NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg) attenuated the responses of systemic arterial pressure (SAP) and nitric oxide (NO) evoked with L-arginine (L-Arg) in the same site of nucleus tractus solitarius. (A) Microinjection of L-Arg (30 nmol) produced decrease in mean SAP (MSAP; 25 mmHg) and increase in NO (1.8 nM). (B) Twenty minutes after the infusion of L-NAME, microinjection of L-Arg produced a smaller decrease in MSAP (12 mmHg) and increase in NO (0.9 nM).
Effects of MB, ODQ, and Hb Pretreatment with MB (4 –5 nmol in 50 nl) antagonized responses evoked by L-Arg in NTS (⌬MSAP: from ⫺27.4 ⫾ 8.5 to ⫺13.4 ⫾ 7.3 mmHg; ⌬HR: from ⫺28.5 ⫾ 6.8 to ⫺12.8 ⫾ 4.8
bpm; ⌬NO: from 1.8 ⫾ 0.4 to 0.7 ⫾ 0.2 nM; p ⬍0.05, n ⫽ 9, Fig. 7) in five cats. Similarly, microinjection of ODQ (5– 6 nmol in 50 nl) decreased responses evoked by L-Arg in NTS (⌬MSAP: from ⫺24.7 ⫾ 6.8 to ⫺13.6 ⫾ 3.5 mmHg; ⌬HR: from ⫺30.4 ⫾ 7.4 to
FIG. 5. Prior intravenous administration of L-arginine (L-Arg; 20 mg/kg) attenuated the responses of systemic arterial pressure (SAP) and nitric oxide (NO) evoked with of NG-nitro-L-arginine methyl ester (L-NAME) in the same site of nucleus tractus solitarius. (A) Microinjection of L-NAME (30 nmol) produced increase in mean (MSAP; 43 mmHg), heart rate (HR; 25 bpm), and decrease in NO (0.8 nM). (B) Twenty minutes after the infusion of L-Arg, microinjection of L-NAME produced smaller increase in MSAP (18 mmHg), HR (7 bpm), and decrease in NO (0.5 nM).
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FIG. 6. Effects of intravenous infusion of L-arginine (L-Arg; 20 mg/kg) and of NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg) on the cardiovascular responses and nitric oxide (NO) concentration. (A) L-Arg induced decreases in mean systemic arterial pressure (MSAP; 40 mmHg), heart rate (HR; 50 bpm) and increase in NO (0.3 nM). (B) L-NAME -induced increase in MSAP (65 mmHg), HR (10 bpm), and decrease in NO concentration (0.2 nM). Please note that the effects of L-Arg were opposite to the effects of L-NAME.
⫺16.1 ⫾ 4.3 bpm; ⌬NO from 1.9 ⫾ 0.4 to 0.5 ⫾ 0.2 nM; p ⬍0.05, n ⫽ 7) in four animals. In another six cats studied, pretreatment with Hb (0.7– 0.8 pmol in 50 nl) decreased responses evoked by L-Arg in NTS (⌬MSAP: from ⫺21.7 ⫾ 9.3 to ⫺10.2 ⫾ 4.4 mmHg; ⌬HR: from ⫺32.6 ⫾ 8.4 to ⫺19.3 ⫾ 5.2 bpm; ⌬NO: from 1.7 ⫾ 0.3 to 0.6 ⫾ 0.2 nM; p ⬍0.05, n ⫽ 8, Fig. 8). These data suggested that the cardiovascular responses and NO release produced by L-Arg in NTS were significantly attenuated by prior microinjection of MB, ODQ, or Hb.
DISCUSSION In the present study, we used voltammetry and selective NO microsensors to measure extracellular NO levels in vivo. We found that local application of L-Arg induced NO release in NTS, hypotension, and bradycardia. Previous studies have indicated that NOS co-localized with N-methyl-D-aspartate (NMDA) receptors in NTS [2,8,20,23]. Exogenously applied L-Arg can be metabolized to NO by NOS in NTS. NO then excites adjacent sympatho-
FIG. 7. Methylene blue (MB, 5 nmol) blocked the cardiovascular responses and nitric oxide (NO) level evoked by L-arginine (L-Arg) at the same site in nucleus tractus solitarius. (A) Microinjection of L-Arg (30 nmol) produced decrease in mean systemic arterial pressure (MSAP; 40 mmHg), heart rate (HR; 35 bpm), and increase in NO (1.8 nM). (B) Microinjection of MB produced no change in SAP and small decrease in NO (0.2 nM). (C) Three minutes after MB, microinjection of L-Arg produced smaller decrease in MSAP (22 mmHg), HR (10 bpm), and a smaller increase in NO (0.6 nM).
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FIG. 8. Hemoglobin (Hb, 0.8 pmol) reduced the cardiovascular responses and nitric oxide (NO) level evoked by L-arginine (L-Arg) at the same site in nucleus tractus solitarius. (A) Microinjection of L-Arg (30 nmol) produced decreases in mean systemic arterial pressure (MSAP; 27 mmHg) and heart rate (HR; 30 bpm), and increase in NO (1.6 nM). (B) Microinjection of Hb produced no change in SAP and small decrease in NO (0.2 nM). (C) Three minutes after Hb, microinjection of L-Arg produced smaller decrease in MSAP (15 mmHg), HR (8 bpm), and a much lower increase in NO (0.6 nM).
inhibitory neurons and suppresses cardiac functions in NTS. Results of the present study are consistent with the hypothesis that NO, like NMDA agonist, induces hypotension in NTS [15,27]. Microinjection of L-NAME into the NTS elicited a dosedependent hypertension and decrease in NO levels, suggesting that NOS is tonically activated in NTS. The effects of L-NAME on cardiovascular responses and NO levels are opposite to that of L-Arg. This is compatible with the observations that direct administration of L-NAME into the brain induced a decrease in the concentration of extracellular L-citrulline [21]. Our data indicate that local application of L-Arg increased NO release in NTS and produced hypotension. However, durations of these two responses are different. Such a discrepancy may be due to the direct vs. indirect reactions of NO. Nitric oxide induces accumulation of the second messenger cyclic guanosine monophosphate (cGMP) and then activates presynaptic neuron to release glutamate, which interacts with NMDA receptors and induces hypotension. It is possible that NO may affect sympathetic output through these relatively long-lasting cascade reactions after it has been synthesized. These hypothesis is further supported by the fact that local injection of NO donor SNAP induce relatively long-term glutamate release in NTS [18]. We found that systemically applied L-NAME significantly antagonized the L-Arg-induced release of NO and hypotension in NTS. Our preliminary study indicates that repeat administration of L-Arg to NTS, given 20 min after the initial injection, did not attenuate its pressor response and NO release, suggesting that the decrease of L-Arg-induced pressor and electrochemical responses is not due to tachyphylaxis in NTS. It has been reported that NO is an important neurotransmitter for baroreceptor reflexes [5,13]. An elevated SAP activates the afferent vagal neurons to release glutamate, which, subsequently, stimulates the transformation of L-Arg to NO in NTS [10], and decreases sympathetic outflow [6]. We found that the differences in SAP and NO levels in NTS after systemic infusions of L-Arg and L-NAME were all potentiated by vagotomy, suggesting that there is less baroreceptor control to normalize SAP in vagotomized cats. In this study we found that microinjection of a low dose of L-Arg (25 nmol) into NTS decreased SAP and increased NO
production (2.1 nM). Systemic administration of L-Arg (20 mg/kg) induced similar reductions in SAP, however, much less NO release in NTS (0.3 nM). Similar results were found after central or peripheral administration of L-NAME. Such discrepancies may be due to different sites of reaction. Systemically applied L-Arg or L-NAME altered SAP through direct interaction of NOS in peripheral cardiovascular system [1,4] and indirectly through the production of NO in central nervous system after the redistribution. On the other hand, centrally applied L-Arg generated NO in NTS. Very low level of L-Arg will be redistributed to systemic circulation and interact with vessels. Previous studies have indicated that L-Arg produced central cardiovascular effects through pathways other than NOS [17]. Intracerebroventricular injection of L-Arg transiently increased SAP through renin-angiotensin pathway [19]. We found that NO scavenger Hb attenuated L-Arg-induced NO formation and hypotension. Because Hb did not bind to L-Arg, the hypotensive reaction induced by L-Arg is, thus, mainly derived from NO. We found that NOS/guanylate cyclase inhibitor MB and ODQ also antagonized L-Arg-mediated responses. These data suggest that L-Arg increases NO production in NTS, which activates NO/ cGMP transduction and induces hypotension. In conclusion, our data indicate that the electrochemical signal of NO can be recorded in real time after exogenous application of L-Arg or L-NAME in NTS. Alternation of NO levels in NTS changes blood pressure and modulates baroreceptor reflex function. ACKNOWLEDGEMENTS
The authors thank Dr. K. H. Lee, Kenan Professor of Medicinal Chemistry of University of North Carolina (USA) for comments on this paper. This study was support in parts by the Foundation of Biomedical Sciences, Shih-Chun Wang Memorial Fund and the National Science Council, ROC, No. NSC 89-2320-B-001-023.
REFERENCES 1. Aisaka, S.; Gross, S. S.; Griffith, O. W.; Levi, R. NG-methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent
NO IN NTS
2. 3. 4.
5.
6. 7.
8. 9. 10. 11. 12.
13.
14. 15. 16. 17.
pressor agent in the guinea pig: Does nitric oxide regulate blood pressure in vivo? Biochem. Biophys. Res. Commun. 160:881– 886; 1989. Broussard, D. L.; Bao, X.; Li, X.; Altschuler, S. M. Co-localization of NOS and NMDA receptor in esophageal premotor neurons of the rat. Neuroreport 6:2073–2076; 1995. Calver, A.; Collier, J.; Vallance, P. Nitric oxide and cardiovascular control. Exp. Physiol. 78:303–326; 1993. Cernadas, M. R.; Lopez-Farre, A.; Riesco, A.; Gallego, M. J.; Espinosa, G.; Digiuni, E.; Hernando, L.; Casado, S.; Caramelo, C. Renal and systemic effects of aminoacids administered separately: Comparison between L-arginine and non-nitric oxide donor aminoacids. J. Pharmacol. Exp. Ther. 263:1023–1029; 1992. Chan, R. K. W.; Sawchenko, P. E. Organization and transmitter specificity of medullary neurons activated by sustained hypertension: Implications for understanding baroreceptor reflex circuitry. J. Neuroscience 18:371–387; 1998. Dampney, R. A. L. Functional organization of central pathways regulating the cardiovascular system. Physiol. Rev. 74:323–364; 1994. Feelisch, M. The biochemical pathways of nitric oxide formation from nitrovasodilators: Appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions. J. Cardiovasc. Pharmacol. 17:S25–S33; 1991. Forster, E. R.; Southam, E. The intrinsic and vagal extrinsic innervation of the rat stomach contains nitric oxide synthase. Neuroreport 4:275–278; 1993. Friedemann, M. N.; Robinson, S. W.; Gerhardt, G. A. o-Phenylenediamine-modified carbon fiber electrodes for the detection of nitric oxide. Anal. Chem. 68:2621–2628; 1996. Garthwaite, J.; Garthwaite, G.; Palmer, R. M. J.; Moncada, S. NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur. J. Pharmacol. 172:413– 416; 1989. Gerhardt, G. A.; Oke, A. F.; Nagy, G.; Moghaddam, B.; Adams, R. N. Nafion-coated electrodes with high selectivity for CNS electrochemistry. Brain Res. 290:390 –395; 1984. Harada, S.; Tokunaga, S.; Momohara, M.; Masaki, H.; Tagawa, T.; Imaizumi, T.; Takeshita, A. Inhibition of nitric oxide formation in the nucleus tractus solitarius increases renal sympathetic nerve activity in rabbits. Circ. Res. 72:511–516; 1993. Hironaga, K.; Hirooka, Y.; Matsuo, I.; Shihara, M.; Tagawa, T.; Harasawa, Y.; Takeshita, A. Role of endogenous nitric oxide in the brain stem on the rapid adaptation of baroreflex. Hypertension 31:27– 31; 1998. Lin, S. Z.; Chiou, A. L.; Wang, Y. Ketamine antagonizes nitric oxide release from cerebral cortex after middle cerebral artery ligation in rats. Stroke 27:747–752; 1996. Lo, W. C.; Lin, H. C.; Ger, L. P.; Tung, C. S.; Tseng, C. J. Cardiovascular effects of nitric oxide and N-methyl-D-aspartate receptors in the nucleus tractus solitarii of rats. Hypertension 30:1499 –1503; 1997. Malinski, T.; Taha, Z. Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor. Nature 358:676 – 678; 1992. Matsumura, K.; Tsuchihashi, T.; Kagiyama, S.; Abe, I.; Fujishima, M.
177
18.
19.
20.
21. 22.
23.
24.
25.
26.
27. 28. 29.
30.
Role of nitric oxide in the nucleus of the solitary tract of rats. Brain Res. 798:232–238; 1998. Matsuo, I.; Hirooka, Y.; Hironaga, K.; Eshima, K.; Shigematsu, H.; Shihara, M.; Sakai, K.; Takeshita, A. Glutamate release via NO production evoked by NMDA in the NTS enhances hypotension and bradycardia in vivo. Am. J. Physiol. 280:R1285–R1291; 2001. Nishimura, M.; Takahashi, H.; Nanbu, A.; Sakamoto, M.; Yoshimura, M. Cardiovascular regulation by L-arginine in the brain of rats. Role of the brain renin-angiotensin system and nitric oxide. Am. J. Hypertension 10:389 –396; 1997. Ohta, A.; Takagi, H.; Matsui, T.; Hamai, Y.; Iida, S.; Esumi, H. Localization of nitric oxide synthase-immunoreactive neurons in the solitary nucleus and ventrolateral medulla oblongata of the rat: Their relation to catecholaminergic neurons. Neurosci. Lett. 158:33–35; 1993. Ohta, K.; Shimazu, K.; Komatsumoto, S.; Araki, N.; Shibata, M.; Fukuuchi, Y. Modification of striatal arginine and citrulline metabolism by nitric oxide synthase inhibitors. Neuroreport 5:766 –768; 1994. Paola, E. D. D.; Vidal, M. J.; Nistico, G. L-Glutamate evokes the release of an endothelium-derived relaxing factor-like substance from the rat nucleus tractus solitarius. J. Cardiovasc. Pharmacol. 17:S269 – S272; 1991. Ruggiero, D. A.; Mtui, E. P.; Otake, K.; Anwar, M. Central and primary visceral afferents to nucleus tractus solitarii may generate nitric oxide as a membrane-permeant neuronal messenger. J. Comp. Neurol. 364:51– 67; 1996. Sakuma, I.; Togashi, H.; Yoshioka, M.; Saito, H.; Yanagida, M.; Tamura, M.; Kobayashi, T.; Yasuda, H.; Gross, S. S.; Levi, R. NGmethyl-L-arginine, an inhibitor of L-arginine-derived nitric oxide synthesis, stimulates renal sympathetic nerve activity in vivo: A role for nitric oxide in the central regulation of sympathetic tone? Circ. Res. 70:607– 611; 1992. Tagawa, T.; Imaizumi, T.; Harada, S.; Endo, T.; Shiramoto, M.; Hirooka, Y.; Takeshita, A. Nitric oxide influences neuronal activity in the nucleus tractus solitarius of rat brainstem slices. Circ. Res. 75:70 – 76; 1994. Togashi, H.; Sakuma, I.; Yoshioka, M.; Kobayashi, T.; Yasuda, H.; Kitabake, A.; Saito, H.; Gross, S. S.; Levi, T. A central nervous system action of nitric oxide in blood pressure regulation. J. Pharmacol. Exp. Ther. 262:343–347; 1992. Tseng, C. J.; Liu, H. Y.; Lin, H. C.; Ger, L. P.; Tung, C. S.; Yen, M. H. Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 27:36 – 42; 1996. Wang, Y.; Lin, S. Z.; Chiou, A. L.; Williams, L. R.; Hoffer, B. J. Glial cell line-derived neurotrophic factor protects against ischemia-induced injury in the cerebral cortex. J. Neurosci. 17:4341– 4348; 1997. Wu, W. C.; Kuo, J. S.; Wang, Y.; Chai, C. Y. Glycine increases arterial pressure and augments NMDA-induced pressor responses in the dorsomedial and ventrolateral medulla of cats. J. Auton. Nerv. Syst. 67:145–155; 1997. Zanzinger, J.; Czachurski, J.; Seller, H. Inhibition of sympathetic vasoconstriction is a major principle of vasodilation by nitric oxide in vivo. Circ. Res. 75:1073–1077; 1994.
PII S0361-9230(01)00737-7
Nitric oxide reduces blood pressure in the nucleus tractus solitarius: A real time electrochemical study W. C. Wu,1 Y. Wang,2 L. S. Kao,3 F. I. Tang3 and C. Y. Chai1* 1
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 2National Institute on Drug Abuse, Baltimore, MD, USA; and 3National Yang-Ming University, Taipei, Taiwan [Received 2 May 2001; Accepted 26 September 2001]
ABSTRACT: Increasing evidence has demonstrated that nitric oxide (NO) is involved in central cardiovascular regulation. In this study, we directly measured extracellular NO levels, in real-time, in the nucleus tractus solitarius (NTS) of anesthetized cats using Nafion/Porphyrine/o-Phenylenediamine-coated NO sensors. We found that local application of L-arginine (L-Arg) induced NO overflow in NTS and hypotension. These responses were potentiated in the vagotomized animals. Pretreatment with NO synthase (NOS)/guanylate cyclase inhibitor methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or NO scavenger hemoglobin attenuated L-Arg-induced hypotension, suggesting that exogenous supplement of NO suppressed cardiac functions through the NOS/cyclic guanosine monophosphate mechanism. The role of endogenous NO was examined after local application of NG-nitro-L-arginine methyl ester (L-NAME). We found that L-NAME suppressed endogenous NO levels in NTS and elicited hypertension and tachycardia. Taken together, our data suggest that NO is tonically released in the NTS to inhibit blood pressure. © 2002 Elsevier Science Inc.
Our data indicate that NO is tonically released in the NTS and that increasing NO level in NTS results in hypotension. MATERIALS AND METHODS Animals Adult cats of either sex, weighing 2.5–3.7 kg, were anesthetized intraperitoneally with urethane (400 mg/kg) and ␣-chloralose (40 mg/kg). The procedures of experimentation have been described previously [29]. These include cannulation of the right femoral artery for monitoring SAP, mean SAP (MSAP), and heart rate (HR), cannulation of the right femoral vein for drug injections, and tracheal intubation for artificial ventilation to maintain the end-tide CO2 concentration at 4%. The body temperature was maintained at 37.5°C. A subgroup of animals received vagotomy. Bilateral cervical vagal and carotid sinus nerves were cut in these animals. Drug Administration
KEY WORDS: Nucleus tractus solitarius, Nitric oxide, L-Arginine, L-NAME, Methylene blue, ODQ, Hemoglobin.
The head of each animal was fixed in a David-Kopf stereotaxic apparatus. The dorsal surface of the brain stem was exposed and the obex was used as the reference point. A three barrel-glass micropipette (outside tip diameter of 30 – 40 m) was glued together with a NO electrode. The distance between the tips of pipette and electrode was 150 –250 m. This electrode/pipette, inclined at 34° from the stereotaxic frame, was inserted to the NTS (0.0 –2.0 mm anterior to the obex, 1.0 –2.0 mm lateral to the midline and 0.5–1.5 mm ventral to the dorsal surface of the medulla). Each barrel containing different a chemical was connected to separate pneumatic pressure pump (PPS-2, PPM-2, Medical Systems Corp., Great Neck, NY, USA) for microinjection. All drugs, except 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), were dissolved in artificial cerebrospinal fluid (aCSF). ODQ was first dissolved in dimethyl sulfoxide (1% DMSO) and then diluted in aCSF. The injection volume was measured by monitoring the movement of the fluid meniscus in the micropipette through a stereomicroscope. At the end of each experiment, animals were euthanized by an overdose of pentobarbital. Brains were removed and immersed in 10% formalin saline for 8 h. After fixation, frozen transverse sections (50 m) were stained with cresyl violet to identify the electrode tracks.
INTRODUCTION Nitric oxide (NO) is an important neurotransmitter in central cardiovascular regulation [3,24,26]. Local application of L-arginine (L-Arg), a NO donor, to the nucleus tractus solitarius (NTS) induced hypotension [25]. Administration of NO synthase (NOS) inhibitors; NG-nitro-L-arginine methyl ester (L-NAME) or NGmonomethyl-L-arginine, produced hypertension [12,27,30] and attenuated the excitatory amino acid -induced hypotension in NTS [15,22]. These data suggest that systemic blood pressure is regulated by the activity of NOS in NTS. Because these studies did not measure the NO level in the NTS, the roles of NO in regulating systemic arterial pressure (SAP) in NTS is still not clearly identified. We, and others, have previously reported that extracellular NO levels can be monitored real-time in rats through Porphryinecoated NO sensors [14,28]. The selectivity to NO is greatly enhanced by Nafion and o-Phenylenediamine [9]. In the present study, we measured NO levels in NTS using these NO sensors.
* Address for correspondence: Dr. Chok-Yung Chai, Institute of Biomedical Science, Academia Sinica, Taipei 11529, Taiwan. Fax: ⫹886-2-2782-9224; E-mail: [email protected]
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NO Measurement Voltammetric measurements of extracellular NO concentration in vivo were performed with microcomputer-controlled apparatus (IVEC-10, Medical systems Co., Greenvale, NY, USA) as described previously [14,28]. Remote from the NTS, a miniature Ag/AgCl reference electrode was inserted into the brain and cemented in place with dental acrylic. The working electrode, a double carbon fiber filaments (each 30 m in diameter; Textron, Lowell, MA, USA), was placed into the NTS. The sensor was first coated with Nafion (5% solution; Aldrich Chemical Co., Milwaukee, WI, USA) at 65°C to decrease any interference from extracellular ascorbic acid (AA) [11]. The electrodes were then electropolymerized with 2 mM Ni-meso-tetra (N-methyl-4-pyridyl) porphyrine-tetratosylate in 0.1 M NaOH at ⫹0.9 V for 25–50 min [16] and then with 5 mM o-Phenylenediamine solution in 0.1 M phosphate buffer solution (PBS, pH 7.4). Each electrode was tested for selectivity and sensitivity to NO in vitro. Calibration of NO was made using 1.0 to 3.0 M S-nitroso-N-acetyl-DL-penicillamine (SNAP) in 0.1 mM PBS. One M of SNAP can generate 1 nM NO in vitro [7]. Only electrodes showing selectivity for NO, compared with AA greater than 100,000:1 in vitro were used in the in vivo recordings. Data Analysis All values were presented as mean ⫾ SEM. Student’s t-test was used to determine the effect of drugs induced into the NTS or intravenously on cardiovascular responses and changes in NO levels. A one-way analysis of variance was used to evaluate the significance (p ⬍ 0.05) of the mean differences between groups of different animals. RESULTS Selectivity, Sensitivity, and Reproducibility of NO Sensors In Vitro and In Vivo We found that our NO electrodes were sensitive to SNAP (2 M), but not to other chemicals, i.e., norepinephrine (2 M), dopamine (2 M), serotonin (2 M), tyrosine (2 M), nitrite (2 M), glycine (2 M), glutamate (2 M), L-NAME (185 M), L-Arg (287 M), methylene blue (MB; 89 M), ODQ (116 M), hemoglobin (Hb; 15 nM), and AA (2 M) in vitro. Moreover, microinjection of AA (100 M ⫻ 100 nl, n ⫽ 5) or 1% DMSO (100 nl, n ⫽ 5) to the NTS did not induce changes in NO level. The sensitivity of NO sensors was examined in vitro before and after 3-h in vivo recording; NO signal generated by exogenous application of SNAP in vitro was not reduced (85 ⫾ 14% of control, n ⫽ 6). The reproducibility of NO sensor was also examined in vivo. Thirty minutes after the first injection of L-Arg in NTS, the ⌬MSAP and peak NO signals induced by the second injection of same dose of L-Arg were not altered (87 ⫾ 5% and 92 ⫾ 6% of the control, n ⫽ 7). These data suggest that our NO sensors have high selectivity and sensitivity to NO. L-Arg and L-NAME Interactions A total of 45 NTS sites recorded from 27 cats were used to study NO release in vivo (Fig. 1). Drug-evoked NO release (⌬NO) was measured by comparing the peak NO value before and after each injection. Microinjection of L-Arg (9 –10, 25–30, 45–50 nmol in 30 –175 nl) produced dose-dependent decreases in ⌬MSAP (12.3 ⫾ 4.5, 24.5 ⫾ 5.4, 33.7 ⫾ 6.5 mmHg; p ⬍0.05, n ⫽ 5) and ⌬HR (23.2 ⫾ 4.6, 42.3 ⫾ 5.2, 49.6 ⫾ 5.4 beats per minute [bpm]; p ⬍0.05, n ⫽ 5) and increases in ⌬NO (1.1 ⫾ 0.3, 2.1 ⫾ 0.4, 3.0 ⫾ 0.4 nM; p ⬍0.05, n ⫽ 5), respectively (Fig. 2). Microinjection of
FIG. 1. The reactive sites of nucleus tractus solitarius (NTS) showing the changes of systemic arterial pressure, heart rate, and nitric oxide level following microinjection of L-arginine (L-Arg; 25–30 nmol) and of NGnitro-L-arginine methyl ester (L-NAME; 25–30 nmol). Dot (●) in the brain drawing shows the point of stimulation. The drawing slices are coronal sections of medulla, 1 mm and 2 mm anterior to the obex. Scale bar: 2 mm. Abbreviations: AN, nucleus of ambiguus; CX, external cuneate nucleus; ION, inferior olivary nucleus; NTS, nucleus tractus solitarius; P, pyramidal tract; VIN, inferior vestibular nucleus; 5sp, spinal trigeminal nucleus; 5st, spinal trigeminal tract; 12N, hypoglossal nucleus; 12n, hypoglossal nerve.
L-NAME (9 –10, 25–30, 45–50 nmol in 50 –270 nl), on the other hand, increased ⌬MSAP (13.3 ⫾ 4.8, 31.4 ⫾ 6.4, 39.3 ⫾ 7.5 mmHg; p ⬍0.05, n ⫽ 5) and ⌬HR (11.3 ⫾ 3.8, 15.2 ⫾ 4.3, 21.6 ⫾ 5.4 bpm; p ⬍0.05, n ⫽ 5) and decreases in ⌬NO levels (0.3 ⫾ 0.2, 0.9 ⫾ 0.3, 1.5 ⫾ 0.5 nM; p ⬍0.05, n ⫽ 5, Fig. 3). Based on the dose response relationship, we selected doses of 25–30 nmol as the microinjected dose of L-Arg and L-NAME in the following study. The interaction between L-Arg and L-NAME was further studied in nine cats. Pretreatment with L-NAME (20 mg/kg, intravenous [i.v.]) significantly antagonized the L-Arg-induced electrochemical (⌬NO levels, 2.0 ⫾ 0.4 nM vs. 0.8 ⫾ 0.3 nM; p ⬍0.05, n ⫽ 5, Fig. 4) and hemodynamic responses (⌬MSAP: ⫺23.2 ⫾ 8.5 vs. ⫺12.4 ⫾ 5.6 mmHg; ⌬HR: ⫺26.4 ⫾ 6.5 vs. ⫺15.8 ⫾ 7.6 bpm; p ⬍0.05) in NTS. In another 4 cats, pretreatment with L-Arg (20 mg/kg, i.v.) reduced the responses evoked by L-NAME in NTS (⌬MSAP: 38.3 ⫾ 7.5 to 16.4 ⫾ 5.8 mmHg; ⌬HR: 28.2 ⫾ 5.9 to 10.9 ⫾ 4.3 bpm; ⌬NO: ⫺0.8 ⫾ 0.3 to ⫺0.4 ⫾ 0.2 nM; p ⬍0.05, Fig. 5). Effects of Baroreceptors To further examine if the changes in ⌬NO in NTS were affected by the activation of baroreceptor reflex, L-Arg and LNAME were injected systemically to nine vagotomized and nine control cats. No significant difference in SAP and HR was found between control and vagotomized animals before injection. In five control animals studied, intravenous administration of L-Arg (20 mg/kg) induced an increase in ⌬NO (0.3 ⫾ 0.1 nM) in NTS, a
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FIG. 2. An example of dose-dependent changes in cardiovascular responses and nitric oxide (NO) release elicited by microinjection of L-arginine (L-Arg) at the same site in nucleus tractus solitarius. Microinjection of 10 (A), 30 (B), and 50 nmole (C) L-Arg elicited decreases in mean systemic arterial pressure (MSAP; 15, 25, 35 mmHg) and heart rate (HR; 30, 45, 50 bpm) associated with increase in NO (1.2, 2.0, 2.8 nM). Please note that the decreases in MSAP and HR, and increase in NO were proportional to the dose of L-Arg. Arrowhead (1) indicates the time of microinjection of chemicals. Bar (—) indicates the time scale of SAP, MSAP, and HR tracings. Please note that the time scale of NO is two times of that of cardiovascular responses (SAP, MSAP, HR).
21.9% decrease in MSAP (from 129.6 ⫾ 16.7 to 101.2 ⫾ 12.5 mmHg; p ⬍0.05), and a 18.3% decrease in HR (from 194.5 ⫾ 25.8 to 159 ⫾ 19.6 bpm; p ⬍0.05, Fig. 6A). L-NAME (20 mg/kg, i.v.) produced a 35.5% increases in MSAP (p ⬍0.05), a 9.1% increase in HR (p ⬍0.05), and a decrease in ⌬NO (⫺0.2 ⫾ 0.1 nM, Fig. 6B) in another four control animals. These L-Arg and L-NAMEinduced responses were all potentiated by vagotomy. In five va-
gotomized cats, L-Arg (20 mg/kg) increased ⌬NO (0.6 ⫾ 0.2 nM) and reduced blood pressure from 138.5 ⫾ 19.7 to 94.9 ⫾ 15.7 mmHg (31.5%, p ⬍0.05). A much greater increase in MSAP (53.8%, p ⬍0.05) and decrease in ⌬NO (⫺0.4 ⫾ 0.1 nM), induced by L-NAME, were also noted in four vagotomized cats. These data suggest that NOS activity in NTS and NOS-mediated depressor responses can be modulated by baroreceptor reflex.
FIG. 3. An example of dose-related changes in cardiovascular response and nitric oxide (NO) concentration elicited by of NG-nitro-L-arginine methyl ester (L-NAME) at the same site in nucleus tractus solitarius. Microinjection of 10 (A), 30 (B), and 50 nmol (C) L-NAME elicited a dose-dependent increases in mean systemic arterial pressure (MSAP) (13, 35, 45 mmHg) and heart rate (HR) (10, 12, 15 bpm) associated with decreases in NO (0.4, 0.9, 1.4 nM). Please note that the increases in MSAP and HR, and decrease in NO were proportional to the dose of L-NAME.
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FIG. 4. Prior intravenous administration of NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg) attenuated the responses of systemic arterial pressure (SAP) and nitric oxide (NO) evoked with L-arginine (L-Arg) in the same site of nucleus tractus solitarius. (A) Microinjection of L-Arg (30 nmol) produced decrease in mean SAP (MSAP; 25 mmHg) and increase in NO (1.8 nM). (B) Twenty minutes after the infusion of L-NAME, microinjection of L-Arg produced a smaller decrease in MSAP (12 mmHg) and increase in NO (0.9 nM).
Effects of MB, ODQ, and Hb Pretreatment with MB (4 –5 nmol in 50 nl) antagonized responses evoked by L-Arg in NTS (⌬MSAP: from ⫺27.4 ⫾ 8.5 to ⫺13.4 ⫾ 7.3 mmHg; ⌬HR: from ⫺28.5 ⫾ 6.8 to ⫺12.8 ⫾ 4.8
bpm; ⌬NO: from 1.8 ⫾ 0.4 to 0.7 ⫾ 0.2 nM; p ⬍0.05, n ⫽ 9, Fig. 7) in five cats. Similarly, microinjection of ODQ (5– 6 nmol in 50 nl) decreased responses evoked by L-Arg in NTS (⌬MSAP: from ⫺24.7 ⫾ 6.8 to ⫺13.6 ⫾ 3.5 mmHg; ⌬HR: from ⫺30.4 ⫾ 7.4 to
FIG. 5. Prior intravenous administration of L-arginine (L-Arg; 20 mg/kg) attenuated the responses of systemic arterial pressure (SAP) and nitric oxide (NO) evoked with of NG-nitro-L-arginine methyl ester (L-NAME) in the same site of nucleus tractus solitarius. (A) Microinjection of L-NAME (30 nmol) produced increase in mean (MSAP; 43 mmHg), heart rate (HR; 25 bpm), and decrease in NO (0.8 nM). (B) Twenty minutes after the infusion of L-Arg, microinjection of L-NAME produced smaller increase in MSAP (18 mmHg), HR (7 bpm), and decrease in NO (0.5 nM).
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FIG. 6. Effects of intravenous infusion of L-arginine (L-Arg; 20 mg/kg) and of NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg) on the cardiovascular responses and nitric oxide (NO) concentration. (A) L-Arg induced decreases in mean systemic arterial pressure (MSAP; 40 mmHg), heart rate (HR; 50 bpm) and increase in NO (0.3 nM). (B) L-NAME -induced increase in MSAP (65 mmHg), HR (10 bpm), and decrease in NO concentration (0.2 nM). Please note that the effects of L-Arg were opposite to the effects of L-NAME.
⫺16.1 ⫾ 4.3 bpm; ⌬NO from 1.9 ⫾ 0.4 to 0.5 ⫾ 0.2 nM; p ⬍0.05, n ⫽ 7) in four animals. In another six cats studied, pretreatment with Hb (0.7– 0.8 pmol in 50 nl) decreased responses evoked by L-Arg in NTS (⌬MSAP: from ⫺21.7 ⫾ 9.3 to ⫺10.2 ⫾ 4.4 mmHg; ⌬HR: from ⫺32.6 ⫾ 8.4 to ⫺19.3 ⫾ 5.2 bpm; ⌬NO: from 1.7 ⫾ 0.3 to 0.6 ⫾ 0.2 nM; p ⬍0.05, n ⫽ 8, Fig. 8). These data suggested that the cardiovascular responses and NO release produced by L-Arg in NTS were significantly attenuated by prior microinjection of MB, ODQ, or Hb.
DISCUSSION In the present study, we used voltammetry and selective NO microsensors to measure extracellular NO levels in vivo. We found that local application of L-Arg induced NO release in NTS, hypotension, and bradycardia. Previous studies have indicated that NOS co-localized with N-methyl-D-aspartate (NMDA) receptors in NTS [2,8,20,23]. Exogenously applied L-Arg can be metabolized to NO by NOS in NTS. NO then excites adjacent sympatho-
FIG. 7. Methylene blue (MB, 5 nmol) blocked the cardiovascular responses and nitric oxide (NO) level evoked by L-arginine (L-Arg) at the same site in nucleus tractus solitarius. (A) Microinjection of L-Arg (30 nmol) produced decrease in mean systemic arterial pressure (MSAP; 40 mmHg), heart rate (HR; 35 bpm), and increase in NO (1.8 nM). (B) Microinjection of MB produced no change in SAP and small decrease in NO (0.2 nM). (C) Three minutes after MB, microinjection of L-Arg produced smaller decrease in MSAP (22 mmHg), HR (10 bpm), and a smaller increase in NO (0.6 nM).
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FIG. 8. Hemoglobin (Hb, 0.8 pmol) reduced the cardiovascular responses and nitric oxide (NO) level evoked by L-arginine (L-Arg) at the same site in nucleus tractus solitarius. (A) Microinjection of L-Arg (30 nmol) produced decreases in mean systemic arterial pressure (MSAP; 27 mmHg) and heart rate (HR; 30 bpm), and increase in NO (1.6 nM). (B) Microinjection of Hb produced no change in SAP and small decrease in NO (0.2 nM). (C) Three minutes after Hb, microinjection of L-Arg produced smaller decrease in MSAP (15 mmHg), HR (8 bpm), and a much lower increase in NO (0.6 nM).
inhibitory neurons and suppresses cardiac functions in NTS. Results of the present study are consistent with the hypothesis that NO, like NMDA agonist, induces hypotension in NTS [15,27]. Microinjection of L-NAME into the NTS elicited a dosedependent hypertension and decrease in NO levels, suggesting that NOS is tonically activated in NTS. The effects of L-NAME on cardiovascular responses and NO levels are opposite to that of L-Arg. This is compatible with the observations that direct administration of L-NAME into the brain induced a decrease in the concentration of extracellular L-citrulline [21]. Our data indicate that local application of L-Arg increased NO release in NTS and produced hypotension. However, durations of these two responses are different. Such a discrepancy may be due to the direct vs. indirect reactions of NO. Nitric oxide induces accumulation of the second messenger cyclic guanosine monophosphate (cGMP) and then activates presynaptic neuron to release glutamate, which interacts with NMDA receptors and induces hypotension. It is possible that NO may affect sympathetic output through these relatively long-lasting cascade reactions after it has been synthesized. These hypothesis is further supported by the fact that local injection of NO donor SNAP induce relatively long-term glutamate release in NTS [18]. We found that systemically applied L-NAME significantly antagonized the L-Arg-induced release of NO and hypotension in NTS. Our preliminary study indicates that repeat administration of L-Arg to NTS, given 20 min after the initial injection, did not attenuate its pressor response and NO release, suggesting that the decrease of L-Arg-induced pressor and electrochemical responses is not due to tachyphylaxis in NTS. It has been reported that NO is an important neurotransmitter for baroreceptor reflexes [5,13]. An elevated SAP activates the afferent vagal neurons to release glutamate, which, subsequently, stimulates the transformation of L-Arg to NO in NTS [10], and decreases sympathetic outflow [6]. We found that the differences in SAP and NO levels in NTS after systemic infusions of L-Arg and L-NAME were all potentiated by vagotomy, suggesting that there is less baroreceptor control to normalize SAP in vagotomized cats. In this study we found that microinjection of a low dose of L-Arg (25 nmol) into NTS decreased SAP and increased NO
production (2.1 nM). Systemic administration of L-Arg (20 mg/kg) induced similar reductions in SAP, however, much less NO release in NTS (0.3 nM). Similar results were found after central or peripheral administration of L-NAME. Such discrepancies may be due to different sites of reaction. Systemically applied L-Arg or L-NAME altered SAP through direct interaction of NOS in peripheral cardiovascular system [1,4] and indirectly through the production of NO in central nervous system after the redistribution. On the other hand, centrally applied L-Arg generated NO in NTS. Very low level of L-Arg will be redistributed to systemic circulation and interact with vessels. Previous studies have indicated that L-Arg produced central cardiovascular effects through pathways other than NOS [17]. Intracerebroventricular injection of L-Arg transiently increased SAP through renin-angiotensin pathway [19]. We found that NO scavenger Hb attenuated L-Arg-induced NO formation and hypotension. Because Hb did not bind to L-Arg, the hypotensive reaction induced by L-Arg is, thus, mainly derived from NO. We found that NOS/guanylate cyclase inhibitor MB and ODQ also antagonized L-Arg-mediated responses. These data suggest that L-Arg increases NO production in NTS, which activates NO/ cGMP transduction and induces hypotension. In conclusion, our data indicate that the electrochemical signal of NO can be recorded in real time after exogenous application of L-Arg or L-NAME in NTS. Alternation of NO levels in NTS changes blood pressure and modulates baroreceptor reflex function. ACKNOWLEDGEMENTS
The authors thank Dr. K. H. Lee, Kenan Professor of Medicinal Chemistry of University of North Carolina (USA) for comments on this paper. This study was support in parts by the Foundation of Biomedical Sciences, Shih-Chun Wang Memorial Fund and the National Science Council, ROC, No. NSC 89-2320-B-001-023.
REFERENCES 1. Aisaka, S.; Gross, S. S.; Griffith, O. W.; Levi, R. NG-methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent
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2. 3. 4.
5.
6. 7.
8. 9. 10. 11. 12.
13.
14. 15. 16. 17.
pressor agent in the guinea pig: Does nitric oxide regulate blood pressure in vivo? Biochem. Biophys. Res. Commun. 160:881– 886; 1989. Broussard, D. L.; Bao, X.; Li, X.; Altschuler, S. M. Co-localization of NOS and NMDA receptor in esophageal premotor neurons of the rat. Neuroreport 6:2073–2076; 1995. Calver, A.; Collier, J.; Vallance, P. Nitric oxide and cardiovascular control. Exp. Physiol. 78:303–326; 1993. Cernadas, M. R.; Lopez-Farre, A.; Riesco, A.; Gallego, M. J.; Espinosa, G.; Digiuni, E.; Hernando, L.; Casado, S.; Caramelo, C. Renal and systemic effects of aminoacids administered separately: Comparison between L-arginine and non-nitric oxide donor aminoacids. J. Pharmacol. Exp. Ther. 263:1023–1029; 1992. Chan, R. K. W.; Sawchenko, P. E. Organization and transmitter specificity of medullary neurons activated by sustained hypertension: Implications for understanding baroreceptor reflex circuitry. J. Neuroscience 18:371–387; 1998. Dampney, R. A. L. Functional organization of central pathways regulating the cardiovascular system. Physiol. Rev. 74:323–364; 1994. Feelisch, M. The biochemical pathways of nitric oxide formation from nitrovasodilators: Appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions. J. Cardiovasc. Pharmacol. 17:S25–S33; 1991. Forster, E. R.; Southam, E. The intrinsic and vagal extrinsic innervation of the rat stomach contains nitric oxide synthase. Neuroreport 4:275–278; 1993. Friedemann, M. N.; Robinson, S. W.; Gerhardt, G. A. o-Phenylenediamine-modified carbon fiber electrodes for the detection of nitric oxide. Anal. Chem. 68:2621–2628; 1996. Garthwaite, J.; Garthwaite, G.; Palmer, R. M. J.; Moncada, S. NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur. J. Pharmacol. 172:413– 416; 1989. Gerhardt, G. A.; Oke, A. F.; Nagy, G.; Moghaddam, B.; Adams, R. N. Nafion-coated electrodes with high selectivity for CNS electrochemistry. Brain Res. 290:390 –395; 1984. Harada, S.; Tokunaga, S.; Momohara, M.; Masaki, H.; Tagawa, T.; Imaizumi, T.; Takeshita, A. Inhibition of nitric oxide formation in the nucleus tractus solitarius increases renal sympathetic nerve activity in rabbits. Circ. Res. 72:511–516; 1993. Hironaga, K.; Hirooka, Y.; Matsuo, I.; Shihara, M.; Tagawa, T.; Harasawa, Y.; Takeshita, A. Role of endogenous nitric oxide in the brain stem on the rapid adaptation of baroreflex. Hypertension 31:27– 31; 1998. Lin, S. Z.; Chiou, A. L.; Wang, Y. Ketamine antagonizes nitric oxide release from cerebral cortex after middle cerebral artery ligation in rats. Stroke 27:747–752; 1996. Lo, W. C.; Lin, H. C.; Ger, L. P.; Tung, C. S.; Tseng, C. J. Cardiovascular effects of nitric oxide and N-methyl-D-aspartate receptors in the nucleus tractus solitarii of rats. Hypertension 30:1499 –1503; 1997. Malinski, T.; Taha, Z. Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor. Nature 358:676 – 678; 1992. Matsumura, K.; Tsuchihashi, T.; Kagiyama, S.; Abe, I.; Fujishima, M.
177
18.
19.
20.
21. 22.
23.
24.
25.
26.
27. 28. 29.
30.
Role of nitric oxide in the nucleus of the solitary tract of rats. Brain Res. 798:232–238; 1998. Matsuo, I.; Hirooka, Y.; Hironaga, K.; Eshima, K.; Shigematsu, H.; Shihara, M.; Sakai, K.; Takeshita, A. Glutamate release via NO production evoked by NMDA in the NTS enhances hypotension and bradycardia in vivo. Am. J. Physiol. 280:R1285–R1291; 2001. Nishimura, M.; Takahashi, H.; Nanbu, A.; Sakamoto, M.; Yoshimura, M. Cardiovascular regulation by L-arginine in the brain of rats. Role of the brain renin-angiotensin system and nitric oxide. Am. J. Hypertension 10:389 –396; 1997. Ohta, A.; Takagi, H.; Matsui, T.; Hamai, Y.; Iida, S.; Esumi, H. Localization of nitric oxide synthase-immunoreactive neurons in the solitary nucleus and ventrolateral medulla oblongata of the rat: Their relation to catecholaminergic neurons. Neurosci. Lett. 158:33–35; 1993. Ohta, K.; Shimazu, K.; Komatsumoto, S.; Araki, N.; Shibata, M.; Fukuuchi, Y. Modification of striatal arginine and citrulline metabolism by nitric oxide synthase inhibitors. Neuroreport 5:766 –768; 1994. Paola, E. D. D.; Vidal, M. J.; Nistico, G. L-Glutamate evokes the release of an endothelium-derived relaxing factor-like substance from the rat nucleus tractus solitarius. J. Cardiovasc. Pharmacol. 17:S269 – S272; 1991. Ruggiero, D. A.; Mtui, E. P.; Otake, K.; Anwar, M. Central and primary visceral afferents to nucleus tractus solitarii may generate nitric oxide as a membrane-permeant neuronal messenger. J. Comp. Neurol. 364:51– 67; 1996. Sakuma, I.; Togashi, H.; Yoshioka, M.; Saito, H.; Yanagida, M.; Tamura, M.; Kobayashi, T.; Yasuda, H.; Gross, S. S.; Levi, R. NGmethyl-L-arginine, an inhibitor of L-arginine-derived nitric oxide synthesis, stimulates renal sympathetic nerve activity in vivo: A role for nitric oxide in the central regulation of sympathetic tone? Circ. Res. 70:607– 611; 1992. Tagawa, T.; Imaizumi, T.; Harada, S.; Endo, T.; Shiramoto, M.; Hirooka, Y.; Takeshita, A. Nitric oxide influences neuronal activity in the nucleus tractus solitarius of rat brainstem slices. Circ. Res. 75:70 – 76; 1994. Togashi, H.; Sakuma, I.; Yoshioka, M.; Kobayashi, T.; Yasuda, H.; Kitabake, A.; Saito, H.; Gross, S. S.; Levi, T. A central nervous system action of nitric oxide in blood pressure regulation. J. Pharmacol. Exp. Ther. 262:343–347; 1992. Tseng, C. J.; Liu, H. Y.; Lin, H. C.; Ger, L. P.; Tung, C. S.; Yen, M. H. Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 27:36 – 42; 1996. Wang, Y.; Lin, S. Z.; Chiou, A. L.; Williams, L. R.; Hoffer, B. J. Glial cell line-derived neurotrophic factor protects against ischemia-induced injury in the cerebral cortex. J. Neurosci. 17:4341– 4348; 1997. Wu, W. C.; Kuo, J. S.; Wang, Y.; Chai, C. Y. Glycine increases arterial pressure and augments NMDA-induced pressor responses in the dorsomedial and ventrolateral medulla of cats. J. Auton. Nerv. Syst. 67:145–155; 1997. Zanzinger, J.; Czachurski, J.; Seller, H. Inhibition of sympathetic vasoconstriction is a major principle of vasodilation by nitric oxide in vivo. Circ. Res. 75:1073–1077; 1994.