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Mechanisms of action of nitric oxide in the brain stem: role of oxidative stress
Autonomic Neuroscience: Basic and Clinical 98 (2002) 24 – 27 www.elsevier.com/locate/autneu
Mechanisms of action of nitric oxide in the brain stem: role of oxidative stress Johannes Zanzinger * Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent, CT13 9NJ England, UK
Abstract The exact mechanisms by which NO mediates its neuromodulatory effects within the central control of cardiovascular functions are still unclear. Both excitatory and inhibitory actions of NO in different regions of the brainstem have been reported, and that it could be caused by direct actions of NO on neurones and/or by NO-mediated changes in local cerebral blood flow. Microinjection studies suggest that direct modulation of neuronal activity by NO through cyclic 3V – 5V guanosine monophosphate (cGMP)-dependent mechanisms predominates. In contrast, endogenous NO produces only minor changes in local cerebral blood flow, and potentiation of NO-dependent vasodilation with an inhibitor of phosphodiesterase V (PDE5i) has no significant effect on sympathetic activity. Activation of the NO-system in the lower brain stem modulates various central and reflex-activated neuronal pathways. To a large extent, this appears to be mediated by NO-induced GABAand glutamate-release within the ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). In addition, NO has been shown to reduce local generation of angiotensin II (AII) in all areas. Recent studies suggest that the NO-mediated modulation of autonomic function is severely impaired in cardiovascular diseases. Possibly in conjunction with AII, which triggers and promotes superoxide radical generation, chronic oxidative stress (COS) could act as a key mediator of this process. Evidence supporting this hypothesis comes from studies on pigs that were chronically treated with organic nitrates to pharmacologically induce COS. In these animals, microinjection of superoxide dismutase into the rostral VLM (RVLM) diminished sympathetic activity by up to 70%, whereas peroxynitrite, a key mediator of NO-related oxidative stress, had excitotoxic effects. Antagonism of neuronal COS may therefore represent a novel approach to counteract neurohumoral activation in diseases such hypertension, obesity and heart failure. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Autonomic nervous system; Sympathetic activity; Pig; Phosphodiesterase V; Peroxynitrite
It is now widely recognised that neuronal production of NO influences cardiovascular functions through neuromodulatory actions within the autonomic nervous system. Research during the last decade revealed that this free radical is probably implicated in the regulation of neuronal excitability, as well as in neurotransmitter release (Prast and Philippu, 2001). NO synthesis is actively regulated in response to external influences and disease processes. For example, restraint stress and high blood pressure seem to activate NO-producing neurones in many autonomic centres including the hypothalamus, nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) (Krukoff, 1998; Nishida et al., 2001; Ye et al., 2000), whereas reductions in the number of NO-producing neurones have been reported in animals with heart failure or experimentally induced chronic oxida-
*
Tel.: +44-1304-642180; fax: +44-1304-656691. E-mail address: Johannes _ [email protected] (J. Zanzinger).
tive stress (Patel et al., 1996; Zanzinger and Czachurski, 2000). This short review will briefly discuss current views on the mechanism of action of NO within the brain stem areas that regulate autonomic functions, particularly within the VLM and the NTS with special emphasis on the potential role of oxidative stress as a pathogenic factor.
1. Biosynthesis and cellular mechanisms of action NO is produced by neurones in many parts of the brain and, in addition, the possibility exists that NO derived from endothelial sources may contribute to the modulation of neuronal function by NO. The enzymology of the NOsynthases and the intracellular actions of NO have been reviewed extensively elsewhere (Andrew and Mayer, 1999; Krukoff, 1999; Zhang and Snyder, 1995). Although direct modulation of channel proteins and interaction with enzymes of the mitochondrial respiratory chain have been discussed, it appears that soluble guanylate cyclase (sGC) is
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 2 5 - 5
J. Zanzinger / Autonomic Neuroscience: Basic and Clinical 98 (2002) 24–27
25
Fig. 1. Mechanisms of action of NO within the medulla oblongata. NO acts primarily through activation of soluble guanylate cyclase in neurones. Inhibition of cGMP-degradation in the vasculature of the brain stem but not in neurones (selective PDE5-inhibition; Wallis et al., 1999) has no effect. The graph shows pressor responses to activation of somatosympathetic reflexes that are relayed in the RVLM. All compounds were administered to the ventral surface of the medulla in pigs and dogs (PDE5i only dogs, n = 5 – 8). The experiments were carried out under general anaesthesia using the methods described in Zanzinger et al. (1997) (n = 5 – 8, */ ** P < 0.05/0.01 vs. control). Compounds were administered to the ventral surface of the medulla: L-NNA (NOS-inhibitior, 0.3 mM), MeTC (nNOS-inhibitor, 0.1 mM), 7-NI (nNOS-inhibitor, 1 mM), ODQ (sGC-inhibitor, 1 mM), SNAP (NO-donor, 0.1 mM), YC-1 (sGC-activator, 0.1 mM) and PDE5-inhibitior (UK-343,664: 0.3 mg, IC50: 1.1 nM, > 3000-fold selective for PDE5 over PDE1).
by far the most important target for NO under physiological conditions (Koesling, 1999). NO activates sGC already at sub-nanomolar concentrations, whereas all other effects of NO require much higher concentrations to become effective (Davis et al., 2001). As a consequence, effects of NO within the brain can be mimicked in vivo by administration of activators/inhibitors of sGC (Fig. 1). Activation of sGC results in formation of cyclic 3V– 5V guanosine monophosphate (cGMP), which in turn modulates ion channel activities and intracellular signalling pathways including gene transcription through activation of Proteinkinase G (Hawkins et al., 1998). NO-release in neurones is calciumdependent and can be triggered by calcium influx through NMDA-receptors and other cation channels. Through a number of feedback mechanisms, NO downregulates its own release thus preventing excessive activation under physiological conditions (Forstermann et al., 1995).
2. Modulation of transmitter release NO modulates neurotransmitter release. In fact, in vivo and in vitro studies have shown that throughout the whole brain, endogenous NO can be found to modulate the release of almost all neurotransmitters including acetylcholine, catecholamines, excitatory and inhibitory amino acids, serotonin, histamine and adenosine (Prast and Philippu, 2001). In most cases, enhanced NO level in the tissue increases the release of neurotransmitters, although decreasing effects have also been observed. Within the brain stem, a stimulatory effect of NO on glutamate release has been described in
the NTS (Matsuo et al., 2001), and recently, in the RVLM as well (Kishi et al., 2001). Furthermore, NO has been shown to increase the release of GABA in different autonomic centres (Kishi et al., 2001; Zhang and Patel, 1998), and to decrease the release and/or formation of angiotensin II (AII) presumably in all areas of the brain stem (Eshima et al., 2000; Tagawa et al., 1999). The most widely studied mechanism for modulation of transmitter release by NO is the retrograde signalling pathway, in which NO released upon NMDA-receptor stimulation diffuses back to the presynaptic terminal to increase glutamate release via a cGMP-dependent mechanism. However, indiscriminate activation of transmitter release through a positive feedback loop would not be a sensible physiological function; and indeed, there are several studies showing that NO may be able to modulate vesicular release of neurotransmitter in either direction, or not at all, depending on the coincident level of presynaptic activity and NO concentration (for review see Garthwaite and Boulton, 1995). Furthermore, due to the complex function of the brain stem neuronal network, it is difficult to determine whether, for example, an inhibitory effect of NO on sympathetic activity is mediated by direct inhibition of excitatory pathways, excitation of inhibitory (inter)neurones or both. Nevertheless, although excitatory and inhibitory pathways can be simultaneously activated by NO, this does not mean that NO-effects simply cancel out each other. For example, NO-induced glutamate release in the NTS causes pronounced sympathoinhibitory effects through activation of baroreceptor reflex pathways (Matsuo et al., 2001). In the rostral VLM (RVLM), where the bulbospinal sympathoexcitatory neurones represent only
26
J. Zanzinger / Autonomic Neuroscience: Basic and Clinical 98 (2002) 24–27
a relatively small fraction of the neuronal population, direct or indirect stimulation of GABA-ergic neurones may be more important leading to an overall inhibition of glutamate effects in this region (Zanzinger et al., 1997).
3. Indirect effects through modulation of cerebral blood flow Another possible way that NO could alter sympathetic activity is through modulation of local blood flow. The RVLM is known to have intrinsic chemosensitivity and changes in sympathetic outflow in response to pronounced increases in blood pCO2 and/or decreases in pO2 have been demonstrated (Reis et al., 1994; Seller et al., 1990). However, it seems unlikely that endogenous NO acts through this mechanism because in other studies, even substantial acute systemic hypoxia did not alter renal sympathetic nerve activity (SNA) in chemoreceptor denervated animals (Zanzinger et al., 1998b). Furthermore, local NOS-inhibition does not cause major changes in cerebral blood flow (Fabricius et al., 1996), and potentiation of NO-dependent vasodilation, with a specific inhibitor of phosphodiesterase V (PDE5i) that inhibits vascular PDEs (Wallis et al., 1999), has no significant effect on sympathetic excitability (Fig. 1).
4. Disease-related changes in NO-action: role of oxidative stress Oxidative stress is a key event in cardiovascular diseases such as hypertension, diabetes, atherosclerosis and heart failure that occurs not only on the level of heart and blood vessels but also within the CNS. In neurones, oxidative
stress can have excitatory and/or damaging effects (Dawson and Dawson, 1996). We studied the effects of RVLMmicroinjections of superoxide dismutase in a model of chronic oxidative stress (COS). In pigs that were chronically treated with organic nitrates to pharmacologically induce COS, microinjection of superoxide dismutase into the RVLM diminished sympathetic activity by up to 70% (Zanzinger and Czachurski, 2000). On the other hand, severe oxidative stress that leads to formation of significant amounts of peroxynitrite may cause neuronal damage. Microinjections of peroxynitrite into the RVLM caused dose-dependent transient excitatory responses, followed by marked and irreversible depression of SNA in the same model (Zanzinger et al., unpublished data). The reduction of NOS-activity within the lower brainstem and the increase in sympathetic outflow that occurs in heart failure (Sakai et al., 2001) and in models of COS (i.e. experimental nitrate tolerance) (Zanzinger et al., 1998a) may thus be functional consequences of oxidative stress within the brain. Fig. 2 schematically summarizes the major effects and interactions of NO and oxidative stress within the brain stem areas that primarily regulate sympathetic outflow. There is increasing evidence that AII may be most important in this process (Boscan et al., 2001; Eshima et al., 2000; Liu and Zucker, 1999). When central AII-formation increases, a positive feedback can be established in which AII enhances sympathetic activity, which potentiates AII-formation and so on. The disruption of the antagonistic NO-system by AII-induced increases in superoxide formation (Rajagopalan et al., 1996) and oxidative stress may facilitate this process and lead to general disinhibition of other sympathoexcitatory influences that culminate in the so-called neurohumoral activation in cardiovascular diseases.
5. Conclusions Despite (or perhaps because) of the intensive research on it, NO remains one of the most elusive biological factors known. Nevertheless, it appears that within the brain stem, NO acts primarily through cGMP-mediated effects on neurones, presumably via modulation of transmitter release and excitability. Its overall inhibitory effect on the generation of sympathetic activity within the brain stem can become reduced or even disrupted by oxidative stress. Antagonism of the local renin –angiotensin system within the brain and counteraction of neuronal oxidative stress therefore represent rationale novel approaches for the treatment of cardiovascular diseases. Fig. 2. Possible effects of oxidative stress within the brain stem. NO modulates transmitter release in all areas involved in autonomic control. Oxidative stress reduces the amount of free NO and may directly excite and/or damage neurones. Abbreviations: AII = angiotensin II; Glu = glutamate; GABA = g-aminobutyric acid; ROS = reactive oxygen species; ONOO = peroxynitrite.
Acknowledgements The cited work by the author was supported by grants from the German Research Foundation (DFG).
J. Zanzinger / Autonomic Neuroscience: Basic and Clinical 98 (2002) 24–27
References Andrew, P.J., Mayer, B., 1999. Enzymatic function of nitric oxide synthases. Cardiovasc. Res. 43, 521 – 531. Boscan, P., Allen, A.M., Paton, J.F., 2001. Baroreflex inhibition of cardiac sympathetic outflow is attenuated by angiotensin II in the nucleus of the solitary tract. Neuroscience 103, 153 – 160. Davis, K.L., Martin, E., Turko, I.V., Murad, F., 2001. Novel effects of nitric oxide. Annu. Rev. Pharmacol. Toxicol. 41, 203 – 236. Dawson, V.L., Dawson, T.M., 1996. Nitric oxide neurotoxicity. J. Chem. Neuroanat. 10, 179 – 190. Eshima, K., Hirooka, Y., Shigematsu, H., Matsuo, I., Koike, G., Sakai, K., Takeshita, A., 2000. Angiotensin in the nucleus tractus solitarii contributes to neurogenic hypertension caused by chronic nitric oxide synthase inhibition. Hypertension 36, 259 – 263. Fabricius, M., Rubin, I., Bundgaard, M., Lauritzen, M., 1996. NOS activity in brain and endothelium: relation to hypercapnic rise of cerebral blood flow in rats. Am. J. Physiol. 271, H2035 – H2044. Forstermann, U., Kleinert, H., Gath, I., Schwarz, P., Closs, E.I., Dun, N.J., 1995. Expression and expressional control of nitric oxide synthases in various cell types. Adv. Pharmacol. 34, 171 – 186. Garthwaite, J., Boulton, C.L., 1995. Nitric oxide signaling in the central nervous system. Annu. Rev. Physiol. 57, 683 – 706. Hawkins, R.D., Son, H., Arancio, O., 1998. Nitric oxide as a retrograde messenger during long-term potentiation in hippocampus. Prog. Brain Res. 118, 155 – 172. Kishi, T., Hirooka, Y., Sakai, J., Shigematsu, H., Itoh, H., Shimokawa, H., Takeshita, A., 2001. Gabaergic mechanism is involved in hypotension of the rat with eNOS gene transfer into the RVLM. FASEB J. 15, 647.14 (Abstract). Koesling, D., 1999. Studying the structure and regulation of soluble guanylyl cyclase. Methods 19, 485 – 493. Krukoff, T.L., 1998. Central regulation of autonomic function: no brakes? Clin. Exp. Pharmacol. Physiol. 25, 474 – 478. Krukoff, T.L., 1999. Central actions of nitric oxide in regulation of autonomic functions. Brain Res. Brain Res. Rev. 30, 52 – 65. Liu, J.L., Zucker, I.H., 1999. Regulation of sympathetic nerve activity in heart failure: a role for nitric oxide and angiotensin II. Circ. Res. 84, 417 – 423. Matsuo, I., Hirooka, Y., Hironaga, K., Eshima, K., Shigematsu, H., Shihara, M., Sakai, K., Takeshita, A., 2001. Glutamate release via NO production evoked by NMDA in the NTS enhances hypotension and bradycardia in vivo. Am. J. Physiol.: Regul., Integr. Comp. Physiol. 280, R1285 – R1291. Nishida, Y., Chen, Q.H., Tandai-Hiruma, M., Terada, S., Horiuchi, J., 2001. Neuronal nitric oxide strongly suppresses sympathetic outflow in highsalt Dahl rats. J. Hypertens. 19, 627 – 634. Patel, K.P., Zhang, K., Zucker, I.H., Krukoff, T.L., 1996. Decreased gene expression of neuronal nitric oxide synthase in hypothalamus and brainstem of rats in heart failure. Brain Res. 734, 109 – 115.
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Prast, H., Philippu, A., 2001. Nitric oxide as modulator of neuronal function. Prog. Neurobiol. 64, 51 – 68. Rajagopalan, S., Kurz, S., Munzel, T., Tarpey, M., Freeman, B.A., Griendling, K.K., Harrison, D.G., 1996. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J. Clin. Invest. 97, 1916 – 1923. Reis, D.J., Golanov, E.V., Ruggiero, D.A., Sun, M.K., 1994. Sympathoexcitatory neurons of the rostral ventrolateral medulla are oxygen sensors and essential elements in the tonic and reflex control of the systemic and cerebral circulations. J. Hypertens. (Suppl. 12) S159 – S180. Sakai, K., Hirooka, Y., Shigematsu, A., Kishi, T., Ito, K., Takeshita, A., 2001. Reduced nitric oxide synthase of the brain stem in heart failure. FASEB J. 15, 410.4 (Abstract). Seller, H., Konig, S., Czachurski, J., 1990. Chemosensitivity of sympathoexcitatory neurones in the rostroventrolateral medulla of the cat. Pfluegers Arch. 416, 735 – 741. Tagawa, T., Horiuchi, J., Potts, P.D., Dampney, R.A., 1999. Sympathoinhibition after angiotensin receptor blockade in the rostral ventrolateral medulla is independent of glutamate and gamma-aminobutyric acid receptors. J. Auton. Nerv. Syst. 77, 21 – 30. Wallis, R.M., Corbin, J.D., Francis, S.H., Ellis, P., 1999. Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function, and the contractile responses of trabeculae carneae and aortic rings in vitro. Am. J. Cardiol. 83, 3C – 12C. Ye, S., Mozayeni, P., Gamburd, M., Zhong, H., Campese, V.M., 2000. Interleukin-1beta and neurogenic control of blood pressure in normal rats and rats with chronic renal failure. Am. J. Physiol.: Heart Circ. Physiol. 279, H2786 – H2796. Zanzinger, J., Czachurski, J., 2000. Chronic oxidative stress in the RVLM modulates sympathetic control of circulation in pigs. Pfluegers Arch. 439, 489 – 494. Zanzinger, J., Czachurski, J., Seller, H., 1997. Neuronal nitric oxide reduces sympathetic excitability by modulation of central glutamate effects in pigs. Circ. Res. 80, 565 – 571. Zanzinger, J., Czachurski, J., Seller, H., 1998a. Impaired modulation of sympathetic excitability by nitric oxide after long-term administration of organic nitrates in pigs. Circulation 97, 2352 – 2358. Zanzinger, J., Czachurski, J., Seller, H., 1998b. Nitric oxide in the ventrolateral medulla regulates sympathetic responses to systemic hypoxia in pigs. Am. J. Physiol. 275, R33 – R39. Zhang, K., Patel, K.P., 1998. Effect of nitric oxide within the paraventricular nucleus on renal sympathetic nerve discharge: role of GABA. Am. J. Physiol. 275, R728 – R734. Zhang, J., Snyder, S.H., 1995. Nitric oxide in the nervous system. Annu. Rev. Pharmacol. Toxicol. 35, 213 – 233.
Mechanisms of action of nitric oxide in the brain stem: role of oxidative stress Johannes Zanzinger * Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent, CT13 9NJ England, UK
Abstract The exact mechanisms by which NO mediates its neuromodulatory effects within the central control of cardiovascular functions are still unclear. Both excitatory and inhibitory actions of NO in different regions of the brainstem have been reported, and that it could be caused by direct actions of NO on neurones and/or by NO-mediated changes in local cerebral blood flow. Microinjection studies suggest that direct modulation of neuronal activity by NO through cyclic 3V – 5V guanosine monophosphate (cGMP)-dependent mechanisms predominates. In contrast, endogenous NO produces only minor changes in local cerebral blood flow, and potentiation of NO-dependent vasodilation with an inhibitor of phosphodiesterase V (PDE5i) has no significant effect on sympathetic activity. Activation of the NO-system in the lower brain stem modulates various central and reflex-activated neuronal pathways. To a large extent, this appears to be mediated by NO-induced GABAand glutamate-release within the ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). In addition, NO has been shown to reduce local generation of angiotensin II (AII) in all areas. Recent studies suggest that the NO-mediated modulation of autonomic function is severely impaired in cardiovascular diseases. Possibly in conjunction with AII, which triggers and promotes superoxide radical generation, chronic oxidative stress (COS) could act as a key mediator of this process. Evidence supporting this hypothesis comes from studies on pigs that were chronically treated with organic nitrates to pharmacologically induce COS. In these animals, microinjection of superoxide dismutase into the rostral VLM (RVLM) diminished sympathetic activity by up to 70%, whereas peroxynitrite, a key mediator of NO-related oxidative stress, had excitotoxic effects. Antagonism of neuronal COS may therefore represent a novel approach to counteract neurohumoral activation in diseases such hypertension, obesity and heart failure. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Autonomic nervous system; Sympathetic activity; Pig; Phosphodiesterase V; Peroxynitrite
It is now widely recognised that neuronal production of NO influences cardiovascular functions through neuromodulatory actions within the autonomic nervous system. Research during the last decade revealed that this free radical is probably implicated in the regulation of neuronal excitability, as well as in neurotransmitter release (Prast and Philippu, 2001). NO synthesis is actively regulated in response to external influences and disease processes. For example, restraint stress and high blood pressure seem to activate NO-producing neurones in many autonomic centres including the hypothalamus, nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) (Krukoff, 1998; Nishida et al., 2001; Ye et al., 2000), whereas reductions in the number of NO-producing neurones have been reported in animals with heart failure or experimentally induced chronic oxida-
*
Tel.: +44-1304-642180; fax: +44-1304-656691. E-mail address: Johannes _ [email protected] (J. Zanzinger).
tive stress (Patel et al., 1996; Zanzinger and Czachurski, 2000). This short review will briefly discuss current views on the mechanism of action of NO within the brain stem areas that regulate autonomic functions, particularly within the VLM and the NTS with special emphasis on the potential role of oxidative stress as a pathogenic factor.
1. Biosynthesis and cellular mechanisms of action NO is produced by neurones in many parts of the brain and, in addition, the possibility exists that NO derived from endothelial sources may contribute to the modulation of neuronal function by NO. The enzymology of the NOsynthases and the intracellular actions of NO have been reviewed extensively elsewhere (Andrew and Mayer, 1999; Krukoff, 1999; Zhang and Snyder, 1995). Although direct modulation of channel proteins and interaction with enzymes of the mitochondrial respiratory chain have been discussed, it appears that soluble guanylate cyclase (sGC) is
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 2 5 - 5
J. Zanzinger / Autonomic Neuroscience: Basic and Clinical 98 (2002) 24–27
25
Fig. 1. Mechanisms of action of NO within the medulla oblongata. NO acts primarily through activation of soluble guanylate cyclase in neurones. Inhibition of cGMP-degradation in the vasculature of the brain stem but not in neurones (selective PDE5-inhibition; Wallis et al., 1999) has no effect. The graph shows pressor responses to activation of somatosympathetic reflexes that are relayed in the RVLM. All compounds were administered to the ventral surface of the medulla in pigs and dogs (PDE5i only dogs, n = 5 – 8). The experiments were carried out under general anaesthesia using the methods described in Zanzinger et al. (1997) (n = 5 – 8, */ ** P < 0.05/0.01 vs. control). Compounds were administered to the ventral surface of the medulla: L-NNA (NOS-inhibitior, 0.3 mM), MeTC (nNOS-inhibitor, 0.1 mM), 7-NI (nNOS-inhibitor, 1 mM), ODQ (sGC-inhibitor, 1 mM), SNAP (NO-donor, 0.1 mM), YC-1 (sGC-activator, 0.1 mM) and PDE5-inhibitior (UK-343,664: 0.3 mg, IC50: 1.1 nM, > 3000-fold selective for PDE5 over PDE1).
by far the most important target for NO under physiological conditions (Koesling, 1999). NO activates sGC already at sub-nanomolar concentrations, whereas all other effects of NO require much higher concentrations to become effective (Davis et al., 2001). As a consequence, effects of NO within the brain can be mimicked in vivo by administration of activators/inhibitors of sGC (Fig. 1). Activation of sGC results in formation of cyclic 3V– 5V guanosine monophosphate (cGMP), which in turn modulates ion channel activities and intracellular signalling pathways including gene transcription through activation of Proteinkinase G (Hawkins et al., 1998). NO-release in neurones is calciumdependent and can be triggered by calcium influx through NMDA-receptors and other cation channels. Through a number of feedback mechanisms, NO downregulates its own release thus preventing excessive activation under physiological conditions (Forstermann et al., 1995).
2. Modulation of transmitter release NO modulates neurotransmitter release. In fact, in vivo and in vitro studies have shown that throughout the whole brain, endogenous NO can be found to modulate the release of almost all neurotransmitters including acetylcholine, catecholamines, excitatory and inhibitory amino acids, serotonin, histamine and adenosine (Prast and Philippu, 2001). In most cases, enhanced NO level in the tissue increases the release of neurotransmitters, although decreasing effects have also been observed. Within the brain stem, a stimulatory effect of NO on glutamate release has been described in
the NTS (Matsuo et al., 2001), and recently, in the RVLM as well (Kishi et al., 2001). Furthermore, NO has been shown to increase the release of GABA in different autonomic centres (Kishi et al., 2001; Zhang and Patel, 1998), and to decrease the release and/or formation of angiotensin II (AII) presumably in all areas of the brain stem (Eshima et al., 2000; Tagawa et al., 1999). The most widely studied mechanism for modulation of transmitter release by NO is the retrograde signalling pathway, in which NO released upon NMDA-receptor stimulation diffuses back to the presynaptic terminal to increase glutamate release via a cGMP-dependent mechanism. However, indiscriminate activation of transmitter release through a positive feedback loop would not be a sensible physiological function; and indeed, there are several studies showing that NO may be able to modulate vesicular release of neurotransmitter in either direction, or not at all, depending on the coincident level of presynaptic activity and NO concentration (for review see Garthwaite and Boulton, 1995). Furthermore, due to the complex function of the brain stem neuronal network, it is difficult to determine whether, for example, an inhibitory effect of NO on sympathetic activity is mediated by direct inhibition of excitatory pathways, excitation of inhibitory (inter)neurones or both. Nevertheless, although excitatory and inhibitory pathways can be simultaneously activated by NO, this does not mean that NO-effects simply cancel out each other. For example, NO-induced glutamate release in the NTS causes pronounced sympathoinhibitory effects through activation of baroreceptor reflex pathways (Matsuo et al., 2001). In the rostral VLM (RVLM), where the bulbospinal sympathoexcitatory neurones represent only
26
J. Zanzinger / Autonomic Neuroscience: Basic and Clinical 98 (2002) 24–27
a relatively small fraction of the neuronal population, direct or indirect stimulation of GABA-ergic neurones may be more important leading to an overall inhibition of glutamate effects in this region (Zanzinger et al., 1997).
3. Indirect effects through modulation of cerebral blood flow Another possible way that NO could alter sympathetic activity is through modulation of local blood flow. The RVLM is known to have intrinsic chemosensitivity and changes in sympathetic outflow in response to pronounced increases in blood pCO2 and/or decreases in pO2 have been demonstrated (Reis et al., 1994; Seller et al., 1990). However, it seems unlikely that endogenous NO acts through this mechanism because in other studies, even substantial acute systemic hypoxia did not alter renal sympathetic nerve activity (SNA) in chemoreceptor denervated animals (Zanzinger et al., 1998b). Furthermore, local NOS-inhibition does not cause major changes in cerebral blood flow (Fabricius et al., 1996), and potentiation of NO-dependent vasodilation, with a specific inhibitor of phosphodiesterase V (PDE5i) that inhibits vascular PDEs (Wallis et al., 1999), has no significant effect on sympathetic excitability (Fig. 1).
4. Disease-related changes in NO-action: role of oxidative stress Oxidative stress is a key event in cardiovascular diseases such as hypertension, diabetes, atherosclerosis and heart failure that occurs not only on the level of heart and blood vessels but also within the CNS. In neurones, oxidative
stress can have excitatory and/or damaging effects (Dawson and Dawson, 1996). We studied the effects of RVLMmicroinjections of superoxide dismutase in a model of chronic oxidative stress (COS). In pigs that were chronically treated with organic nitrates to pharmacologically induce COS, microinjection of superoxide dismutase into the RVLM diminished sympathetic activity by up to 70% (Zanzinger and Czachurski, 2000). On the other hand, severe oxidative stress that leads to formation of significant amounts of peroxynitrite may cause neuronal damage. Microinjections of peroxynitrite into the RVLM caused dose-dependent transient excitatory responses, followed by marked and irreversible depression of SNA in the same model (Zanzinger et al., unpublished data). The reduction of NOS-activity within the lower brainstem and the increase in sympathetic outflow that occurs in heart failure (Sakai et al., 2001) and in models of COS (i.e. experimental nitrate tolerance) (Zanzinger et al., 1998a) may thus be functional consequences of oxidative stress within the brain. Fig. 2 schematically summarizes the major effects and interactions of NO and oxidative stress within the brain stem areas that primarily regulate sympathetic outflow. There is increasing evidence that AII may be most important in this process (Boscan et al., 2001; Eshima et al., 2000; Liu and Zucker, 1999). When central AII-formation increases, a positive feedback can be established in which AII enhances sympathetic activity, which potentiates AII-formation and so on. The disruption of the antagonistic NO-system by AII-induced increases in superoxide formation (Rajagopalan et al., 1996) and oxidative stress may facilitate this process and lead to general disinhibition of other sympathoexcitatory influences that culminate in the so-called neurohumoral activation in cardiovascular diseases.
5. Conclusions Despite (or perhaps because) of the intensive research on it, NO remains one of the most elusive biological factors known. Nevertheless, it appears that within the brain stem, NO acts primarily through cGMP-mediated effects on neurones, presumably via modulation of transmitter release and excitability. Its overall inhibitory effect on the generation of sympathetic activity within the brain stem can become reduced or even disrupted by oxidative stress. Antagonism of the local renin –angiotensin system within the brain and counteraction of neuronal oxidative stress therefore represent rationale novel approaches for the treatment of cardiovascular diseases. Fig. 2. Possible effects of oxidative stress within the brain stem. NO modulates transmitter release in all areas involved in autonomic control. Oxidative stress reduces the amount of free NO and may directly excite and/or damage neurones. Abbreviations: AII = angiotensin II; Glu = glutamate; GABA = g-aminobutyric acid; ROS = reactive oxygen species; ONOO = peroxynitrite.
Acknowledgements The cited work by the author was supported by grants from the German Research Foundation (DFG).
J. Zanzinger / Autonomic Neuroscience: Basic and Clinical 98 (2002) 24–27
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