(Hypertension. 2000;36:1089.)
© 2000 American Heart Association, Inc.
Colin Johnston - A Celebration |
Nitric Oxide Limits Pressor Responses to Sympathetic Activation in Rat Spinal Cord
From the Cardiovascular Neuroscience Group, Cardiovascular Medicine and Centre for Neuroscience, Flinders University, SA 5042, Australia.
Correspondence to Assoc Prof Leonard Arnolda, Department of Cardiology, Division of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. E-mail Leonard.Arnolda{at}flinders.edu.au
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Abstract |
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Abstract—N-methyl D-aspartate (NMDA) receptor stimulation is known to activate nitric oxide (NO) synthase, an enzyme present in a high proportion of sympathetic preganglionic neurons. In this study, we have examined the possibility that NO modulates the pressor responses elicited by NMDA receptor stimulation in the spinal cord. In experiments on anesthetized rats, we determined whether intrathecal administration of either 3-morpholinylsydnoneimine chloride (SIN-1), an NO donor, or NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, affected the response to stimulation of spinal NMDA receptors by NMDA (1 pmol to 1 µmol in 10-µL intrathecal administration). Intrathecal NMDA resulted in dose-dependent increases in blood pressure. SIN-1 (100 nmol) attenuated the pressor responses to NMDA (F1,70=12, P=0.001). Conversely, L-NAME (1 nmol to 1 µmol) augmented the pressor response to NMDA in a dose-dependent manner (F3,161=28.3, P<0.001). The effect of L-NAME to amplify the pressor response to NMDA was reversed by L-arginine but not by D-arginine. These results indicate that endogenous synthesis of NO in the spinal cord limits the pressor response to stimulation of spinal NMDA receptors.
Key Words: blood pressure • sympathetic nervous system • nitric oxide synthase • L-NAME
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Introduction |
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Nitric oxide (NO), synthesized from L-arginine by NO synthase (NOS), mediates endothelium-dependent vasodilation, acts as a neurotransmitter, and is used by macrophages to kill tumor cells and bacteria.1 Inhibition of NOS increases arterial pressure by causing peripheral vasoconstriction and by increasing sympathetic nerve activity by central nervous system (CNS) effects.2 Inhibition of NOS has been reported to produce diverse actions at CNS sites involved in cardiovascular regulation. NOS inhibitors increase arterial pressure when injected into the rostral ventrolateral medulla3 or the hypothalamic paraventricular nucleus4 and lower arterial pressure when injected into the caudal ventrolateral medulla.5 NOS has been identified in most sympathetic preganglionic neurons (SPN) in the intermediolateral column of the spinal cord,6 7 but its function at this site is not yet established. The NO precursor L-arginine has been reported to excite renal sympathetic nerves and the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) to silence renal sympathetic nerves when injected into the spinal intrathecal space in the rabbit.8 If NO activated SPN, then L-NAME might be expected to lower arterial pressure. To date, studies on anesthetized rats have been inconsistent, with some studies reporting pressor responses9 10 and others reporting depressor responses11 to intrathecal administration of L-NAME.
Because NOS is activated by an increase in intracellular calcium, such as that caused by stimulation of N-methyl D-aspartate (NMDA) receptors, we tested the possibility that NO synthesis modulates the pressor response elicited by NMDA in the spinal cord.
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Methods |
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Fifty-two male Wistar-Kyoto rats (weight 300 to 400 g) were obtained from the Flinders Medical Center Animal House. All experimental procedures conformed to guidelines laid down by the National Health and Medical Research Council of Australia and were approved by the Animal Welfare Committee at Flinders University of South Australia.
Effect of 3-Morpholinylsydnoneimine Chloride on Pressor Response to
Intrathecal NMDA in Anesthetized Rats
Studies were performed under barbiturate anesthesia
(60 mg/kg IP sodium pentobarbital and supplementary doses of 30 mg/kg
if required). A femoral artery was cannulated for measurement of
arterial pressure and a femoral vein was cannulated for
administration of drugs (vinyl tube, outer diameter 0.50 mm, Dural
Plastics SV 8). Through a small incision in the atlanto-occipital
membrane, a stretched polyethylene tube (outer diameter 0.80 mm,
Dural Plastics SP 31) was inserted into the spinal subarachnoid
space and threaded caudally 8.5 cm so that the tip lay at the
thoraco-lumbar junction. Pancuronium bromide (0.4 mg) was administered
intravenously, and an additional dose was given if NMDA
evoked muscle twitching. The need for supplemental doses of barbiturate
was assessed before each dose of pancuronium was administered. The
spinal cord was exposed at the C1 level and sectioned. Drugs were
administered intrathecally in 10 µL of artificial
cerebrospinal fluid (an aqueous solution of 1.4 mmol/L
CaCl2, 1.0 mmol/L
MgCl2, 2.6 mmol/L KCl, and 128.6 mmol/L
NaCl, phosphate buffered to pH 7.2). Drug solutions were made up fresh
on the day of the experiment. Increasing doses of NMDA (1 pmol to
1 µmol in 10-µL intrathecal administration) were
coadministered with 3-morpholinylsydnoneimine chloride (SIN-1) (100
nmol/L) or vehicle. Blood pressure was measured for 20 minutes after
each dose of NMDA. SIN-1 or vehicle was administered between doses of
NMDA+SIN-1 (or NMDA+vehicle), and another 20 minutes was allowed to
pass before the next dose of NMDA.
Effect of L-NAME on Pressor Response to Intrathecal
NMDA in Anesthetized Rats
These experiments were carried out in a fashion similar to those
described above except that increasing doses of NMDA were
coadministered with 1 of 4 doses of L-NAME (1 nmol, 10 nmol, 100 nmol,
or 1 µmol), and L-NAME alone was given between doses of
NMDA+L-NAME.
Effect of L- or D-Arginine Plus L-NAME on
Pressor Response to Intrathecal NMDA in Anesthetized
Rats
In these experiments, increasing doses of NMDA were
coadministered with one of L-arginine (5 µmol) and
L-NAME (100 nmol) or D-arginine (5 µmol) and L-NAME
(100 nmol/L). L-NAME was administered together with L- or
D-arginine between doses of NMDA.
At the end of each experiment, 10 µL of methylene blue was injected into the intrathecal catheter followed by 10 µL of artificial cerebrospinal fluid, and rats were killed with an overdose of sodium pentobarbital. An autopsy was performed to confirm the location of the intrathecal catheter and to establish the spread of dye in the intrathecal space.
Statistics
Results are shown as mean±SEM. Changes in mean
arterial pressure (ie, mean arterial pressure
after administration of drugs compared with pretreatment mean
arterial pressure) were compared between groups by means of
1- or 2-way ANOVA.12 Differences between groups were
analyzed by the Ryan-Einot-Gabriel-Welsch (REGW)
test.12
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Results |
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Effects of SIN-1 on Pressor Response to Intrathecal NMDA in Anesthetized Rats
In anesthetized and paralyzed rats spinally transected to exclude supraspinal effects, pretreatment arterial pressure was 54±3 mm Hg in rats subsequently given NMDA alone and 55±7 mm Hg in rats subsequently given NMDA together with SIN-1 (n=6 per group, P=NS). Intrathecal. administration of NMDA (1 pmol to 1 µmol) resulted in dose-dependent increases in blood pressure in both groups of rats (Figure 1, significant NMDA effect, F6,70=19.8, n=6 per group, P<0.001). SIN-1 (100 nmol) attenuated the pressor responses to NMDA (significant SIN-1 effect, F1,70=12, n=6 per group, P=0.001).
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MAP) in anesthetized and paralyzed rats spinally transected to exclude supraspinal effects given escalating doses of NMDA (1 pmol to 1 µmol) coadministered with SIN-1 (100 nmol, •) or vehicle (
). SIN-1 (100 nmol/L) attenuated pressor responses to NMDA (significant SIN-1 effect, F1,70=12, n=6 per group, P=0.001).
Effect of L-NAME on Pressor Response to Intrathecal
NMDA in Anesthetized Rats
Arterial pressure was similar in anesthetized,
paralyzed, spinally transected rats subsequently given different doses
of L-NAME (1 nmol; 61±5 mm Hg, n=9; 10 nmol, 65±2 mm Hg,
n=7; 100 nmol, 64±2 mm Hg, n=6; 1 µmol, 67±4
mm Hg, n=6, F3,24=0.5, P=NS).
Intrathecal administration of NMDA (1 pmol/L to 1
µmol/L) resulted in dose-dependent increases in blood pressure in
rats receiving L-NAME (Figure 2, significant NMDA effect, F6,161=63.4,
P<0.001). L-NAME amplified the pressor response to NMDA in
a dose-responsive manner (Figure 2, F3,161=28.3, P<0.001). Post hoc
testing with the REGW test revealed significant differences between
groups. The pressor responses to intrathecal NMDA were
similar in rats receiving 1 nmol and 10 nmol L-NAME. The NMDA-induced
pressor responses in rats receiving 100 nmol L-NAME were significantly
larger than the responses in rats receiving lower doses of L-NAME. Rats
receiving 1 µmol L-NAME showed significantly larger NMDA-induced
pressor responses than those in any of the other groups.
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, n=9; 10 nmol/L, 
, n=7; 100 nmol/L,
Effect of NOS Inhibition in Presence of L- or
D-Arginine in Anesthetized Rats
Pretreatment arterial pressure was 67±4 mm Hg
in rats allocated to receive L-arginine (5 µmol)
coadministered with L-NAME (100 nmol) and 63±2 mm Hg in rats
allocated to receive D-arginine (5 µmol/L)
coadministered with L-NAME (100 nmol). Increasing doses of NMDA
produced dose-related pressor responses in both
L-arginine–treated and D-arginine–treated
rats (Figure 3, significant NMDA effect,
F6,70=72.4, P<0.001).
L-Arginine (5 µmol) coadministered with
L-NAME (100 nmol) significantly attenuated the pressor response to NMDA
compared with coadministration of D-arginine
(5 µmol) coadministered with L-NAME (100 nmol) (Figure 3, significant L-arginine effect,
F1,70=8.8, P<0.005, significant NMDA
by L-arginine interaction,
F6,70=3.3, P<0.01).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Many neurons in the CNS contain NOS, a calmodulin-dependent enzyme, and generate NO in response to increased intracellular calcium.1 NO in turn binds to soluble guanylate cyclase, eliciting large increases in the second messenger, cyclic GMP.13 In the CNS, activation of NMDA receptors is likely to be an important mechanism leading to NOS activation. Indeed, it was the observation that the excitatory neurotransmitter glutamate elicited large increases in cyclic GMP that led to the suggestion that NO might function as an intercellular messenger in the CNS.14
In this study, we have explored possible interactions between NMDA receptor activation and NO in the spinal mechanisms controlling blood pressure. The major finding of this study is that intrathecal administration of the NO donor SIN-1 attenuated the pressor responses induced by intrathecal NMDA, whereas the intrathecal administration of the NOS inhibitor L-NAME amplified these responses. The effect of L-NAME was a result of inhibition of NOS, since it was attenuated by L-arginine but not by D-arginine. These data demonstrate that endogenously generated NO limits the pressor response elicited by activation of spinal NMDA receptors.
NOS is found in more than half of all sympathetic preganglionic neurons,6 7 but the functional significance of NO in controlling cardiovascular and other autonomic functions is uncertain. Hakim et al8 reported that the NO precursor L-arginine, excited renal sympathetic nerves and that the NOS inhibitor L-NAME silenced renal sympathetic nerves when injected into the spinal intrathecal space in the rabbit. No significant change in arterial pressure was found in this study, a finding that was attributed to the small injection volumes that may have only affected a few SPN. If NO activated SPN projecting to cardiovascular targets, then NOS inhibitors would be expected to lower arterial pressure and NO donors would be expected to raise arterial pressure. Experiments in which NO donors or NOS inhibitors have been administered intrathecally to anesthetized rats have yielded inconsistent results. Lee et al11 reported that administration of a NOS inhibitor lowered arterial pressure. In contrast, Garcia et al9 and Koga et al10 reported that intrathecal administration of L-NAME elicited pressor responses. These inconsistencies in response suggest that the effects of NO are complex and might be critically dependent on the experimental conditions. This situation could arise if NO effects are mediated by modulation of the actions of other neurotransmitters rather than by direct neuronal activation or inhibition. Inhibition by an NMDA antagonist of the pressor response to L-NAME9 and inhibition by L-NAME of the pressor response and tachycardia elicited by intrathecal administration of carbachol15 are observations consistent with a modulatory rather than a direct role for NO in blood pressure control. In the present study, the dose-response relation of NMDA administered intrathecally was determined in the presence of a NO donor, SIN-1, or a range of doses of the NOS inhibitor L-NAME. The NO donor SIN-1 attenuated the pressor response to NMDA, an effect that was most prominent at relatively low doses of NMDA. Because activation of NMDA receptors is a potent stimulus for NO synthesis,16 an inhibitory effect of exogenous NO may be less apparent when endogenous NO synthesis is increased by NMDA receptor activation. Blockade of NO synthesis with L-NAME produced a marked shift in the dose-response relation to NMDA. The lowest dose of NMDA (1 pmol/L) elicited a 29–mm Hg pressor response when coadministered with L-NAME (1 µmol), whereas a 1000-fold-higher NMDA dose (1 nmol) only increased arterial pressure by 25 mm Hg when administered without L-NAME. The effect of L-arginine is probably a result of inhibition of NOS because L-arginine reverses the amplification of the pressor response to NMDA induced by L-NAME.
The mechanism by which NO might inhibit pressor responses induced by NMDA is unclear. Soluble guanylate cyclase is a major target for NO.17 However, this enzyme is not activated in neurons that produce NO because the increases in Ca2+ necessary to activate NOS inhibit soluble guanylate cyclase.18 NO is freely diffusible, and significant NO concentrations are likely to be observed up to 200 µmol/L from its site of production.19 Hence, NO generated within a neuron could act on terminals impinging on that neuron as well as nearby neurons. NO has been shown to influence neurotransmitter release,20 and it is probably by this mechanism that NO can enhance either excitatory21 or inhibitory22 synaptic potentials in SPN. Under conditions in which inhibitory synaptic inputs predominate, enhancement of inhibitory synaptic potentials by NO might decrease sympathetic outflow.
A direct effect of NO on NMDA receptor function is, perhaps, a more likely explanation for our findings. NMDA is capable of activating SPN directly. Consequently, the pressor response to intrathecal NMDA is more likely to be a result of activation of NMDA receptors on SPN rather than an indirect effect from activation of unidentified spinal interneurons. NO inhibits NMDA receptors in other neurons. The NO donor SIN-1 attenuates NMDA-induced increases in intracellular calcium and inhibits NMDA-induced currents in striatal neurons.23 This is a cyclic GMP–independent effect of NO on the redox modulatory site of the NMDA receptor24 that appears to be mediated by nitrosylation of a specific cysteine residue on the NR2A subunit of the NMDA receptor.25 Our results are consistent with a NO-mediated inhibition of NMDA receptors. In our experimental paradigm, this inhibition attenuates the sympathetic activation and pressor response elicited by NMDA receptor activation in the spinal cord. Thus, the effect of NO on NMDA-mediated responses in SPN that we have observed may affect the baroreflex and other cardiovascular responses that are dependent on the activity of RVLM neurons.26
In summary, we have demonstrated that NO attenuates the pressor responses to NMDA receptor–mediated neuronal excitation and is likely to modulate descending excitatory signals mediating cardiovascular responses to diverse stimuli.
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Acknowledgments |
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This work was supported by a grant-in-aid from the National Health and Medical Research Council of Australia. Natalie Hulbert provided expert technical assistance.
Received April 6, 2000; first decision May 2, 2000; accepted June 30, 2000.
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References |
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|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic messenger: review. Ann Intern Med. 1994;120:227–237.
2.
Zanzinger J, Czachurski J, Seller H. Inhibition of
sympathetic vasoconstriction is a major principle of vasodilation by
nitric oxide in vivo. Circ Res. 1994;75:1073–1077.
3.
Zanzinger J, Czachurski J, Seller H. Inhibition of
basal and reflex-mediated sympathetic activity in the RVLM by nitric
oxide. Am J Physiol. 1995;268:R958–R962.
4.
Zhang K, Mayhan WG, Patel KP. Nitric oxide within the
paraventricular nucleus mediates changes in renal
sympathetic nerve activity. Am J Physiol. 1997;273:R864–R872.
5. Lage RC, Campagnole-Santos MJ, Fontes MA, Santos RA. Cardiovascular effects produced by nitric oxide-related drugs in the caudal ventrolateral medulla. Neuroreport. 1999;10:731–735.[Medline] [Order article via Infotrieve]
6. Anderson CR. NADPH diaphorase-positive neurons in the rat spinal cord include a subpopulation of autonomic preganglionic neurons. Neurosci Lett. 1992;139:280–284.[Medline] [Order article via Infotrieve]
7. Blottner D, Baumgarten H. Nitric oxide synthetase (NOS)-containing sympathoadrenal cholinergic neurons of the rat IML-cell column: evidence from histochemistry, immunohistochemistry, and retrograde labeling. J Comp Neurol. 1992;316:45–55.[Medline] [Order article via Infotrieve]
8. Hakim MA, Hirooka Y, Coleman MJ, Bennett MR, Dmapney RAL. Evidence for a critical role of nitric oxide in the tonic excitation of rabbit renal sympathetic preganglionic neurons. J Physiol. 1995;482:2:401–407.
9. Garcia MC, Celuch SM, Adler-Graschinsky E. Possible participation of spinal nitric oxide in the control of the blood pressure in anesthetized rats. Brain Res. 1997;764:67–74.[Medline] [Order article via Infotrieve]
10. Koga N, Takano Y, Honda K, Saito R, Kamiya H. Roles of nitric oxide in the spinal cord in cardiovascular regulation in rats. Neurosci Lett. 1999;267:173–176.[Medline] [Order article via Infotrieve]
11. Lee SB, Koh HC, Kim ON, Sung KW, Kim SY. Intrathecal administration of sodium nitroprusside, a nitric oxide donor, increases blood pressure in anesthetized rats. Neurosci Lett. 1996;203:53–56.[Medline] [Order article via Infotrieve]
12. Anonymous. SPSS Base 7.5 for Windows: Users Guide. Chicago, Ill: SPSS Inc; 1997.
13. Southam E, Garthwaite J. The nitric oxide-cyclic GMP signalling pathway in rat brain. Neuropharmacology. 1993;32:1267–1277.[Medline] [Order article via Infotrieve]
14. Garthwaite J, Charles SL, Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature. 1988;336:385–388.[Medline] [Order article via Infotrieve]
15. Feldman DS, Terry AVJ, Buccafusco JJ. Spinal muscarinic cholinergic and nitric oxide systems in cardiovascular regulation. Eur J Pharmacol. 1996;313:211–220.[Medline] [Order article via Infotrieve]
16. Garthwaite J. Glutamate, nitric oxide and cell-cell signalling in the nervous system: review. Trends Neurosci. 1991;14:60–67.[Medline] [Order article via Infotrieve]
17. Garthwaite J, Boulton CL. Nitric oxide signaling in the central nervous system: review. Annu Rev Physiol. 1995;57:683–706.[Medline] [Order article via Infotrieve]
18.
Knowles RG, Palacios M, Palmer RM, Moncada S. Formation
of nitric oxide from L-arginine in the central nervous system: a
transduction mechanism for stimulation of the soluble guanylate
cyclase. Proc Natl Acad Sci U S A. 1989;86:5159–5162.
19. Wood J, Garthwaite J. Models of the diffusional spread of nitric oxide: implications for neural nitric oxide signalling and its pharmacological properties. Neuropharmacology. 1994;33:1235–1244.[Medline] [Order article via Infotrieve]
20. Dawson TM, Dawson VL. Nitric oxide synthase: role as a transmitter/mediator in the brain and endocrine system. Annu Rev Med. 1996;47:219–227.[Medline] [Order article via Infotrieve]
21.
Wu SY, Dun NJ. Calcium-activated release of
nitric oxide potentiates excitatory synaptic potentials in immature rat
sympathetic preganglionic neurons. J Neurophysiol. 1995;74:2600–2603.
22.
Dun NJ, Mo N. Inhibitory postsynaptic
potentials in neonatal rat sympathetic preganglionic neurones in vitro.
J Physiol(Lond). 1989;410:267–281.
23. Manzoni O, Prezeau L, Marin P, Deshager S, Bockaert J, Fagni L. Nitric oxide-induced blockade of NMDA receptors. Neuron. 1992;8:653–662.[Medline] [Order article via Infotrieve]
24. Lei SZ, Pan ZH, Aggarwal SK, Chen HS, Hartman J, Sucher, NJ, Lipton SA. Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron. 1992;8:1087–1099.[Medline] [Order article via Infotrieve]
25. Choi YB, Tenneti L, Le DA, Ortiz J, Bai G, Chen HS, Lipton SA. Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation. Nat Neurosci.. 2000;3:15–21.[Medline] [Order article via Infotrieve]
26. Dampney RAL. The subretrofacial vasomotor nucleus: anatomical, chemical and pharmacological properties and role in cardiovascular regulation. Prog Neurobiol. 1994;42:197–227.[Medline] [Order article via Infotrieve]
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