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(Hypertension. 2001;37:246.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Nitric Oxide and Central Antihypertensive Drugs
One More Difference Between Catecholamines and Imidazolines
From the Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire, Faculté de Médecine, Université Louis Pasteur, Strasbourg, France.
Correspondence to Dr P. Bousquet, Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire, Faculté de Médecine, Université Louis Pasteur, 11 rue Humann, 67000 Strasbourg, France. E-mail Pascal.Bousquet{at}medecine.u-strasbg.fr
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Abstract |
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NO is known to be involved in the peripheral and central regulation of the cardiovascular function. It plays a neuromodulatory role via a direct action on presynaptic nerve terminals, stimulating the release of
-aminobutyric acid, glutamate,
and norepinephrine. Our aim was to study the possible role
of NO in the cardiovascular effects of the central
antihypertensive drugs clonidine, rilmenidine, and
-methyl-norepinephrine (-MNA). Sites and mechanisms
of the hypotensive action of these drugs were different; clonidine and
rilmenidine acted on imidazoline receptors in the nucleus reticularis
lateralis, whereas -MNA acted upon
2-adrenoceptors in the nucleus tractus
solitarius. The influence of
NG-nitro-L-arginine,
an NO synthase inhibitor, on the central hypotensive
effects of these drugs was investigated in
pentobarbital-anesthetized rabbits. The intracisternal (IC)
administration of -MNA (30 µg/kg) induced hypotension (79±2
versus 103±4 mm Hg) and bradycardia (222±8 versus 278±4 bpm)
(P<0.05) (n=5). Clonidine
(0.07 µg/kg IC) also induced hypotension (69±5 versus 99±4
mm Hg) and bradycardia (266±7 versus 306±10 bpm)
(P<0.05) (n=5). In addition to
clonidine, rilmenidine (1 µg/kg IC) induced hypotension (64±4 versus
97±4 mm Hg) and bradycardia (264±11 versus 310±4 bpm)
(P<0.05) (n=5). Pretreatment
with
NG-nitro-L-arginine
(900 µg/kg IC) completely prevented the hypotensive effect of -MNA
but influenced the cardiovascular effects of neither
clonidine nor rilmenidine. These results confirm that imidazoline
drugs, such as clonidine, rilmenidine, and the
catecholamine 2-adrenoceptor
agonist -MNA, have distinct mechanisms of action.
Key Words: nitric oxide • blood pressure • clonidine • norepinephrine • central nervous system
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The free radical NO is known to be involved in the central and peripheral regulation of the cardiovascular function (for a review, see Krukoff1 ). In the central nervous system, NO has a neuromodulatory role, influencing the peripheral autonomic function.2 3 In neurons, the synthesis of NO from L-arginine is catalyzed by the NO synthase enzyme (nNOS), which is calcium/calmodulin dependent. This enzyme is stimulated by glutamate-induced activation of N-methyl-D-aspartate receptors, which increases the intracellular influx of calcium; in turn, the diffusible NO activates soluble guanylyl cyclase, producing cGMP in neighboring neurons and astrocytes to trigger its effect.1 4 Immunohistochemical studies have shown the existence of cellular groups containing nNOS that are constitutive of many important autonomic structures of the hypothalamus, brain stem, and spinal cord.5 6 7 8 9
We are particularly interested in the brain stem structures
of the baroreflex arch, including the nucleus tractus solitarius (NTS)
and the rostroventrolateral medulla/nucleus reticularis lateralis
(RVLM/NRL). The NTS is the site of the hypotensive action of
-methyl-norepinephrine (-MNA), the active metabolite
of -methyl-dopa, which is a catecholamine, full agonist
at
2-adrenoceptors.10 11
The RVLM/NRL is the major site of the hypotensive effect of imidazoline
drugs such as clonidine and rilmenidine, which requires specific
imidazoline receptors, insensitive to
catecholamines.12 13 14 15 16 17
Ma et al18 showed
that
NG-nitro-L-arginine
methyl ester, an NOS inhibitor applied directly into the
NTS, inhibited the electrical activity of the
cardiovascular neurons. According to Chan and
Sawchenko,19 there is a
large amount of NOS mRNA in the NTS. These authors also reported that
NOS expression was enhanced in the RVLM region during the
baroreflex activation. They described a direct NOergic pathway from the
NTS to the RVLM, which does not relay in the caudal
ventrolateral medulla. Microinjections of L-arginine into the
RVLM/NRL induced an inhibition of the sympathetic tone, which was
prevented by nNOS
blockers.20 21
Agmatine, which was described as an endogenous ligand of
the imidazoline receptors, as well as NO, derives from L-arginine.22
Agmatine is also a competitive inhibitor of NOS and thus
could be an endogenous regulator of the production
of NO.23 A large body of
evidence supports that NO plays a neuromodulatory role and influences
the release of several neurotransmitters, such as glutamate,
-aminobutyric acid (GABA), and
norepinephrine.24 25 26 27 28 29
Our aim in the present work was to study the possible
role of NO in the cardiovascular effects of
antihypertensive imidazoline drugs, such as clonidine, rilmenidine, and
the catecholamine -MNA. The central
cardiovascular effects of -MNA are due to
stimulation of the 2-adrenoceptors of the
NTS, whereas those of clonidine and rilmenidine involve the specific
imidazoline receptors of the
NRL.12 13 14 15 16 17
To study whether NOergic pathways are involved in the
cardiovascular effects of clonidine, rilmenidine,
and/or -MNA, we used
NG-nitro-L-arginine
(L-NNA) as an NOS inhibitor.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Animals and Hemodynamic Measurements
Normotensive male rabbits (Zika strain) weighing 2.5 to 3.5 kg were anesthetized with 40 mg/kg sodium pentobarbitone injected through the marginal vein of the ear. Rectal temperature was maintained at 38±0.5°C with the aid of a warming blanket as soon as anesthesia was established (Harvard Apparatus Ltd). The animals were tracheotomized, immobilized with pancuronium bromide (1 mg/kg IV), and artificially ventilated with room air (model 6025; Hugo Sachs Elektronic). The ventilation parameters were adjusted to maintain PaO2 at 100 mm Hg and PaCO2 at <40 mm Hg. The right femoral vein was catheterized to allow intravenous injections, and the instantaneous arterial pressure was measured through a catheter inserted in the abdominal aorta via the right femoral artery and connected to a pressure processor and recorder (model BS-272; Gould Electronics). Mean arterial pressure (MAP) was calculated as diastolic pressure plus one third of the differential pressure. The heart rate (HR) was also continuously monitored from the pressure signal with a Gould Biotach amplifier (model 13-4615-66).
Drugs
The following drugs were used: sodium pentobarbitone
(Sanofi), pancuronium bromide (Pavulon), L-NNA (Sigma), clonidine
(Catapressan; Boehringer-Ingelheim), -MNA (Sigma), and
rilmenidine (Servier).
Intracerebral
Injections
The animal’s head was placed in a
stereotaxic frame (La Précision Cinématographique
Française). At the beginning of each experiment, before any drug
injection, an equal volume of cerebrospinal fluid was withdrawn. Single
doses of clonidine (0.07 µg/kg), rilmenidine (1 µg/kg), or -MNA
(30 µg/kg) were injected intracisternally (100 µL) directly within
the cisterna magna after a pretreatment with vehicle or L-NNA. In
pretreated animals, L-NNA was injected 15 minutes before the single
dose of clonidine, rilmenidine, or -MNA. The effects of the drugs
were measured only when the steady-state effect was achieved (ie, 5 to
10 minutes after central injection). Repeated injections of vehicle
never produced any significant change of the
hemodynamic parameters
(n=5).
Statistics and Calculations
Data are given as mean±SEM. Effects of drugs in the
presence or absence of L-NNA were compared. The homogeneity of the
initial cardiovascular parameters in all
groups was checked with a 1-way ANOVA. Results were then compared with
an intergroup ANOVA with repeated measures followed by a post hoc test.
A value of P<0.05 was used as
the criterion of significance, and n represented the number
of experiments. The calculations were made with computer-assisted
analyses with the StatView software (Abacus
Concepts).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In all groups of animals, the initial cardiovascular parameters were checked for their homogeneity and were not significantly different (P>0.05).
Central Cardiovascular Effects
of -MNA in the Presence or Absence of L-NNA
The intracisternal (IC) administration of a single dose
of -MNA (30 µg/kg) induced hypotension and bradycardia; MAP varied
from 103±4 to 79±2 mm Hg
(P<0.05), and HR varied from
278±4 to 222±8 bpm (P<0.05)
(n=5).
L-NNA (900 µg/kg IC) had no significant effect on the
cardiovascular parameters by its own: MAP
varied from 96±4 to 106±2 mm Hg (NS) and HR varied from 290±7
to 294±4 bpm after 15 minutes of observation (NS, n=5). Pretreatment
with L-NNA (900 µg/kg IC) completely prevented the hypotensive effect
of -MNA but only partially its bradycardic effect. MAP varied from
96±4 to 97±3 mm Hg (NS), and HR varied from 290±7 to 256±10
bpm (P<0.05). Compared with
the maximal bradycardic effect observed with -MNA alone (
HR
56 bpm), -MNA–induced bradycardia was significantly weakest
in animals pretreated with L-NNA (HR 34 bpm)
(P<0.05)
(Figure).
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Central Cardiovascular Effects
of Clonidine in the Presence or Absence of L-NNA
The IC administration of a single dose of clonidine
(0.07 µg/kg) reduced MAP from 99±4 to 69±5 mm Hg and HR from
306±10 to 266±7 bpm (P<0.05)
(n=5). In another series of 5 experiments, L-NNA (900 µg/kg IC) had
no significant effect on the cardiovascular
parameters by itself: MAP varied from 101±2 to 107±2
mm Hg (NS) and HR varied from 302±10 to 308±10 bpm (NS) within 15
minutes of injection. In these animals, L-NNA (900 µg/kg IC)
prevented neither the hypotensive nor the bradycardic effects of
clonidine subsequently administered intracisternally: MAP still varied
from 100±2 to 64±3 mm Hg, and HR varied from 302±10 to 248±17
bpm (P<0.05) (n=5). These
values were not significantly different from control values observed
with clonidine alone
(Figure).
Central Cardiovascular Effects
of Rilmenidine in the Presence or Absence of L-NNA
The IC administration of a single dose of rilmenidine
(1 µg/kg) induced hypotension and bradycardia; MAP varied from 97±4
to 64±4 mm Hg, and HR varied from 310±4 to 264±11 bpm
(P<0.05) (n=5). L-NNA (900
µg/kg IC) did not significantly change the
cardiovascular parameters: MAP varied from
96±2 to 106±1 mm Hg and HR varied from 318±9 to 334±10 bpm
after 15 minutes of observation (NS, n=5). As observed with clonidine,
the pretreatment with L-NNA did not prevent the hypotensive and
bradycardic effects of rilmenidine. MAP varied from 96±2 to 70±3
mm Hg, and HR varied from 318±9 to 270±4 bpm
(P<0.05,
n=5).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Based on the data from preliminary experiments, we selected the dose of each drug (clonidine, rilmenidine,
-MNA) that
reduced blood pressure by 
30% when injected
intracisternally.
This study shows that a central pretreatment with the NOS
inhibitor L-NNA completely prevented the hypotensive effect
of -MNA, whereas under the same experimental conditions, the
cardiovascular effects of clonidine and rilmenidine
were not affected by this pretreatment. This observation provides
further evidence that the pharmacological mechanisms of action of
-MNA on the one hand and of clonidine and rilmenidine on the other
hand and/or the central structures that they target are different.
-MNA is a catecholamine with high affinity and
selectivity for 2-adrenoceptors, whereas
clonidine and rilmenidine require nonadrenergic
imidazoline-specific receptors to reduce blood
pressure.12 13 14 15 16
It is established that the site of the hypotensive action of the
-MNA is the NTS, where imidazoline-like drugs are inactive, whereas
the NRL is the site of the hypotensive action of imidazoline-like drugs
but not of
catecholamines.10 13 14 15 30 31 32
Rilmenidine is a centrally acting antihypertensive drug that is more
selective for imidazoline receptors than
clonidine.33 Like
clonidine, the cardiovascular effects of rilmenidine
were insensitive to the NOS blocker L-NNA. The present data provide
additional evidence that show clonidine and rilmenidine injected
intracisternally do not act on the
2-adrenoceptors of the NTS, which are
normally targeted by -MNA injected in the same
manner.31 They also indicate
that the central hypotensive effect of imidazoline-like drugs does not
involve NO.
The most attractive hypothesis concerning the mechanisms
beyond the present data are that an interaction may occur at the
presynaptic level on neuronal pathways that project into the NRL.
Stimulation of the NTS by -MNA is known to elicit a presynaptic GABA
release within the
NRL.34 35 This
GABA release might be modulated by
NO.28 29 In vitro
studies carried out in isolated brain nerve endings demonstrated that
the depolarizing effect of NO was due to a direct action on the
presynaptic membrane; this depolarization was mediated by a decrease in
potassium permeability and an inhibition of the sodium
pump.36 In addition,
electrophysiological experiments performed
on rat nodose ganglions nerve endings have shown that NO donors such as
the diethylamine-NO and
S-nitroso-N-acetylpenicillamine
induced a dose-dependent depolarization; a pretreatment with LY83583,
an inhibitor of guanylate cyclase, prevented
the depolarizing effect of these drugs, suggesting that the
depolarizing effect of NO was mediated by
cGMP.37 38 The
NOS inhibitor could therefore block the NO-stimulated GABA
release in the NRL and consequently prevent the hypotensive effect of
-MNA.
Further experimental studies are needed to test these hypotheses, which are not exclusive.
The hypotensive effect of clonidine, which is known to
originate in the NRL, where -MNA is inactive, is insensitive to NOS
blockers. Therefore, any direct interaction among clonidine,
rilmenidine, and NO in the NRL can be ruled out. We also observed a
difference between the bradycardic effects of -MNA and clonidine in
the presence of L-NNA. The bradycardic effect of clonidine and
rilmenidine was not prevented by the pretreatment with L-NNA, whereas
that of -MNA was diminished by L-NNA.
In conclusion, the present study provides further
evidence that the central sites and/or mechanisms of the
cardiovascular effects of clonidine, rilmenidine, and
-MNA are different. We also show for the first time that the central
hypotensive effect of the 2-adrenoceptor full
agonist -MNA is NO
dependent.
Received August 3, 2000; first decision August 7, 2000; accepted August 28, 2000.
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