From the Institute of Physiology I, University of Heidelberg (Germany).
Correspondence to Dr J. Zanzinger, I. Physiologisches Institut, Im Neuenheimer Feld 326, D-69120 Heidelberg, FRG. E-mail zanzing{at}novsrv1.pio1.uni-heidelberg.de
Methods and Results—Nitrate tolerance was induced by oral
administration of isosorbide dinitrate (ISDN, 4 mg/kg per day for 4
weeks) in eight pigs. Four of them were chronically instrumented for
the measurement of mean arterial blood pressure and cardiac
output in the conscious state. ISDN treatment caused
hemodynamic tolerance to NO donors and significantly
increased the hypotensive responses to pharmacologic ganglionic
blockade in conscious pigs. In general anesthesia,
ISDN-treated animals and age-matched controls (n=5) had similar
baseline renal sympathetic nerve activity and in both groups neither
inhibition of NO synthases (NOS) nor administration of NO donors to the
brain stem by intracerebroventricular
(ICV) infusions caused significant changes in baseline renal
sympathetic nerve activity. However, whereas
sympathoexcitatory responses to glutamate (0.5
mL, 0.1 mol/L, ICV) or electrical stimulation of somatic nerve
afferents were significantly potentiated by central NOS inhibition and
attenuated by NO donors in controls, these treatments no longer had
significant effects in ISDN-treated pigs. Furthermore, reflex
sympathetic activation in response to intravenous NO donor
treatment was more pronounced in nitrate tolerant animals, which
suggests loss of central sympathoinhibitory effects of NO.
Subsequent histology on brain stem slices with
NADPH-diaphorase as NOS marker revealed significant
reduction of NOS density in ISDN-treated pigs.
Conclusions—Long-term administration of organic nitrates reduces
the number of NO-producing neurons in the brain stem and causes loss of
inhibitory effects of NO on sympathetic excitability. This
component of tolerance to organic nitrates may be important in patients
confronted frequently with sympathetic activation caused by mental
and/or physical stressors.
Throughout the study the pigs were fed with a standard diet (400 to 500
g/d of a cereal-based diet containing 12.6 MJ metabolizable energy/kg
with 15.5% crude protein) and had free access to tap water. For
induction of nitrate tolerance, pigs received 4 mg/kg per day ISDN with
the diet. Two other groups of pigs were housed similarly but were not
chronically instrumented. One group (n=4) received ISDN at the same
dosage and the other group (n=5, control) was fed with a similar diet
without drugs for the same duration. The mean weight gain throughout
the study period was 428±39 g/d. All animals were studied in acute
experiments (see below) subsequent to the chronic observations. The
care of the pigs and the execution of the experimental protocol were
supervised by an independent veterinarian in accordance with German
laws and the animal welfare regulations of the University of
Heidelberg.
Acute Experiments
Detection of NO Synthases Within the Brain Stem
Drugs and Infusions
Data Analysis
Changes in Sympathetic Functions Associated With Nitrate
Tolerance
Effects of Central NOS Inhibition on Hemodynamic
and Sympathetic Responses to Systemic Administration of NO
Donors
NOS Density Within the Brain Stem
Impaired Inhibition of Sympathetic Excitability Versus
Neurohumoral Activation
Another factor contributing to nitrate tolerance, often called
pseudotolerance,25 is the enhancement of
sympathetic activity during long-term nitrate administration. Our
results are largely in accordance with those of Stewart et
al,25 who similarly observed in conscious dogs
much greater decreases in MAP in response to ganglionic blockade by
hexamethonium in nitrate-tolerant animals, suggesting
that overall sympathetic activity may be markedly higher during nitrate
tolerance. These authors also reported that during
anesthesia, vasodilator effects of NO donors became
considerably greater in tolerant dogs because of the reduced
sympathetic activity under these conditions, which is in accordance
with the present findings. However, the term pseudotolerance
probably does not correctly describe the mechanisms underlying
activation of sympathetic activity in nitrate tolerance. Together with
our previous observations,15 the results of this
study instead suggest that nitrates cause specific impairment
(tolerance) of the modulatory effects of NO on
sympathoexcitatory afferents to the vasomotor
center in the brain stem, that is, the RVLM. A term such as
"sympathetic tolerance" therefore would be more appropriate to
describe this phenomenon. Sympathoexcitation by afferents to the RVLM
is most important in the awake state when sympathetic activation can be
caused by influences such as emotional stress, pain, exercise, or
ventilatory dysfunctions.18 The relevance of an
enhancement of the effects of these influences in nitrate tolerance are
difficult to study and may become only fully apparent in normal living
conditions. We used ICV-injections of glutamate and activation of
somatosympathetic reflexes to study the role of these excitatory
afferent influences in acute experiments during
anesthesia.15
Relevance of the Central Effects of NO in the Vasodilator Effects
of NO Donors In Vivo
Structural Changes in the Brain Stem Caused by Long-term ISDN
Treatment
Perspectives
Received October 14, 1997;
revision received December 29, 1997;
accepted January 9, 1998.
© 1998 American Heart Association, Inc.
Basic Science Reports
Impaired Modulation of Sympathetic Excitability by Nitric Oxide After Long-term Administration of Organic Nitrates in Pigs
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Endogenous
nitric oxide (NO) reduces sympathetic vasoconstriction by attenuating
neuronal excitability in the brain stem and inhibition of
postganglionic neurotransmission. We studied whether this modulation of
sympathetic circulatory control by NO may be altered during chronic
administration of NO donor drugs in pigs.
Key Words: nitric oxide • brain • nervous system
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Endogenous NO
regulates vascular tone by direct actions on smooth
muscle1 2 and, in addition, to a significant
extent by inhibition of sympathetic vasoconstrictor
mechanisms.3 4 5 6 Organic nitrates that are known
to act through the release of NO7 are widely used
as vasodilator drugs. The efficacy of these drugs, however, can be
severely impaired by the development of nitrate
tolerance.8 9 Although it has been shown that NO
release from organic nitrates is preserved during tolerance in
vivo,10 reduced vasodilator effects of NO caused
by enhanced endothelial superoxide
production11 and endothelin-1
release12 may be causes of true vascular
tolerance. In addition, neurohumoral activation and pseudotolerance
associated with increased sympathetic activity have been proposed to
contribute the phenomenon.13 Under physiologic
conditions, NO inhibits sympathetic vasoconstrictor influences by both
reducing the release of noradrenaline from postganglionic
sympathetic fibers and by attenuation of neuronal sympathetic
excitability within the medullary areas that regulate sympathetic
outflow from the brain stem.3 14 15 Because
impaired modulation of sympathetic functions by NO could contribute to
nitrate tolerance, we studied whether long-term organic nitrate
treatment affects basal and activated SNA in pigs and whether
vasoconstrictor effects of SNA may be altered in nitrate tolerance in
vivo. In addition, we studied the distribution of NOS within the lower
brain stem in both control and nitrate-tolerant pigs by
NADPH-diaphorase staining.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Long-term Experiments
Young farm pigs (n=4, 16 to 20 kg body wt) were sedated with
ketamine (10 mg/kg IM) and anesthetized with
pentobarbital (12 to 15 mg/kg IV). Anesthesia was
maintained after intubation with isoflurane (1.0% to 1.5%) in the
inspired air consisting of 2:1
N2O/O2. An aseptic left
thoracotomy was performed through the fourth intercostal space under
positive pressure respiration. The pulmonary artery was
instrumented with an transient time ultrasonic flow probe (Triton,
ART2) for measurement of cardiac output and with a chronic catheter for
infusion of drugs. For measurement of arterial BP, another
catheter was implanted in the ascending aorta. Catheters and wires were
tunneled subcutaneously to the dorsal neck. The pigs received
antibiotics (cephalosporin 1 mg/kg) and analgesia (metamizol 50 mg/kg)
for 1 week after surgery and were allowed to recover from surgery for
at least 7 days. During the recovery period, the pigs were made
familiar with the laboratory and trained to rest quietly in a straw
filled cage for experimental periods of 
1 hour. Measurements were
subsequently made in conscious animals at least twice per week during
continuous registration of all parameters. To monitor the
development of nitrate tolerance, dose-response relations for the NO
donor SNAP were performed by intravenous infusion after
periods of registration of baseline hemodynamic
parameters.
Acute experiments were performed with similar general
anesthesia as described for long-term experiments. For
infusion of drugs and for measurement of blood pressure, catheters were
placed into a femoral vein and artery (advanced in the abdominal
aorta). Antibiotics (100 mg/kg ampicillin) were given to prevent
possible influences of infections such as the induction of expression
of inducible NOS by bacterial endotoxins. The pigs were paralyzed by
0.2 mg/kg per hour pancuronium bromide and artificially ventilated by a
tracheal tube. End-tidal CO2 was kept at normal
levels by adjustment of ventilatory depth and rate.
Arterial blood gases were monitored with a blood gas
analyzer (AVL 990, AVL List) and maintained in the normal range
by administration of sodium bicarbonate solutions or adjustment of
ventilation. Rectal temperature was maintained at 38.5°C by a
thermostatically controlled infrared lamp. For measurements of CO in
animals that were not instrumented long term, a biluminal (right
atrium, pulmonary artery) 5F Swan-Ganz thermodilution catheter
(Baxter) was inserted through a jugular vein and advanced through the
right ventricle in the A pulmonalis under blood pressure control. In
the other pigs CO was measured by the chronically implanted transient
time flow probes (ART, Triton). The similarity of CO data has been
checked in previous studies.15 For
recording of RSNA, the left renal nerve was retroperitoneally
exposed, placed on bipolar platinum electrodes, and kept in a mixture
of petroleum jelly and paraffin oil. Neural signals were amplified
(x20 000 to 50 000; Tektronix AM 502), filtered (2 to 3 kHz), and
stored and analyzed with a CED 1401 interface connected to an
80486 PC computer. RSNA was full-wave rectified and then
resistance-capacity integrated with a time constant between 7 to 10 ms.
To activate somatosympathetic reflex responses, the left great
sciatic nerve (N ischiadicus) was placed on bipolar platinum
electrodes, embedded in petroleum jelly, and connected to an isolated
stimulator (Digitimer). Stimulation was performed to produce
reproducible submaximal excitatory effects on RSNA by using consecutive
trains of 20-second length with 10 to 20 V, 30 Hz, and 0.5- to 1-ms
pulse duration at intervals of 120 seconds.
Brain stems were removed from the skull and placed in 0.1 mol/L
PBS (pH 7.4) containing 4% paraformaldehyde for at
least 24 hours (at 4°C). After fixation, tissue from all animals was
rinsed with pure PBS and placed in 15% sucrose containing PBS for
cryoprotection. Cryotome sections were cut 30 µm thick, and
the free-floating sections were stained as follows. They were incubated
in PBS containing 0.75 mg/mL of reduced nicotinamide adenine
dinucleotide phosphate (NADPH, Boehringer,
Mannheim) and 0.375 mg/mL nitro blue tetrazolium at 37°C for 3 hours.
After incubation, the sections were rinsed in PBS, put on
gelatin-coated glass slides, dried in air, dehydrated, and coverslipped
in Permount. Mapping of the brain stem was done in analogy to Berman's
atlas of the cat brain,16 and slices derived from
similar rostrocaudal locations normalized to the overall size of the
medulla were used for the study. The RVLM region was also functionally
identified by microinjections of glutamate (500 nL, 0.5 mol/L) in three
pigs in vivo, yielding maximal
sympathoexcitatory responses on microinjections
at the following distances relative to the obex: 4.5 to 5.0 mm
lateral, 3.5 to 4.0 mm rostral, 5.0 to 6.0 mm deep (from the
dorsal surface of the medulla). For statistical comparison of NOS
density, NOS-positive cells on two slices (3.0 and 4.0 mm
rostrally to the obex, respectively) were counted on a total area of
50 mm2 per animal including both the
ventrolateral and the dorsal medullary region. As similarly observed in
a previous study on different laboratory animal
species,17 variation of NOS density between
regions was small. Therefore, these data were pooled. NOS density is
expressed in number of NOS-positive neurons/mm2
slice.
SNAP was from Alexis Chemicals. All other drugs were from Sigma.
All drugs were dissolved in distilled water. For preparation of the
final concentrations, the substances were further diluted in Ringer
solution shortly before administration. For ICV-administrations, a
catheter was inserted into the cerebroventricular space
from the dorsal surface of the medulla at the level of the obex and
advanced to the ventral surface of the medulla. The position of the
catheter was functionally verified by instantaneous excitatory
sympathetic responses to injections (0.3 mL) of glutamate, which are
finally integrated within the brain stem by presympathetic neurones
within the RVLM.15 18 Central NOS inhibition was
carried out by short-term infusion (within 5 minutes, 1 mL/min ICV) of
L-NNA (0.3 mmol/L). Effects of exogenous NO were tested by
short-term ICV-infusion of SNAP (100 µmol/L) after NOS
inhibition. As a test for central sympathetic excitability, 0.5 mL of
0.1 mol/L glutamate was injected (ICV) subsequent to the above
pretreatments or sham control.
HR was derived from the BP signal. TPR was calculated as
(MAP-CVP)/CO, where central venous pressure (CVP) was assumed to be
2 mm Hg when no measured data were available (during CO
measurements with flow probes). RSNA was resistance-capacity integrated
and measured in arbitrary units (aU). CO and TPR measurements in
chronically instrumented animals were normalized with respect to the
increase in metabolic body mass
(W0.75) during the study period. For
presentation in summarizing figures, RSNA data were
normalized and expressed in percentage of control values (%RSNA). All
direct measurements were stored on a linear recorder (Gould) and on
DAT tape for further computing. Responses to drugs were separately
calculated as individual differences from pretreatment values.
Responses to electrical stimulation were calculated as average peak
values derived from three consecutive stimuli, respectively. All data
were analyzed by ANOVA (for repeated measurements where
appropriate). A comparison of the data obtained from chronically
instrumented and noninstrumented ISDN-treated pigs (n=4 each) in the
acute experiments revealed no significant differences, suggesting that
the instrumentation procedure had no effect on the outcome of the
study. Therefore these data were subsequently pooled for statistical
evaluation. Comparison of means was carried out with Tukey's
studentized range test. Differences of P<0.05 were
considered to be significant. Values are reported as mean±SEM.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Assessment of Nitrate Tolerance in Conscious Pigs
Measurements of MAP, CO, and HR obtained from conscious pigs
throughout the study period are shown in Figure 1
. Chronic organic nitrate treatment (4
mg/kg per day) had only small hemodynamic effects.
While significant increases in HR occurred during the first week of
ISDN treatment, the other parameters were not significantly
affected. However, as shown in Figure 2, significant impairment of the arterial vasodilatory effects
of additional exogenous NO (SNAP, 30 to 300 µg/kg), as indicated by
reduced changes in vascular resistance occurred already after the first
week of ISDN treatment and was further enhanced until the fourth week
of treatment. To get an estimate of the relevance of autonomic
influences on blood pressure in awake animals, ganglionic
neurotransmission was blocked by hexamethonium (0.5
mg/kg IV) in the four chronically instrumented pigs during control
conditions and in the fourth week of ISDN treatment.
Hexamethonium caused significantly less hypotension
during control conditions (MAP fell from 92.1±3.1 to 80.4±4.5
mm Hg) than in the nitrate-tolerant state (99.2±2.9 versus
70.8±1.8 mm Hg).
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Figure 1. Baseline hemodynamic
parameters during long-term ISDN treatment (4 mg/kg per
day) in conscious pigs (n=4). Course of MAP (in mm Hg), HR (in
bpm), and normalized CO (L/min/kg W0.75) during the
observation period of 5 weeks. *P<0.05 vs control
values (week 0).
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Figure 2. Development of nitrate tolerance during long-term
ISDN treatment as indicated by reduced dilator effects of exogenous NO
in conscious pigs (n=4). Effects of intravenous
administration of the NO donor SNAP (30 to 300 µg/kg) on normalized
TPR (in mm Hg/[L/min per kilogram W0.75]) are
shown.
A comparison of baseline hemodynamics and
RSNA between control and ISDN-treated pigs under general
anesthesia in acute experiments is given in the
Table. Figure 3 shows
original tracings of baseline hemodynamics and RSNA and
responses to activation of somatosympathetic reflexes by electrical
stimulation of the great sciatic nerve in a control and a
nitrate-tolerant pig during similar general anesthesia.
Baseline sympathetic activity was slightly higher in the tolerant pig,
but comparable RSNA increases and hemodynamic responses
to sciatic nerve stimulation could be evoked in both animals, which
suggests maintained integrity of theses reflexes. A summary of the
effects of reflex activation on MAP and RSNA for all animals and the
effects of acute
intracerebroventricular inhibition of
NO synthesis or NO donor treatment in control and nitrate-tolerant
animals is shown in Figure 4. In contrast
to controls, ISDN-treated pigs no longer responded to the pharmacologic
variation of NO availability within the brain stem. When, as shown in
Figure 5, glutamate was injected
intracerebroventricularly as a test for
tonic excitation of medullary vasomotor neurons, the hypertensive
effects were, albeit not significantly, greater in ISDN-treated pigs.
The disappearence of sympathoinhibitory effects of NO after
ISDN treatment was similarly observed.
View this table:
[in a new window]
Table 1. Baseline Sympathetic Activity and Hemodynamics
in Normal (n=5) and Nitrate-Tolerant Pigs (n=8) During Acute
Experiments With General Anesthesia
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[in a new window]
Figure 3. Representative tracings of effects
of somatosympathetic reflex activation by electrical stimulation of the
left greater sciatic nerve on renal sympathetic nerve activity (RSNA,
RN), blood pressure (BP), cardiac output (CO), and heart rate (HR)
during control conditions in an anesthetized untreated and a
chronically ISDN-treated pig (4 mg/kg per day), respectively.
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Figure 4. Disappearance of the
sympathoinhibitory effects of central NO on
somatosympathetic reflexes in nitrate tolerance. Summary of the effects
of ICV treatment with sham control, L-NNA (0.3 mmol/L ICV), or
SNAP (100 µmol/L ICV) on the responses of blood pressure
( 
MAP) and %RSNA to electrical stimulation of the left greater
sciatic nerve (10 to 20 V, 1-ms pulse duration, 30 Hz for 20 seconds,
every 2 minutes) in anesthetized control (n=5) and ISDN-treated
pigs (n=8). Asterisks denote significant changes from control.
*P<0.05, **P<0.01.
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Figure 5. Tolerance to inhibitory effects of NO
on glutamate response in the ventrolateral medulla oblongata after
long-term ISDN treatment. Effects of ICV treatment with sham (control),
L-NNA (0.3 mmol/L ICV), or SNAP (100 µmol/L ICV) on the
responses of blood pressure ( MAP) and %RSNA to ICV injections of
glutamate (0.5 mL, 0.1 mol/L) in anesthetized control (n=5) and
ISDN-treated pigs (n=8). Asterisks denote significant changes from
control. *P<0.05, **P<0.01.
To test whether nitrate tolerance may alter the impact of
centrally acting NO on sympathetic and/or hemodynamic
responses to systemically administered NO donors, SNAP (40 µg/kg) was
given intravenously after NOS inhibition in the brain stem
(ICV) in five control and four ISDN-treated pigs. The results are shown
in Figure 6. Whereas SNAP caused
significantly smaller decreases in blood pressure in nitrate-tolerant
animals, the correspondent increases in sympathetic activity in
response to baroreceptor unloading were even slightly greater than
during control conditions.
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Figure 6. Hypotensive and
sympathoexcitatory effects of
intravenous administration of NO donors in centrally
NOS-blocked pigs. Effects of SNAP (40 µg/kg IV) on the responses of
blood pressure ( MAP) and %RSNA in pigs (n=4 each) that were
pretreated by ICV infusion of L-NNA (0.3 mmol/L ICV) are shown.
Asterisks denote significant changes from control
(*P<0.05).
Figure 7A shows NADPH-positive
structures within the brain stem at the level of the RVLM in a control
and a nitrate-tolerant pig. A comparison of NOS density between control
and nitrate-tolerant pigs is given in Figure 7B. Nitrate-tolerant pigs
had significantly fewer NOS-positive neurons, which indicates that
nitrate administration reduced NOS density by 50% in these
animals.
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Figure 7. Distribution of NOS within the lower brain stem in
normal and nitrate-tolerant pigs. A, Representative
photographs of NOS-positive neurons within the RVLM in a control and an
ISDN-treated pig, respectively. B, Statistical comparison of NOS
density in the lower brain stem (3 to 4 mm rostrally to the obex)
expressed in numbers of NOS-positive neurons/mm2 slice.
*P<0.05 vs control. C, Cross section of the lower brain
stem (left side) of a pig at the level of the RVLM showing the position
from which the magnified pictures in "A" were taken.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The major finding of this study is that long-term administration
of exogenous NO in the form of organic nitrates may reduce
endogenous NO synthesis within the brain stem and cause
almost complete disappearance of the NO-mediated inhibition of
sympathetic excitability observed in normal pigs. Such loss of
sympathoinhibitory effects of both endogenous
and exogenous NO after long-term nitrate administration may contribute
to the clinical phenomenon of nitrate tolerance in patients receiving
long-term nitrate therapy.
Our experiments suggest that the mechanisms underlying the
observed changes in the modulation of sympathetic functions by NO
during long-term nitrate treatment may be different from the so-called
neurohumoral activation, which may be caused by stimulation of the
renin-angiotensin system associated with blood volume
expansion and increased circulating aldosterone levels in
response to long-term treatment with organic
nitrates.13 19 20 However, throughout the 4-week
period of ISDN treatment in the pigs studied here, BP probably has
always been higher than the threshold for significant activation of
renin release. Moreover, increases in sympathetic activity that
probably occurred in the animals are known to further increase this
threshold.21 Furthermore, it has been recently
shown in dogs22 and in
patients23 24 that activation of the
renin-angiotensin system may not be critically involved in
nitrate tolerance. Nevertheless, the fact that heart rate was
significantly increased during the first week of ISDN treatment could
mean that some baroreflex-mediated increase in sympathetic activity may
have initially contributed to the responses to ISDN.
We have recently characterized the mechanisms by which NO reduces
central sympathetic excitability in normal
pigs.15 In this and in a number of other
studies,26 27 28 29 it has been shown that NO probably
has no physiologically significant effects on
the function of the sympathetic baroreceptor reflex. Therefore it is
likely that the potentiation after long-term ISDN treatment of
increases in sympathetic activity in response to
intravenous administration of an NO donor (Figure 6
)
reflect the removal of centrally mediated sympathoinhibition by NO. In
other words, in nontolerant animals, NO donors may have acted to a
significant extent through central inhibition of sympathetic activity.
In addition, the "sympathetic tolerance" may have prevented greater
decreases in BP in response to the acute administration of the NO donor
SNAP in the nitrate-tolerant animals. Tolerance to the effects of
nitrates on sympathetic functions probably will be most important in
vascular beds that are under strong control of sympathetic nerves. One
can therefore expect that "sympathetic tolerance" may be primarily
apparent in resistance vessels and less in large arterial
vessel or veins. Previous observations suggest that this may be the
case. In veins and large (epicardial) arterial vessels, the
true vascular tolerance to nitrates appears to be more important,
whereas tolerance on the level of resistance vessels may be more
clearly dominated by sympathetic
influences.8 9 25 However, despite the fact that
nitrates are primarily given to reduce preload (effect on veins) and to
dilate epicardial arteries, tolerance to the effects of NO on
sympathetic functions may be nevertheless clinically relevant. Strong
sympathoexcitation in patients with coronary artery disease may
cause impaired myocardial perfusion and concomitantly increased
myocardial oxygen consumption because of increased cardiac work
(augmented TPR) and HR. Situations causing strong sympathetic
activation may be more frequent in patients living at home than in
patients in hospitals or in otherwise healthy experimental animals
trained to rest quietly during laboratory measurements. Therefore it is
possible that the relevance of "sympathetic tolerance" to organic
nitrates may be higher in normal clinical situations than under study
conditions.
Within a period of 4 weeks, nitrate therapy reduced the
number of NO-producing cells within the brain stem by 50%. In
addition, the activity of nNOS may also be reduced in nitrate tolerance
through NO-induced inhibition of nNOS.30 Recent
studies suggest that nNOS expression and activity within the brain can
be relatively rapidly upregulated and
downregulated.31 We used the relatively long
period of treatment to allow the development of structural changes, to
minimize the effects of initial short-term counterregulatory responses,
and to achieve steady-state conditions. On the other hand, the
long-term ISDN treatment protocol was chosen to simulate real long-term
nitrate therapy that sometimes lasts several months or even years in
patients. ISDN doses used in this study (4 mg/kg per day) would be in
the upper range of therapeutic doses in patients. However, in the pigs
studied here, these doses were still nonhypotensive. Furthermore,
overall metabolism in growing young pigs is considerably
higher than in mature humans. We therefore assume that the ISDN effects
observed were within clinically relevant ranges.
We have shown that long-term administration of organic
nitrates causes not only vascular tolerance but also a tolerance to the
inhibitory effects of NO on sympathetic excitability. These
functional alterations are associated with a reduction of NOS density
within the brain stem. NO-induced downregulation of nNOS observed in
the present study could also occur during inflammatory or
infectious diseases associated with expression of inducible NOS when
endogenous storage forms of NO such as
S-nitrosothiols reach the brain through the
bloodstream.32 33 Because sympathetic
excitability of nitrate-tolerant pigs was considerably lower than that
of normal pigs acutely treated (ICV) with NOS inhibitors,
endogenous counterregulatory mechanisms may have partially
replaced the functions of NO on central
sympathoexcitatory neurons. It will be
interesting to study these adaptations that prevent hypersensibility to
sympathoexcitatory stimuli. Another important
question to be studied is to what extent current concepts for the
prevention of nitrate tolerance, for example, intermittent
therapy,34 35 new NO donor
compounds,36 or cotreatment with
antioxidants,11 37 38 prevents or reduces the
structural and functional changes in sympathetic functions caused by
long-term nitrate therapy.
Selected Abbreviations and Acronyms
BP
=
blood pressure
CO
=
cardiac output
HR
=
heart rate
ICV
=
intracerebroventricular
ISDN
=
isosorbide dinitrate
L-NNA
=
nitro-L-arginine
MAP
=
mean arterial pressure
nNOS
=
neuronal NOS
NO
=
nitric oxide
NOS
=
NO synthase
PBS
=
phosphate-buffered saline
RSNA
=
renal sympathetic nerve activity
RVLM
=
rostral ventrolateral medulla
SNAP
=
S-nitroso-N-acetylpenicillamine
TPR
=
total peripheral resistance
Acknowledgments
This work was supported by the German Research Foundation
(D.F.G.) grant Za 176/3–1. We would like to thank Manuela Höfer
and Anita Kühner for skillful technical assistance and Gabi
Froelich for help with the preparation of figures.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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