Hypertension. 1999;34:802-807
(Hypertension. 1999;34:802-807.)
© 1999 American Heart Association, Inc.
Long-Term Nitric Oxide Inhibition and Chronotropic Responses in Rat Isolated Right Atria
Sonia R. Riado;
Angelina Zanesco;
Louis Allen Barker;
Iara M. S. De Luca;
Edson Antunes;
Gilberto De Nucci
From the Department of Pharmacology, Faculty of Medical Sciences
(S.R.A.), the Department of Histology and Embryology, Biology Institute
(I.M.S.D.L.), and the Department of Pharmacology, Faculty of Medical Sciences
(E.A., G.D.N.), UNICAMP, Campinas, Brazil; the Department of Physical
Education, Biosciences Institute, UNESP (A.Z.), Rio Claro, Brazil; and the
Department of Pharmacology, Louisiana State University Medical Center
(L.A.B.), New Orleans.
Correspondence to Angelina Zanesco, PhD, Department of Physical Education, Bioscience Institute, UNESP, Av 24A n° 1515, Bela Vista, CEP 13506-900, Rio Claro (SP), Brazil. E-mail azanesco{at}bestway.com.br
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Abstract
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Abstract—The long-term
administration of nitric oxide
synthesis inhibitors induces
arterial hypertension accompanied
by left
ventricular hypertrophy and myocardial
ischemic lesions.
Because the enhancement of sympathetic drive
has been implicated
in these phenomena, the current study was performed
to determine
the potency of ß-adrenoceptor agonists and muscarinic
agonists
on the spontaneous rate of isolated right atria from rats
given
long-term treatment with the nitric oxide inhibitor
N
-nitro-
L-arginine
methyl
ester (L-NAME). Atrial lesions induced by long-term treatment
with
L-NAME were also evaluated. Long-term L-NAME treatment
caused a
time-dependent, significant (
P<0.05) increase in
tail-cuff
pressure compared with control animals. Our results showed
that
the potency of isoproterenol, norepinephrine,
carbachol, and
pilocarpine in isolated right atria from rats given
long-term
treatment with L-NAME for 7, 15, 30, and 60 days was not
affected
as compared with control animals. Addition of L-NAME in vitro
(100
µmol/L) affected neither basal rate nor chronotropic
response
for isoproterenol and norepinephrine in rat heart.
Stereological
analysis of the right atria at 15 and 30 days
revealed a significant
increase on amount of fibrous tissues in
L-NAME–treated
groups (27±2.3% and 28±1.3% for 15 and 30 days,
respectively;
P<0.05) as compared with the control
group (22±1.1%).
Our results indicate that nitric oxide does not to
interfere
with ß-adrenoceptor–mediated and muscarinic
receptor–mediated
chronotropic responses.
Key Words: adrenergic receptor agonists • receptors, muscarinic • receptors, adrenergic, beta • blood pressure • nitric oxide
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Introduction
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The primary autonomic regulation of sinoatrial
nodal function
is by actions of sympathetic and parasympathetic
systems. The
effects of parasympathetic stimulation on the rat heart
are
mediated by muscarinic
M
2-receptors,
1 whereas the
sympathetic
actions are mediated by ß-adrenoceptors.
ß
1-, ß
2-,
and
ß
3-adrenoceptors are known to coexist in atria
from
several animal species, including humans.
2 3 4 5 In
rats, under
physiological conditions, only
ß
1-adrenoceptors mediate
chronotropic
response.
6
A role for nitric oxide (NO) has been proposed in the modulation
of sympathetic and parasympathetic neurotransmission in different
tissues.7 Regarding the effects of NO on the chronotropic
responses in the heart, little is known. Long-term administration of NO
synthesis inhibitors induces arterial
hypertension8 9 accompanied by left
ventricular hypertrophy and myocardial
ischemic lesions.10 The increase in the activity
of the renin-angiotensin system11 12 and
enhancement of sympathetic drive13 14 have been implicated
in these phenomena. However, the underlying mechanisms by which
long-term NO blockade induces arterial hypertension and
other cardiovascular changes remain unclear. Therefore,
the current study was performed to determine the potency of
ß-adrenoceptor agonists and muscarinic-agonists on the spontaneous
rate of isolated right atria from rats given long-term treatment with
the NO inhibitor
N-nitro-L-arginine
methyl ester (L-NAME). We have also evaluated atria lesions induced by
long-term treatment with L-NAME.
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Methods
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Experimental Design
Experiments were performed in 164 male Wistar rats (150 to
200
g) that were provided by animal care of Paulista State
University
(UNESP, Botucatu). They were randomly divided into 2
experimental
groups: control rats that received tap water alone and
treated
rats that received L-NAME (20 mg/rat per day, dissolved in the
drinking
water). The experiment lasted up to 8 weeks. All procedures
were
designed in accordance with the animal care guidelines of the
State
University of Campinas.
Blood Pressure Measurement
The mean arterial blood pressure was measured
by use of a modified tail-cuff method in awake animals.15
The measurements were performed weekly and 24 hours before the animals
were killed.
Functional Assays With Isolated Right Atria
At 7, 15, 30, or 60 days after L-NAME treatment, the
animals were anesthetized with halothane and euthanized by
stunning and exsanguination. The hearts were rapidly removed. The right
atria were isolated and mounted in a water-jacketed tissue chamber (20
mL volume) containing Krebs-Henseleit buffer, pH 7.3 to 7.5, at 37°C
and gassed with 95% O2-5%
CO2. The composition of the Krebs-Henseleit
buffer was (mmol) NaCl 124; KCl 4.75; MgCl2 1.30;
CaCl2 2.25; NaHCO3 25.0;
NaH2PO4 0.6; dextrose 10.0;
sodium ascorbate 0.3; and disodium EDTA 0.03. Ascorbate and EDTA (Sigma
Chemical Co) were added to inhibit the oxidation of
catecholamines.16 One hour was allowed to
obtain a stable basal rate. Another set of experiments in right atria
from naive rats was performed by the addition of L-NAME (100
µmol/L) in the tissue bath.
Construction of Concentration-Response Curves
Concentration-response curves for the positive
chronotropic actions of isoproterenol and norepinephrine
and negative chronotropic actions of carbachol and pilocarpine (Sigma
Chemical Co) were constructed by the cumulative variation of agonist
concentration at one-half log unit increments.17
All concentration-response data were evaluated for a fit to a
logistics function in the form
where
E is the increase in rate above basal;
E
max is the maximum response
that the agonist can
produce; c is the logarithm of the EC
50,
the
concentration of agonist that produces half-maximal response;
x is the
logarithm of the concentration of agonist; the exponential
term n is a
curve-fitting parameter that defines the slope of
the
concentration-response line; and
is the response observed
in the
absence of added agonist. Nonlinear regression analyses
to
determine the parameters E
max, log
EC
50, and n were done
with the use of GraphPad
Prism (GraphPad Software) with the
constraint that
=zero.
Stereological Procedures
Stereological analyses were performed according to
the method described by Aherne.18 The atria were dissected
and fixed in formalin for 24 hours. The right atria were then embedded
in paraffin, and 5-µm sections were stained with Masson's trichrome.
Analysis of the slides was performed blinded on a light
microscope (Zeiss), and the relative volume occupied by each element of
the right atrium (myocardial fibers, fibrous tissue, or vessels) was
measured with a special ocular containing a 25-point reticulum (5
parallel lines with 5 points each, kpl 8x, Zeiss, Germany). For
counting, 50 microscopic fields were evaluated and the relative volume
(Ppi) occupied by each component was calculated as follows: Ppi=p/P,
where p is the number of reticular points hitting each cardiac element
and P is the total number of reticular points. We assumed fibrous
tissue as the sum of postnecrotic fibrous scars and
interstitial and perivascular fibrosis in treated animals.
In the control animals, fibrous tissue means the normal connective
tissue present in right atria.
Statistical Analysis
All values are expressed as mean±SEM. The program InStat
(GraphPad Software) was used for statistical analyses. Where
appropriate, 1-way ANOVA followed by a Bonferroni multiple comparisons
post hoc test were performed to determine if the treatments had an
effect. In some cases, a paired or unpaired Student's t
test was used. A level of P<0.05 was accepted as
significant.
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Results
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Time Course of L-NAME Treatment on Blood Pressure
Long-term L-NAME treatment caused a time-dependent,
significant
(
P<0.05) increase in tail-cuff pressure, as
evaluated at
7, 15, 30, and 60 days (132±5, 143±2, 150±3,
and
165±9 mm Hg, respectively) compared with control
animals
(128±5 mm Hg at 60 days).
Potency and Efficacy of ß-Adrenergic and Muscarinic
Agonists
L-NAME Treatment
There were no alterations on the potency of isoproterenol
(Figure 1) and norepinephrine
(Figure 2) at 7, 15, 30, and 60 days
after L-NAME treatment. Although there was a tendency of rightward
shift on the concentration-response curves to isoproterenol at 15, 30,
and 60 days after L-NAME treatment, the pEC50
values were not statistically significant (Table 1). Negative chronotropic
responses to muscarinic agonists carbachol and pilocarpine were also
unaffected by L-NAME treatment at all times studied (Figures 3 and 4).
L-NAME treatment had no effect on the spontaneous rate of isolated
right atria. Maximal responses produced by each used agonist were also
not affected by L-NAME treatment (Table 2).
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Figure 1. Concentration-response curves to isoproterenol in
isolated right atria from L-NAME–treated rats at 7, 15, 30, and 60
days.  , Control animals; •, treated animals. Data are mean±SEM
for 5 to 12 experiments.
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Figure 2. Concentration-response curves to
norepinephrine in isolated right atria from L-NAME–treated
rats at 7, 15, 30, and 60 days. , Control animals; •, treated
animals. Data are mean±SEM for 6 experiments.
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Table 1. Effects of Nitric Oxide Inhibition by Long-Term
L-NAME Treatment on Potency of Isoproterenol in Rat Isolated Right
Atria
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Figure 3. Concentration-response curves to carbachol in
isolated right atria from L-NAME–treated rats at 7, 15, 30, and 60
days. , Control animals; •, treated animals. Data are mean±SEM
for 5 to 12 experiments.
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Figure 4. Concentration-response curves to pilocarpine in
isolated right atria from L-NAME–treated rats at 7, 15, 30, and 60
days. , Control animals; •, treated animals. Data are mean±SEM
for 5 to 12 experiments.
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Table 2. Maximal Responses (bpm) to Isoproterenol,
Norepinephrine, Carbachol, and Pilocarpine in Isolated
Right Atria From L-NAME–Treated Rats at 7, 15, 30, and 60 Days
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In Vitro Studies
Addition of L-NAME (100 µmolar/L) in vitro in rat
isolated right atria had no effect on the basal rate (250±10 bpm)
compared with control animals (245±7 bpm). The
pEC50 for isoproterenol and
norepinephrine were also unchanged by L-NAME (8.66±0.07
and 7.92±0.07, respectively) in comparison to control animals
(8.79±0.04 and 7.84±0.03, respectively).
Stereological Studies
Stereological analysis of the right atria at 15 and
30 days revealed significant (P<0.05) atrial lesions as
assessed by the increase on amount of fibrous tissues in
L-NAME–treated groups (27±2.3% and 28±1.3%, respectively) compared
with the control group (22±1.1%).
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Discussion
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Our study shows that although long-term treatment with
L-NAME
induced atrial lesions, there were no repercussions in the
responses
to both sympathetic and parasympathetic agonists, indicating
that
NO is not involved in either receptor regulation and/or
transduction
mechanisms. This is further supported by the finding that
addition
of L-NAME in vitro had no effect on basal rate and
chronotropic
response for isoproterenol and
norepinephrine.
NO is believed to play an important role in the regulation of
sympathetic activity in the brain stem, including the ventrolateral
medulla and nucleus tractus solitarius.19 20 Additionally,
hypertension induced by long-term L-NAME treatment produces increased
plasma levels of epinephrine (but not
norepinephrine) through activation of the adrenal-medullary
system.21 It is well established that overstimulation of
adrenoceptors may induce their desensitization,22 23
leading to a decrease in receptor density and/or reduction of agonist
efficacy.24 The high epinephrine plasma levels
found in this experimental model of hypertension would therefore be
expected to alter the adrenoceptor-mediated chronotropic responses.
However, in the current study, we found that hypertension induced by
long-term L-NAME administration had no effect on the sensitivity
of right atria for positive chronotropic responses to
norepinephrine and isoproterenol. Thus, similar
pEC50 values for positive chronotropic response
to selective ß1-agonist
norepinephrine were seen for both control and treated
groups. The concentration-response curves to the nonselective
ß-agonist isoproterenol showed a slight rightward shift at 15, 30,
and 60 days after L-NAME treatment (approximately 2-fold), but this was
not statistically different when a multiple comparison test was used.
Similarly, we verified that addition of L-NAME to the organ bath failed
to alter basal rate and chronotropic response mediated by
ß-adrenoceptors in right atria from naive rats. These results suggest
that neither long-term nor short-term blockade of NO synthesis affects
the heart ß-adrenoceptor–mediated responses. Our results corroborate
previous studies showing that the substrate L-arginine
and/or the NO donors influence neither chronotropic25 nor
inotropic26 responses in rat right atria.
The influence of NO on the parasympathetic nervous system in the
heart is a controversial matter. L-NAME in vitro acts as a
muscarinic antagonist blocking M2-
and M3-receptors in isolated tissues, displacing
the concentration-response curves.27 In mice lacking
endothelial NO synthase, the myocyte responsiveness
failed to be stimulated by carbachol, suggesting a role for NO
in coupling muscarinic receptor activation in cardiac
myocytes.28 However, our current results show that the
negative chronotropic responses to the full agonist, carbachol,
were not changed in animals given long-term treatment with L-NAME.
Similarly, this treatment had no effect on the potency of the partial
agonist pilocarpine, whose actions are more sensitive to detect changes
in receptor number and/or coupling mechanisms.29 These
findings show that NO does not interfere with muscarinic
receptor–mediated chronotropic responses. A recent study also
demonstrated that mice lacking endothelial NO synthase
do not exhibit any alterations on cardiac muscle function by muscarinic
receptor stimulation, thus refuting the idea that NO plays a role
in parasympathetic control.30
In summary, in this particular hypertension model we did not
observe any changes on the potency or efficacy of ß-adrenoceptors and
muscarinic receptors. Whether the nonstatistically different decrease
in the isoproterenol pEC50 might reflect a
desensitization of ß2-adrenoceptors remains to
be further studied.
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Acknowledgments
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S.R. Riado, A. Zanesco, and E. Antunes thank
Fundação
de Amparo à Pesquisa do Estado de São
Paulo (FAPESP).
Received May 8, 1999;
first decision June 24, 1999;
accepted July 8, 1999.
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Abstract
Introduction