(Hypertension. 1999;34:802-807.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Long-Term Nitric Oxide Inhibition and Chronotropic Responses in Rat Isolated Right Atria
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 M2-receptors,1 whereas the sympathetic actions are mediated by ß-adrenoceptors. ß1-, ß2-, and ß3-adrenoceptors are known to coexist in atria from several animal species, including humans.2 3 4 5 In rats, under physiological conditions, only ß1-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
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is the response observed in the
absence of added agonist. Nonlinear regression analyses to
determine the parameters Emax, log
EC50, 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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, Control animals; •, treated animals. Data are mean±SEM
for 5 to 12 experiments.
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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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