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1 University Laboratory of Physiology, Oxford OX1 3PT; 2 Department of Human Anatomy and Genetics, Oxford OX1 3QX, United Kingdom; and 3 Department of Physiology, Monash University, Victoria 3800, Australia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The role of nitric oxide (NO) in the vagal
control of heart rate (HR) is controversial. We investigated the
cholinergic regulation of HR in isolated atrial preparations with an
intact right vagus nerve from wild-type (nNOS+/+, n = 81) and neuronal NO synthase (nNOS) knockout (nNOS
/,
n = 43) mice. nNOS was immunofluorescently colocalized
within choline-acetyltransferase-positive neurons in nNOS+/+ atria. The
rate of decline in HR during vagal nerve stimulation (VNS, 3 and 5 Hz)
was slower in nNOS/ compared with nNOS+/+ atria in vitro
(P < 0.01). There was no difference between the HR
responses to carbamylcholine in nNOS+/+ and nNOS/ atria. Selective
nNOS inhibitors, vinyl-L-niohydrochloride or
1-2-trifluoromethylphenyl imidazole, or the guanylyl cyclase
inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one significantly (P < 0.05) attenuated the decrease in HR
with VNS at 3 Hz in nNOS+/+ atria. NOS inhibition had no effect in
nNOS/ atria during VNS. In all atria, the NO donor sodium
nitroprusside significantly enhanced the magnitude of the vagal-induced
bradycardia, showing the downstream intracellular pathways activated by
NO were intact. These results suggest that neuronal NO facilitates vagally induced bradycardia via a presynaptic modulation of neurotransmission.
nitric oxide; parasympathetic; sinoatrial node
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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NITRIC OXIDE (NO) is an important signaling molecule in the regulation of vascular resistance and myocardial contraction; however, its role in the cholinergic modulation of cardiac excitability is controversial. Balligand et al. (2) first reported that NO synthase (NOS) inhibition blocked the negative chronotropic effects of cholinergic agonists in spontaneously beating rat neonatal myocytes. It was subsequently found that inhibition of NOS also prevented the cholinergic inhibition of the L-type Ca2+ current (ICa,L) in adrenergically prestimulated sinoatrial node cells (13), an effect absent in endothelial NOS knockout mice (12). This has led to speculation that NO plays an obligatory role in the autonomic control of heart rate (13), although others fail to confirm this idea (38) and have shown that endothelial NOS (eNOS) knockout mice exhibit normal autonomic activation of ICa,L (3, 39). Interpretation of these data are further complicated by the observation that activation of the NO-cGMP pathway can also stimulate the hyperpolarization-activated pacemaking current (If) in sinoatrial node myocytes (24).
Data reporting the heart rate response to cholinergic activation using putatively selective neuronal NOS (nNOS) inhibitors and nonisoform-specific NOS inhibitors are as controversial as that for the cellular studies. Inhibition of nNOS causes a dramatic reduction in the vagally mediated bradycardia in the ferret and guinea pig in vivo (6) and a modest effect in the dog (10), whereas others report no significant effect in the guinea pig in vitro (32) or rabbit in vivo (21, 32). However, NOS inhibition slows the rate of decay in heart rate with vagal stimulation in the guinea pig following adrenergic prestimulation (33), suggesting a small modulatory role for NO in the indirect control of vagally mediated bradycardia (i.e., accentuated antagonism). Increasing the bioavailability of NO with NO donors or cGMP analogs enhances the drop in heart rate caused by vagal nerve stimulation (VNS) in vitro and in vivo (34). This effect is not mimicked by bath-applied acetylcholine (ACh), suggesting that NO may act presynaptically (16, 34).
The degree of variability between many studies may be related to the lack of specificity of some NOS inhibitors for the nNOS isoform (29), significant differences in NOS protein expression due to the developmental state of the animal (16), and additional actions of NOS inhibitors in vivo on vascular resistance and baroreflex activation. Therefore, the aims of this study were twofold. First, to localize nNOS in the vagal innervation of the sinoatrial node region of the adult mouse heart. Second, to determine whether the heart rate response to VNS in the isolated atria is impaired in the nNOS knockout mouse compared with its wild-type control.
Some of these results have been communicated in abstract form (5).
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
All experiments were performed in accordance with Home Office license requirements (PPL 30/16060, Queen Anne's Gate, London, UK) and the United Kingdom's Animals (Scientific Procedures) Act 1986. Mice homozygous for targeted disruption of the nNOS gene (B6,129-NOS1tm1plh, nNOS/) (15) were
purchased from Jackson Laboratories (Bar Harbor, Maine). Because the
nNOS/ came from a mixed background, we used the C57BL/6J as our
homozygous wild-type control (nNOS+/+) (e.g., Refs. 18 and
41). Animals were genotyped postmortem from tail clippings. DNA
extracted from the tail clippings was amplified in a PCR reaction, with
the use of one pair of oligonucleotide primers complimentary to part of
the neomycin resistance sequence (present in nNOS/ and nNOS+/)
and a second pair complimentary to part of the nNOS gene sequence
(present in nNOS+/+ and nNOS+/ mice). Amplified DNA products were
then identified by agarose gel electrophoresis and used to determine
the mouse genotype.
Adult (3-4 mo old) male mice were used in all experiments.
Anatomy
Tissue processing.
Animals (10 nNOS+/+, 4 nNOS/) were terminally anesthetized
(pentobarbitone ip) and then fixed by perfusion of the left ventricle with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/l phosphate buffer solution (PBS, pH 7.1, 20 min). The mouse sinoatrial node has been anatomically located near the crista terminalis at the
junction between the superior vena cava and the intercaval region of
the right atrium (26). To isolate the sinoatrial node region, the posterior aspect of the right atrium was dissected free. To
provide a positive control for the nNOS antibody, slices of the brain
(40 µm thick) were prepared by vibratome. The tissues were treated
for immunohistochemistry as has been previously described (40).
Immunohistochemistry.
After processing was completed, tissue (nNOS+/+, n = 5, nNOS/, n = 2) was incubated in primary antiserum
against nNOS (raised in sheep against whole recombinant rat nNOS, 1:400
dilution, a gift from Dr. P. C. Emson and Dr. I. Charles) for
12 h. The tissue was then washed in 1% chicken egg albumin-PBS
solution (30 min), incubated in a biotinylated secondary antiserum (2 h, 1:200 dilution), and then immersed in an avidin-biotin peroxidase
complex (1 h). Immunoreactivity was revealed by the chromogenic
substrate diaminobenzidene with hydrogen peroxide. As a negative
control, tissues (nNOS+/+, n = 2) were treated without
primary antiserum.
/) was incubated with primary
antisera against nNOS (1:400) and ChAT (dilution 1:250, raised in
goats, obtained from Vector Laboratories). Rhodamine-conjugated
anti-goat (1:200) and fluorescein-conjugated anti-sheep (1:200) were
used as secondary antisera. Immunoreactivity for ChAT was viewed using
565 nm/590 nm filters and for nNOS using 485 nm/520 nm filters and a
laser-scanning confocal microscope (Leica).
Physiology
Mouse atrial preparation.
Mice (n = 73 nNOS+/+, n = 39 nNOS/)
were killed by cervical dislocation. The thorax and mediastinum were
removed, placed in mouse physiological saline (mmol/l: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 0.5 Na2EDTA, 1.2 KH2PO4, 25 NaHCO3, 11 glucose, and
1.75 CaCl2, pH 7.4), and aerated with carbogen (95%
O2-5% CO2) at room temperature (~22°C). A
double atrial-right vagal preparation was dissected free and placed
into an organ bath (5 ml vol) maintained at 37 ± 0.5°C. The
preparation was attached to a silicone resin base in the bath via fine
pins inserted into the inferior vena cava and the pericardium below the
right atrium. A silk suture (0.6 µm diameter, Mersilk) was placed
into the left auricle, and this was attached to an isometric force
transducer (F30, Hugo Sachs Electronik). The force response (mN) was
amplifed, and data were acquired (150 Hz sampling rate) with a Power
Macintosh 7500 computer using a Biopac MP100 data acquisition system
and Acknowledge 3.5 software. Heart rate was triggered from the
upstroke of each contraction. The right vagus was tied onto a pair of
fine silver electrodes connected to a stimulator, and the preparation
was left to equilibrate (45 min) until the heart rate did not alter by
>5 beats/min for 20 min. Postequilibration, the vagus was stimulated at 10 V, 1 ms pulse width, 3 Hz for 30 s at 2- to 3-min intervals, and experimental protocols were commenced when three consistent consecutive vagal heart rate responses were produced.
In Vitro Protocols
Vagal nerve stimulation. The changes in heart rate with right vagal stimulation at 3 and 5 Hz (10 V, 1 ms duration, 30 s, in random order) were determined. These were measured as the difference in average baseline heart rate between a 5-s period before the onset and offset of nerve stimulation. The average of three heart rate responses was determined for each stimulation frequency. To calculate any differences in vagal bradycardia over the time course of nerve stimulation, the time taken to reach 50% of the maximum heart rate response to vagal stimulation (TT50%) was also calculated.
Bath-applied carbamylcholine. To assess changes in the postsynaptic regulation of heart rate, the chronotropic responses to the cumulative bath application of the ACh analog, carbamylcholine (CCh) (0.01-100 µmol/l) were investigated. The responses to the doses were measured in the same way as with VNS and used to calculate the the concentration that produced a half-maximal response (IC50) for heart rate responses to CCh.
EFFECTS OF NNOS GENE KNOCKOUT ON HEART RATE RESPONSES . Heart rate responses to VNS at 3 and 5 Hz were compared in nNOS+/+ (n = 56) and nNOS/ (n = 39) atria,
and IC50 values were calculated for the response to
bath-applied CCh.
EFFECTS OF NOS AND GUANYLYL CYCLASE INHIBITION ON HEART
RATE RESPONSES
. 1-(2-Trifluoromethylphenyl)imidazole (TRIM, 100 µmol/l;
nNOS+/+, n = 8; nNOS/, n = 8) or
vinyl-L-niohydrochloride (L-VNIO, 100 µmol/l;
nNOS+/+, n = 8; nNOS/, n = 8) were
used as selective inhibitors of nNOS. TRIM and L-VNIO were
chosen because they have different specificities for nNOS over eNOS and
have a different mechanism of inhibition {TRIM:
IC50[nNOS] = 27 µmol/l; IC50[eNOS] = 1.06 mmol/l (14).
L-VNIO: inhibitory constant for nNOS
(Ki,[nNOS]) = 90 nmol/l;
Ki,[eNOS] = 12 µmol/l
(1)}. nNOS inhibitors were equilibrated for 20 min and tested on control responses to VNS at 3 and 5 Hz. The substrate
for NOS enzymes L-arginine (1 mmol/l, 20 min equilibration, Sigma) was used to reverse the effect of nNOS inhibition. Higher concentrations of L-arginine were avoided because they can
change baseline heart rate by affecting the pH of the Tyrode's
solution (25). To investigate the proposed presynaptic
action (16) of the nNOS inhibitors, the effects of TRIM on
the chronotropic responses to different doses of carbamylcholine
(0.01-100 µmol/l) were also investigated.
Also, to establish whether the effects of NOS inhibition were likely to
be via the cGMP pathway, the effect of the soluble guanylyl cyclase
inhibitor
1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ,
10 µmol/l, 40 min equilibration, Calbiochem Novabiochem) on the vagal
bradycardia (3 and 5 Hz stimulation) were determined (nNOS+/+,
n = 6).
Time control experiments (nNOS+/+, n = 4) were
performed to ensure that changes in the responses during equilibration
for the drugs were not due to time-dependent rundown.
EFFECTS OF AN NO DONOR
. An NO donor was used to bypass the endogenous production of NO to
determine whether the downstream signaling pathway was intact in
nNOS/ atria. Low doses of sodium nitroprusside (SNP, 10 µmol/l)
release NO and stimulate guanylyl cyclase, rather than causing
nitrosylation or the generation of superoxide radicals, which is a
property of many other donors (30). The effects of SNP on
the vagal bradycardia (1, 3 and 5 Hz stimulation) were therefore
investigated in nNOS+/+ (n = 10) and nNOS/
(n = 7) atria.
EFFECTS OF ATROPINE AND PROPRANOLOL ON BASELINE HEART RATE
. To assess whether background release of ACh or norepinephrine made
significant contributions to the heart rates recorded in atria, doses
of 0.1 µmol/l atropine and 0.1 µmol/l propranolol were added at the
end of each experiment to block cardiac muscarinic and 
-adrenergic
receptors, respectively (nNOS+/+, n = 9; nNOS/, n = 4).
EFFECTS OF BLOCKADE OF IF ON BASELINE
HEART RATE
. From the evidence that NO can activate If via
production of cGMP (24), we assessed whether blockade of
If in the nNOS/ atria had a smaller effect
on baseline heart rate. Cs+ (2 mmol/l) can produce complete
pharmacological blockade of If in isolated
sinoatrial node cells (8) and was therefore administered at this dose in nNOS+/+ (n = 8) and nNOS/
(n = 8) atria.
Statistics
Data are presented as means ± SE. All data were normally distributed. Between-group comparisons were performed by one-way ANOVA. Comparisons among multiple groups were performed by repeated-measures ANOVA (one or two factor) where appropriate, followed by Dunn's multiple comparison post hoc tests. Statistical significance was accepted at P < 0.05.Drugs
The NOS inhibitors and ODQ were obtained from Calbiochem Novabiochem (Nottingham, UK). Carbamylcholine, SNP, and atropine were obtained from Sigma. All solutions were made up immediately before use.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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There were no differences in the body weights of nNOS+/+ (27 ± 1 g; n = 56) and nNOS/ (26 ± 1 g; n = 39) mice. Similarly, the ventricular
weight-to-body weight ratios were not different for nNOS+/+ (5.56 ± 0.33 mg/g) and nNOS/ (5.41 ± 0.39 mg/g) mice.
Localization of nNOS in Mouse Right Atrium and Hypothalamus
nNOS-positive neurons were found in the sinoatrial node region of the right atrium (n = 8, nNOS+/+). These neurons were all unipolar, with an unstained nucleus and a thin axon (see Fig. 1A). At the light and electron microscope level there was no evidence of nNOS staining within the atrial myocytes. Fluorescent staining for ChAT was revealed in neuronal cell bodies of cardiac ganglia (see Fig. 1B) as well as within nerve fibers coursing throughout the atrium. In 16% of the ChAT-positive neuronal cell bodies in 20 different ganglia, nNOS was colocalized (see Fig. 1C).
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In nNOS+/+ mice (n = 3), nNOS stained positive in the
neurons of the supraoptic nucleus, but as expected (40),
not in neurons of the adjacent preoptic area of the hypothalamus. This
observation was used as a positive control for nNOS staining because it
is consistent with the results obtained by other investigators
(28, 40). No positive staining for nNOS was found in
atrial or hypothalamic tissue from nNOS/ mice (n = 4) or in nNOS+/+ tissue (n = 2) in which primary
antiserum was omitted from the immunostaining protocol.
Effects of nNOS Gene Knockout on Heart Rate Responses
nNOS/ atria had significantly higher baseline heart rates
(360 ± 7 beats/min, n = 39) than the nNOS+/+
atria (322 ± 6 beats/min, n = 56) (one-way ANOVA,
P < 0.01).
At both 3 and 5 Hz stimulation frequency, the average rates of decline
in the heart rate response to VNS (measured as the TT50%)
were significantly slower in the nNOS/ (n = 39) compared with the nNOS+/+ (n = 56) atria (one-way
ANOVA, P < 0.01) (see Fig.
2). In contrast, the heart rate responses
to carbamylcholine (0.01-100 µmol/l) were not different for the
nNOS+/+ (n = 16; IC50 = 6.00 ± 0.37 µmol/l) and nNOS/ (n = 12;
IC50 = 6.37 ± 0.39 µmol/l) atria.
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Effects of NOS and Guanylyl Cyclase Inhibition on Heart Rate Responses
Inhibition of nNOS with TRIM (100 µM) or L-VNIO (100 µM) significantly attenuated the magnitude of the decrease in heart rate with vagal stimulation at 3 Hz in nNOS+/+; an effect reversed by excess L-arginine (see Fig. 3, Table 1). All NOS inhibitors used had no significant effect on the response to VNS in nNOS/ atria (Table 1).
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Inhibition of soluble guanylyl cyclase with ODQ also significantly
attenuated the vagal heart rate response (3 and 5 Hz) in nNOS+/+ atria
(n = 6) (see Fig. 4).
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TRIM had no significant effect on the chronotropic responses to
bath-applied carbamylcholine (0.01-100 µmol/l), suggesting that
NO was working on a presynaptic pathway (see Fig.
5).
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Time-control experiments (n = 4) on nNOS+/+ atria
confirmed that the baseline heart rate, vagal bradycardia, and
TT50% values (3 Hz, 30 s) were not altered over a
40-min experimental period (baseline heart rate: 0 min = 339 ± 22 beats/min, 20 min = 326 ± 19 beats/min, 40 min = 323 ± 20 beats/min. Decrease in heart rate with VNS at 3 Hz: 0 min = 64 ± 5 beats/min, 20 min = 67 ± 7 beats/min, 40 min = 61 ± 2 beats/min. TT50%: 0 min = 5.47 ± 1.51 s, 20 min = 6.44 ± 0.88 s, 40 min = 5.93 ± 0.80 s).
Effect of an NO Donor
The NO donor SNP (10 µmol/l) significantly enhanced the magnitude of the heart rate responses to vagal stimulation (see Fig. 6). This effect was similar in magnitude in nNOS+/+ (n = 10) and nNOS/ atria
(n = 7).
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Effects of Atropine and Propranolol on Baseline Heart Rate and Responses
All heart rate responses to VNS and bath-applied CCh were abolished by 0.1 µmol/l atropine, confirming their action through cardiac muscarinic receptors. In addition, atropine significantly increased baseline heart rates, suggesting that there was a small amount of background release of ACh from terminals of the sectioned nerve. Furthermore, the tachycardia induced by atropine was less in nNOS/ (increase in heart rate of 20 ± 1 beats/min or 5.4 ± 0.1%) compared with nNOS+/+ (26 ± 2 beats/min or 8.0 ± 0.1%) atria (P < 0.05, one-way ANOVA), indicating
that background release of ACh from vagal terminals may be impaired in
nNOS/ atria. However, heart rates of nNOS/ atria remained
significantly elevated compared with the nNOS+/+ atria following
muscarinic receptor inhibition. Propranolol (0.1 µmol/l) had no
effect on heart rates.
Effects of Blockade of If on Baseline Heart Rate
As expected (8), heart rate was significantly reduced after blockade of If with 2 mmol/l Cs+ in both nNOS+/+ (from 322 ± 6 beats/min to 249 ± 9 beats/min, n = 8) and nNOS/ (from
361 ± 7 to 286 ± 14, n = 8) atria.
There was no statistical difference between the responses
to Cs+ in nNOS+/+ and nNOS/ atria (one-way ANOVA),
indicating that the contribution of If to the
spontaneous beating rate was unchanged in nNOS/ compared with
nNOS+/+ atria.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The novel findings of this study are the following: 1)
in mouse atria, nNOS is coexpressed only in ChAT-positive neurons
innervating the sinoatrial node region of the right atrium;
2) nNOS/ atria have significantly slower HR responses to
VNS compared with nNOS+/+ atria; 3) HR responses to VNS are attenuated
by nNOS inhibition in nNOS+/+, but not in nNOS/ atria;
4) responses to CCh are unaffected by nNOS inhibition in
nNOS+/+ atria, suggesting the vagal bradycardia is modulated by NO via
a presynaptic mechanism; and 5) increasing NO production
with a NO donor enhances vagal-induced bradycardia in both nNOS+/+ and
nNOS/, showing that the downstream NO-cGMP pathway is intact in
nNOS/ atria.
Location of nNOS in Mouse Atria
We have identified for the first time positive staining for nNOS within intrinsic neurons innervating the sinoatrial node region of the mouse heart. These were the only cells that stained immunopositive for nNOS in the whole mount preparation of the right atrium. No cross-reactivity of the nNOS antibody for other NOS isoforms has been observed using this antibody (28). Immunoelectron microscopy has been able to identify nNOS in the cardiac sarcoplasmic reticulum of ventricular myocytes (42); however, sarcoplasmic reticulum staining was not evident in this study at the electron microscopic level, though size and prevalence of the sarcoplasmic reticulum may be less in atrial tissue.Other immunohistochemical studies have reported nNOS staining in intrinsic neurons of the atria in the rat (31, 35) and guinea pig (35, 36). Vagotomy studies in the guinea pig suggest preganglionic vagal fibers terminate on these neurons (37). Furthermore, Mawe et al. (22) found that in guinea pig atria, all of the cardiac neurons containing nNOS also stained for ChAT. However, these data suggested that only 5% of the population of ChAT cell bodies were nitrergic. From the reports that 37% of right atrial ganglia label positive for NADPH diaphorase, a marker for nNOS (19), 5% may be an underestimate. Also, recent evidence suggests that many of the commercially available nNOS antibodies have a limited specificity (20), and poor penetration of antisera into intact atrial tissue may impair the visualization of nNOS-positive staining. Using laser-scanning confocal microscopy, our estimation that 16% of cholinergic neurons are nitrergic is based on counts of double-labeled neurons at different focal points within the tissue in 20 different ganglia.
Effects of nNOS Gene Knockout
We have shown that isolated nNOS/ atria, devoid of circulating
factors and reflexes, have an elevated heart rate compared with nNOS+/+
atria. This finding is consistent with data from the conscious and
anesthetized nNOS/ mice (18). This previous study also
demonstrated that the nNOS/ mouse has a blunted response to
atropine, suggesting an impaired vagal tone. The small positive chronotropic response to atropine in the nNOS/ compared with nNOS+/+ atria in vitro is also consistent with the idea that background release of ACh from nerve terminals may be impaired in nNOS/ atria.
However, a reduction in peripheral parasympathetic nerve activity alone
was unable to account for the elevated heart rates in nNOS/
compared with nNOS+/+. Further experiments with propranolol and the
sinoatrial node If inhibitor, Cs+
indicated that the activation of -adrenoceptors or
If was unlikely to contribute to the elevated
heart rate in the nNOS/ atria. This suggests that other ionic
pacemaking currents may be upregulated in nNOS/ atria.
Upon vagal nerve stimulation at 3 and 5 Hz, the rate of decrease in
heart rate was significantly slowed in nNOS/ compared with nNOS+/+
atria. The effect of nNOS gene knockout is therefore similar to that of
pharmacological NOS inhibition in the isolated guinea pig atria [see
Sears et al. (33)]. Sears et al. also demonstrated that
transient decreases in HR can be artificially accelerated by inhibition
of If or slowed by inhibition of
ICa,L. Direct evidence for these possibilities
comes from experiments on isolated sinoatrial node cells showing that
postsynaptically, NO-cGMP can activate both If
(24) and inhibit ICa,L
(13).
However, postsynaptic muscarinic (M2)-receptor-coupled NO-cGMP-dependent modulation of HR may not be as physiologically important in the vagal control of heart rate as was first thought, because we saw no effect of NOS inhibitors on the bradycardia induced by bath-applied CCh in nNOS+/+ atria. Similar results have been reported from experiments on guinea pig atria (16, 34).
Bypassing endogenous NOS and amplifying NO production enhanced the HR
response to VNS, showing that the downstream NO signaling mechanism was
intact in nNOS/ and nNOS+/+ atria. In isolated guinea pig atria, NO
donors were without effect on the HR responses to CCh (16,
34). Inhibition of protein kinase A, a mediator through which
NO-cGMP may act (23), diminishes the effect of SNP on
vagally induced bradycardia (16). Conversely, inhibition of phosphodiesterase III, which prevents the breakdown of cAMP, mimics
the effect of SNP (17). This suggests that SNP may work by
activating cAMP-dependent protein kinase A and analogs of cAMP as well
as activators of adenylate cyclase have been shown to increase
exocytotic release of ACh in mouse atria (7). Furthermore, NO donors have recently been reported to increase the release of
radiolabeled ACh from nerve terminals during field stimulation in
isolated guinea pig atria; an effect abolished by inhibition of
guanylyl cyclase (17).
Effects of NOS Inhibition on Vagal Modulation of Heart Rate
Previous studies investigating the effects of NOS inhibition on the vagal control over cardiac function have produced inconsistent results (6, 10, 16, 21, 32, 33). Here we present evidence showing consistent attenuation of vagal bradycardia using two different selective nNOS inhibitors. For both compounds, this was only seen when the nerve was stimulated at 3 Hz, although a nonsignificant trend was observed at 5 Hz. Significant attenuation of responses by NOS inhibitors have been revealed at these stimulation frequencies in the majority of other studies of this kind (16). Our finding is therefore consistent with the idea that pathways involving NOS have a more visible role in pacemaking when cholinergic activation is low (18) and when activation of the acetylcholine-activated potassium current (IKACh), on which NOS inhibitors have no effect (13), is minimized.Nonspecific constitutive NOS inhibition has only a small modulatory
effect on the transient bradycardia in guinea pig atria in vitro at 3 Hz vagal stimulation (33). Though it is known that the
mechanism of inhibition of the constitutive NOS isoforms by
NG-nitro-L-arginine
(L-NMMA) is different, it is not known whether this drug is
more isoform specific for eNOS than was previously thought
(29). Selective nNOS inhibitors L-VNIO or TRIM
caused a more pronounced inhibition of the vagal bradycardia than
L-NMMA did in guinea pig atria (33). TRIM
binds to L-arginine and tetrahydrobiopterin sites
(14), whereas L-VNIO binds to
L-arginine and hemecofactor sites (1).
L-VNIO may be a more potent inhibitor of nNOS than TRIM,
but we found no differences between the decrease in response to nerve
stimulation when using either drug, suggesting that we achieved
complete inhibition in both cases. More importantly, however, all
effects were reversed with excess L-arginine. In addition,
NOS inhibitors caused no change in the HR response to bath-applied CCh
and had no effect on the response to VNS in nNOS/ atria. Our
results therefore suggest that these drugs work presynaptically and
that they are specific for nNOS.
The vagal HR responses after NOS inhibition were quantitatively smaller compared with the responses in the nNOS knockout atria. This could either reflect the nonspecificity of the NOS inhibitors, although all responses were reversed by L-arginine, or that acute administration of pharmacological inhibitors may demonstrate more pronounced effects than gene knockout models, where compensatory changes might be brought about by gene deletion. Evidence suggests that signaling cascades involving atrial natriuretic peptide, which is also coupled to cGMP production (11), and inhibitory G proteins (9) may compensate for redundant NO signaling in transgenic mouse hearts.
In conclusion, we present anatomic and pharmacological evidence that implicates a modulatory role for neuronally derived NO in the peripheral presynaptic facilitation of vagal bradycardia.
The functional differences resulting from the genetic knockout of neuronal NOS support this hypothesis.
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ACKNOWLEDGEMENTS |
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We are grateful to the British Heart Foundation for supporting this study (Grant Code: RG 2000 005).
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FOOTNOTES |
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* J. K. Choate and E. J. F. Danson contributed equally to this work.
E. J. F. Danson is a James Knott Medical Research Scholar at St. Cross College, Oxford, UK.
Address for reprint requests and other correspondence: D. J. Paterson, Univ. Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK (E-mail: david.paterson{at}physiol.ox.ac.uk) or to J. K. Choate (E-mail: julia.choate{at}med.monash.edu.au.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 May 2001; accepted in final form 5 September 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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