Inhibitory effects of NO on carotid body: contribution of
neural and endothelial nitric oxide synthase isoforms
Viviana
Valdés,
Matías
Mosqueira,
Sergio
Rey,
Rodrigo
Del Rio, and
Rodrigo
Iturriaga
Laboratorio de Neurobiología, Facultad de Ciencias
Biológicas, Pontificia Universidad Católica de Chile,
Santiago 1, Chile
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ABSTRACT |
We tested the
hypothesis that nitric oxide (NO) produced within the carotid body is a
tonic inhibitor of chemoreception and determined the contribution of
neuronal and endothelial nitric oxide synthase (eNOS) isoforms to the
inhibitory NO effect. Accordingly, we studied the effect of
NO generated from S-nitroso-N-acetylpenicillamide (SNAP) and compared the effects of the nonselective inhibitor N
-nitro-L-arginine methyl ester
(L-NAME) and the selective nNOS inhibitor
1-(2-trifluoromethylphenyl)-imidazole (TRIM) on chemosensory dose-response curves induced by nicotine and NaCN and responses to
hypoxia (PO2
30 Torr). CBs excised from
pentobarbitone-anesthetized cats were perfused in vitro with Tyrode at
38°C and pH 7.40, and chemosensory discharges were recorded from the
carotid sinus nerve. SNAP (100 µM) reduced the responses to nicotine
and NaCN. L-NAME (1 mM) enhanced the responses to nicotine
and NaCN by increasing their duration, but TRIM (100 µM) only
enhanced the responses to high doses of NaCN. The amplitude of the
response to hypoxia was enhanced by L-NAME but not by TRIM.
Our results suggest that both isoforms contribute to the NO action, but
eNOS being the main source for NO in the cat CB and exerting a tonic
effect upon chemoreceptor activity.
chemoreceptor; nitric oxide
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INTRODUCTION |
THE VENTILATORY
EFFECTS of nitric oxide (NO) and the role played by nitric oxide
synthase (NOS) isoforms are complex (14, 18, 30). In the
nucleus tractus solitarii, NO plays a significant excitatory role in
sustaining the ventilatory response to hypoxia (14, 18,
35). However, several lines of evidence indicate that NO
produced within the carotid body (CB) is an inhibitory modulator of
hypoxic chemoreception (2, 8, 20, 25, 34, 38). The
administration of the precursor L-arginine, NO donor molecules (8, 22, 38), and NO gas (20) to the
cat CB perfused in vitro reduces the chemosensory response to hypoxia. On the other hand, the inhibition of NOS increases the frequency of
carotid chemosensory discharges (fx) in the cat
CB in situ and in vitro (19, 38). However, little is known
about the effects of NO on chemosensory responses induced by other
excitatory stimuli, such as nicotine and NaCN. In a previous paper, we
found that the NO donor sodium nitroprusside (SNP) reversibly reduced chemosensory responses induced by single doses of NaCN and nicotine in
the superfused cat CB (2). In anesthetized cats, the NOS inhibitor N
-nitro-L-arginine
methyl ester (L-NAME) increased basal
fx and enhanced responses to NaCN and dopamine
(19). These results suggest that, besides the well-known
inhibitory effect of NO on hypoxic chemoreception, NO may also modulate
the responses to other stimuli.
In the cat CB, NOS immunoreactivity and diaphorase activities have been
found in endothelial cells (10, 37), autonomic parasympathetic neurons (10, 11, 37, 38), and petrosal sensory C fibers (38) but are absent in glomus cells, type
II cells, and vascular smooth muscle cells (10, 11, 38).
NO is a ubiquitous molecule that may modulate CB chemoreception at different target sites and by several mechanisms (see Ref.
33 for review). NO has been proposed to produce
vasodilatation in the CB (5, 8, 28, 38), retrograde
inhibition of the glomus cell's excitability (39),
inhibition of Ca2+ channels in glomus cells
(36), modulation of petrosal ganglion neuron's
excitability (1), and inhibition of mitochondrial metabolism (22, 28). At least three isoforms of NOS have
been isolated (32): neuronal (nNOS), endothelial (eNOS),
and inducible. In the CB, nNOS is present in neuronal structures
(34, 37), and eNOS in endothelial cells (10,
37). In the rat, Western blots of CB homogenates showed a
relative abundance of eNOS protein compared with nNOS
(13). The NO produced by eNOS may regulate blood flow and
subsequent changes in CB tissue PO2, whereas NO produced by nNOS in terminals of sensory C fibers and parasympathetic neurons may regulate glomus cell excitability and the vascular tone,
respectively (8, 28, 34, 38, 39). The contribution of nNOS
and eNOS isoforms to the production of NO in the CB has been assessed
indirectly by testing the effect of the pharmacological and genetic
suppression of these NOS isoforms' activities on ventilatory responses
induced by hypoxia and NaCN. Gozal et al. (13) found that
the specific nNOS inhibitor
S-methyl-L-thiocitrulline did not modify rat
ventilatory responses induced by NaCN, but the nonspecific NOS
inhibitor L-NAME significantly enhanced them. More
recently, Kline et al. (26), using mutant mice deficient in nNOS and eNOS isoforms, found that mice lacking nNOS exhibited greater ventilatory responses to hypoxia, NaCN, and hyperoxia than
wild-type controls, whereas responses to NaCN and brief hyperoxia were
blunted in mutant mice lacking eNOS compared with the wild type
(27). These observations contradict those reported by
Gozal et al. (13) showing that ventilatory responses to
NaCN were unaffected after the administration of a specific nNOS
inhibitor. Kline et al. (27) speculated that acute
inhibition of eNOS might differ from chronic deficiency of the enzyme.
However, these studies measured the effects of NOS isoforms' blockade
on ventilation, not on CB chemosensory discharges. Thus the
contribution of NOS isoforms to the generation of NO in the CB may be
underestimated. Therefore, in the present paper, we studied the effects
of NO and the pharmacological inhibition of nNOS and eNOS on the cat CB
chemosensory response to hypoxia, nicotine, and NaCN. We compared the
effects of the NO donor,
S-nitroso-N-acetylpenicillamide (SNAP), the
nonspecific NOS inhibitor L-NAME, and the specific nNOS
inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM) on the
chemosensory response to hypoxia (PO2
30 Torr) and to several doses of nicotine and NaCN. We tested the
effect of the NOS inhibitors on the dose-response curves to nicotine
and NaCN, because this analysis provides information about the NOS
inhibitors' effects on the sensitivity and reactivity of the
chemosensory response. Accordingly, we fitted the experimental data to
a logistic expression for comparing the effects of L-NAME and TRIM on the maximal reactivity, the median effective dose (ED50), and the slope factor. Experiments were performed
using an in vitro preparation of the cat CB perfused at constant
pressure (21) to avoid cardiovascular and respiratory
systemic effects produced by NO donor and NOS inhibitors (2,
19). However, local vascular effects are still likely
functioning since the CB is perfused through its vessels
(21).
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METHODS |
Animals.
Experiments were performed on 18 adult cats (2-4 kg) anesthetized
with sodium pentobarbitone (40 mg/kg ip), followed by
additional doses (12 mg iv) to maintain a level of surgical anesthesia.
The experimental protocols were approved by the Ethical Committee of
the Facultad de Ciencias Biológicas of the Pontificia Universidad Católica de Chile and were performed according to the Guiding Principles for the Care and Use of Animals of the American
Physiological Society.
Experimental protocol.
The carotid bifurcation, including the CB and the carotid sinus nerve,
was excised from the cats and perfused in vitro with a Tyrode solution
at 38.5 ± 0.5°C and pH 7.40, as previously described (21). In brief, the carotid bifurcation including the CB
was cannulated through the common carotid artery, excised from the cat,
and placed in a chamber. The CB was perfused by gravity at constant
pressure (
80 Torr) with Tyrode equilibrated with 20% O2
and 5% CO2 and simultaneously superfused with Tyrode
equilibrated with 95% N2 and 5% CO2. The
composition of the Tyrode was (in mM): 154.0 Na+, 4.7 K+, 2.2 Ca2+, 1.1 Mg2+, 120.0 Cl
, 21.0 glutamate, 21.0 HCO
, 5.5 D-glucose, and 5.0 HEPES. Chemosensory discharges were
recorded from the carotid sinus nerve placed on a pair of platinum
electrodes and lifted into mineral oil. The neural signals were
preamplified and amplified, filtered (10 Hz-1 kHz; notch filter,
50 Hz), and fed to an electronic amplitude discriminator, which allowed
the selection of action potentials of a given amplitude above the noise
to be counted with a frequency meter to measure the
fx expressed in Hz. The
fx signal was digitized with an analog-digital
board DIGITADA 1200 (Axon Instruments). Chemosensory responses were expressed as fx or as the summation of
chemosensory discharges over baseline (
fx).
The sensitivity and reactivity of chemosensory responses to nicotine
(0.01-100 µg) and to NaCN (0.01-100 µg) were assessed
before (control) and during the perfusion with Tyrode containing SNAP
(100 µM), L-NAME (1 mM), or TRIM (100 µM). Nicotine and
NaCN were injected into the perfused line in boluses of 0.2 ml.
Chemosensory responses to hypoxic perfusion
(PO2
30 Torr) were measured in the
same CBs before (control) and during perfusion with Tyrode containing
100 µM TRIM for 10-15 min and then during perfusion with Tyrode
plus 1 mM L-NAME for 10-15 min.
NOS inhibitors.
The concentrations of NOS inhibitors used corresponded to their
inhibitory activities [TRIM: IC50 for nNOS
30 µM,
IC50 for eNOS
1 mM; L-NAME IC50
for eNOS
500 nM (16, 32)]. We tested the effect of
TRIM on the NO production mediated by nNOS with the same method used by
Wang et al. (39). Accordingly, we studied the effect of
TRIM on the NO synthesis induced by electrical stimulation of the
carotid sinus nerve in three CBs superfused with Tyrode containing
L-arginine (1 mM). NO was measured with a
chronoamperometric system (IVEC 10; Medical System) as previously
described (22). Briefly, NO-sensitive microelectrodes
consisting of single carbon fibers (30-µm diameter) covered with
porphyry and Nation (Quanteon) were inserted into the CB. A potential
of 0.9 V, with respect to the reference Ag-AgCl electrode, was applied
for 100 ms at a rate of 5 Hz. The resulting oxidation current was
digitally integrated during the last 80 ms of each pulse, averaged for
five cycles, displayed at a rate of 1 Hz, and stored in the computer. The NO-electrochemical signal was expressed in arbitrary units. Figure
1 shows the effects of TRIM on the
increase of the NO-electrochemical signal evoked by the electrical
stimulation of the carotid sinus nerve (5 V, 1 ms, 20 Hz for 5 min).
Perfusion with Tyrode solution containing 100 µM TRIM abolished the
electrically induced generation of NO in all the CBs studied.

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Fig. 1.
Effect of 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on
the nitric oxide (NO)-electrochemical response evoked by electrical
stimulation (5 V, 1 ms, 20 Hz) of the carotid sinus nerve in 1 superfused carotid body (CB). A: control; B:
during superfusion with TRIM (100 µM). Bars, electrical
stimulation.
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Statistical analysis.
Data were expressed as means ± SE. To compare data points from
different experiments, we expressed fx and
fx as percentages of their maximal control
response. The maximal responses correspond to the maximal chemosensory
discharge attained with the larger doses of nicotine and NaCN.
Statistical differences between dose-response curves for all conditions
(control vs. SNAP, L-NAME, or TRIM, and L-NAME
vs. TRIM) were analyzed by a two-way ANOVA with repeated measures, and
post hoc analyses were performed by Bonferroni test. Differences for
two and three samples were respectively assessed by means of one-way
ANOVA and by Student's paired or unpaired t-tests. The
level of significance for all statistical analyses was
P < 0.05. To compare the effects of SNAP,
L-NAME, and TRIM on the maximal reactivity of chemosensory
responses (maxfx or max
fx) and on the ED50 of
dose-response curves, we fitted data of individual experiments to the
following logistic expression: R = maxR + {[basR
maxR]/[1 + (D/ED50)S]}, where R = response, maxR = maximal response, basR = basal response,
D = arithmetic dose, and S = slope factor determining the
steepness of each curve. Correlation coefficients for adjusted curves
were >0.90 (P < 0.01) for all conditions studied.
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RESULTS |
Effects of SNAP on carotid chemosensory responses induced by
nicotine and NaCN.
Figure 2 illustrates the effects of SNAP
(100 µM) on the chemosensory responses induced by several doses of
nicotine (0.1-100 µg). Perfusion with Tyrode containing SNAP
reduced basal fx and the amplitude of the
responses induced by nicotine (Fig. 2B). In this experiment,
SNAP abolished the chemosensory excitation produced by doses of
nicotine <10 µg. As is shown in Fig.
3, the chemosensory responses induced by
NaCN were also reduced by SNAP. Normally, the responses to NaCN
consisted of two phases: an initial one that declines rapidly, followed
by a slow secondary increase of fx. SNAP reduced
the initial phase and almost abolished the secondary increase (Fig.
3B). Upon the removal of SNAP, chemosensory responses to
nicotine and NaCN were restored (data not shown). Figure
4 shows the effects of SNAP on
dose-response curves for nicotine- and NaCN-induced chemosensory
excitation recorded from six CBs. Statistical analyses demonstrate that
SNAP reduced the responses to nicotine and NaCN (P < 0.01, by a two-way ANOVA). To assess the effect of SNAP on parameters
of the dose-response curves, we fitted the data points of individual
experiments (n = 6) to a logistic expression (see
METHODS). The maximal response (maxfx) for nicotine dose-response curves
predicted from the fit model was reduced by SNAP from a control value
of 104.2 ± 1.5 to 80.0 ± 8.3% (P < 0.01, paired t-test), whereas maxfx induced by NaCN was reduced from 99.1 ± 2.3 to 86.5 ± 3.6%
(P < 0.05, paired t-test). However, SNAP
did not modify significantly the ED50 of dose-response
curves induced by nicotine (4.1 ± 2.4 vs. 8.0 ± 4.4 µg)
or NaCN (0.73 ± 0.43 vs. 0.72 ± 0.4 µg).

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Fig. 2.
Effect of
S-nitroso-N-acetylpenicillamide (SNAP) on
chemosensory responses induced by several doses of nicotine in 1 CB.
A: control; B:
S-nitroso-N-acetylpenicillamide (SNAP, 100 µM). fx, Frequency of carotid
chemosensory discharges; arrowheads, nicotine injections.
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Fig. 3.
Effect of SNAP on chemosensory responses induced by several doses
of NaCN in 1 CB. A: control; B: SNAP (100 µM).
Arrowheads, NaCN injections.
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Fig. 4.
Effect of SNAP (100 µM) on chemosensory dose-response
curves for nicotine (A) and NaCN (B) in 4 CBs.
P, statistical differences between SNAP and control
dose-response curves.
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Effects of L-NAME on chemosensory responses induced by
nicotine and NaCN.
The effects of L-NAME (1 mM) on chemosensory responses to
nicotine (0.1-50 µg) and NaCN (0.1-100 µg) are shown in
Figs. 5 and
6, respectively. Perfusion with
Tyrode containing L-NAME increased basal
fx, but the amplitude of
fx induced by nicotine and NaCN was almost the
same. However, L-NAME increased the duration of chemosensory responses induced by nicotine (Fig. 5B) and by
NaCN (Fig. 6A), producing a marked increase in
fx. Figures 7
and 8 summarize the effects of
L-NAME on chemosensory dose-response curves induced by
nicotine and NaCN, respectively, in four CBs. Although the
fx dose-response curve for nicotine was
different in the presence of L-NAME (P < 0.01, by two-way ANOVA), the Bonferroni test did not detect any
significant difference (P > 0.05) attributed to
L-NAME when we compared responses induced by the same doses of nicotine. On the other hand, L-NAME did not modify the
fx dose-response curve induced by NaCN
(P = 0.09, by two-way ANOVA). On the contrary,
fx dose-response curves for nicotine and NaCN
were enhanced by L-NAME (P < 0.01, by two-way ANOVA), and the Bonferroni test showed that responses
to nicotine (Fig. 7B) and NaCN (Fig. 8B), in
doses
10 µg, were significantly augmented by L-NAME
(P < 0.01). The comparison of the parameters of the
logistic fit indicated that the max
fx for
dose-response curves induced by nicotine and NaCN increased in the
presence of L-NAME from 105.6 ± 6.0 to 309.8 ± 26.7% (P < 0.01, paired t-test) and from
93.5 ± 10.6 to 178.9 ± 13.3% (P < 0.01, paired t-test), respectively. L-NAME did not modify the ED50 of dose-response curves for nicotine
(12.5 ± 7.2 vs. 14.6 ± 3.6 µg) or NaCN (3.6 ± 7.3 vs. 7.3 ± 6.1 µg).

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Fig. 5.
Effect of N -nitro-L-arginine
methyl ester (L-NAME, 1 mM) on chemosensory responses
induced by several doses of nicotine in 1 CB. A: control;
B: L-NAME. Arrowheads, nicotine injections.
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Fig. 6.
Effect of L-NAME (1 mM) on chemosensory responses
induced by several doses of NaCN in 1 CB. A: control;
B: L-NAME. Arrowheads, NaCN injections.
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Fig. 7.
Effect of L-NAME (1 mM) on chemosensory
dose-response curves for nicotine in 4 CBs. A:
fx; B: fx,
summation of chemosensory discharges over baseline.
* P < 0.01 (Bonferroni test after 2-way ANOVA),
statistically different between L-NAME and control
dose-response curves.
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Fig. 8.
Effect of L-NAME (1 mM) on chemosensory
dose-response curves for NaCN in 4 CBs. A:
fx; B: fx.
* P < 0.01 (Bonferroni test after 2-way ANOVA)
statistically different between L-NAME and control
dose-response curves.
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Effects of TRIM on chemosensory responses to nicotine and NaCN.
The nNOS inhibitor TRIM produced different effects on the chemosensory
excitation induced by nicotine and NaCN. Figure
9 shows the effect of TRIM (100 µM) on
chemosensory responses induced by nicotine (0.01-100 µg).
Clearly, TRIM did not increase the amplitude of responses induced by
nicotine, and the duration of the responses to large doses appears to
be reduced as shown in Fig. 9B. Contrarily, TRIM enhanced
chemosensory responses induced by NaCN (Fig.
10). Note the marked augmented duration
of the secondary phase of the chemosensory responses to medium and
large doses of NaCN (Fig. 10B). Effects of TRIM on
dose-response curves for nicotine and NaCN measured in seven and six
CBs are summarized in Figs. 11 and
12, respectively. TRIM did not
modify chemosensory responses induced by nicotine, expressed as either
fx or
fx
(P > 0.05, by two-way ANOVA), but enhanced the
NaCN-induced
fx (P < 0.01 by
two-way ANOVA).

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Fig. 9.
Effect of TRIM (100 µM) on chemosensory responses induced by
several doses of nicotine in 1 CB. A: control; B:
TRIM. Arrowheads, nicotine injections.
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Fig. 10.
Effect of TRIM (100 µM) on chemosensory responses evoked by
several doses of NaCN in 1 CB. A: control; B,
TRIM. Arrowheads, NaCN injections.
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Fig. 11.
Effect of TRIM (100 µM) on chemosensory dose-response
curves for nicotine in 7 CBs A: fx;
B: fx. P, statistical
differences between TRIM and control dose-response curves.
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Fig. 12.
Effect of TRIM (100 µM) on chemosensory dose-response
curves for NaCN in 6 CBs. A: fx;
B: fx. * P < 0.01 (Bonferroni test after 2-way ANOVA), statistically different
between TRIM and control dose-response curves.
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Comparison of the effects of TRIM and L-NAME on
dose-response curves induced by nicotine and NaCN.
To determine the contribution of nNOS and eNOS to the inhibitory
modulation of NO on chemoreception, we compared the effects of TRIM and
L-NAME on the adjusted dose-response curves for
fx induced by nicotine and NaCN (Fig.
13). Dose-response curves for nicotine
and NACN were significantly different in the presence of
L-NAME or TRIM (P < 0.01, by two-way
ANOVA). However, L-NAME and TRIM produced different effects
on the reactivity and sensitivity of the dose-response curves. The
dose-response curve induced by nicotine in the presence of
L-NAME has higher reactivity than that in the presence of
TRIM (max
fx of 309.8 ± 26.7 vs.
74.2 ± 12.6%, P < 0.05, unpaired
t-test), and both curves have similar ED50
(8.2 ± 1.2 vs. 1.6 ± 0.6 µg for L-NAME and
TRIM, respectively). Multiple comparisons with the Bonferroni test
showed that chemosensory responses induced by nicotine in doses
10
µg were significantly higher (P < 0.01) in the
presence of L-NAME (Fig. 13A). Thus the blockade
of eNOS and nNOS with L-NAME produced a larger increase in
the reactivity of the dose-response curves induced by nicotine compared
with the blockade of nNOS alone. On the contrary, the reactivity of
dose-response curves for NaCN in the presence of L-NAME or
TRIM was similar. Multiple comparisons with the Bonferroni test
indicate that only the chemosensory response induced by 10 µg of NaCN
was higher in the presence of L-NAME than in the presence of TRIM (P > 0.01). This effect of TRIM on the
reactivity of NaCN-induced responses was due to a shift of the curve to
the right (Fig. 13B). In fact, the ED50 of the
dose-response curve for NaCN in the presence of TRIM and
L-NAME was 54.3 ± 0.6 and 1.1 ± 0.6% µg,
respectively (P < 0.05, t-test).

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Fig. 13.
Comparison of the effects of L-NAME and TRIM
on chemosensory dose-response curves for nicotine (A) and
NaCN (B). * P < 0.01 (Bonferroni test
after 2-way ANOVA), statistically different between L-NAME
and TRIM dose-response curves.
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Effects of TRIM and L-NAME on the chemosensory response
to hypoxia.
Figure 14 shows the effect of TRIM
(Fig. 14B) and L-NAME (Fig. 14C) on
the carotid chemosensory response to hypoxic perfusion (PO2
30 Torr for 2 min) in the same
CB. During perfusion with normoxic Tyrode, switching to a
pre-equilibrated hypoxic Tyrode solution promptly increased
fx to a semisteady plateau (Fig.
14A). During perfusion with Tyrode containing TRIM (100 µm), the maximal chemosensory response to hypoxia was slightly
smaller than the control response (Fig. 14B). However, the
maximal hypoxic response increased during perfusion with Tyrode
containing L-NAME (1 mM). We tested the effect of 100 µM
TRIM on the chemosensory responses to hypoxia in seven CBs. Hypoxic
perfusion for 2 min increased basal fx from
58.2 ± 7.4 to a maxfx of 267.3 ± 15.8 Hz without TRIM and from 54.4 ± 7.0 to 267.1 ± 18.1 Hz
with TRIM. Both basal fx and the
maxfx attained during hypoxia with and without
TRIM were not significantly different (P > 0.05, paired t-test). In four out of these seven CBs, we tested
the effect of 1 mM L-NAME. Figure
15 summarized the effects of TRIM and
L-NAME on the amplitude of the hypoxic response. Multiple
comparisons with the Bonferroni test after one-way ANOVA
(P < 0.01) indicate that the chemosensory response to
hypoxia was higher in the presence of L-NAME than in the
presence of TRIM or during control perfusion (P < 0.05). In fact, in the presence of L-NAME, hypoxia
increased the chemosensory discharge to a maxfx
of 384 ± 23.1 Hz, whereas during control perfusion and in the
presence of TRIM, hypoxia increased maxfx to
344.5 ± 19 and 325.8 ± 23.1 Hz, respectively
(P < 0.01 by one-way ANOVA).

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Fig. 14.
Effect of TRIM and L-NAME on chemosensory response to
hypoxia (PO2 30 Torr). A:
control response; B: after 10 min of perfusion with TRIM
(100 µM); C: after 10 min of perfusion with
L-NAME (1 mM).
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Fig. 15.
Summary of the effects of TRIM and L-NAME on
chemosensory response to hypoxia in 4 CBs during Tyrode perfusion
(control) and after 10 min of perfusion with TRIM (100 µM) and after
10 min of perfusion with L-NAME (1 mM).
fx, expressed as % of maximal control response.
Open bars, basal fx; solid bars, maximal
fx. P < 0.05, Bonferroni test
after 1-way ANOVA; ns, Not significant.
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DISCUSSION |
The present results show that the nonspecific NOS inhibitor
L-NAME significantly enhanced the chemosensory response to
hypoxia and the reactivity of the dose-response curves for nicotine and NaCN, without apparent changes on sensitivity. On the contrary, the
specific inhibitor of the nNOS isoform TRIM enhanced the chemosensory response to higher doses of NaCN but produced no effect on the responses induced by hypoxia and nicotine. These results suggest that
NO produced by the nNOS isoform contributes to modulate the response to
NaCN, but eNOS seems to be the main source of NO in the CB. In
addition, our results showing that SNAP decreased chemosensory dose
responses induced by nicotine and NaCN extend our previous observations
(2) and support the proposal that NO is a broad inhibitor
of CB chemoreception.
In the CB, NO produced by the eNOS may inhibit chemosensory activity by
increasing blood flow and O2 delivery to glomus cells (8, 28). The withdrawal of an inhibitory vascular tone
mediated by NO is expected to increase basal chemosensory discharges
and is compatible with an increased duration of the responses to bolus injections of excitatory stimuli, as we observed here after the inhibition of the eNOS. A major role for a vascularly mediated action
of NO in the CB is supported by the observation of Wang et al.
(38). They found that L-NAME produced a larger
chemosensory excitation in the perfused cat CB than in the superfused
preparation, where vascular effects are absent. Furthermore, Buerk and
Lahiri (5) reported that SNP increased
PO2 in the CB and reduced basal chemosensory
discharges, whereas L-NAME reduced the CB
PO2 and increased chemosensory discharges,
indicating that part of the inhibitory effect of NO on CB
chemoreception is mediated by vasodilatation. More evidence supporting
a crucial role for eNOS in the production of NO was reported by Gozal
et al. (13). They studied the effect of the
pharmacological inhibition of eNOS and nNOS on ventilatory responses
induced by NaCN in freely moving rats. They found that the specific
nNOS inhibitor S-methyl-L-thiocitrullin did not
enhance the ventilatory dose-response curve induced by NaCN. However, L-NAME that blocks both eNOS and nNOS enhanced the
ventilatory responses to NaCN. In a separate study, Gozal et al.
(15) reports that L-NAME markedly increased
basal ventilation in rats, whereas such an effect was minimal after the
injection of S-methyl-L-thiocitrullin. In addition, they found that L-NAME augmented the early
ventilatory response to mild hypoxia, and this effect was absent when
S- methyl-L-thiocitrullin was used. Thus Gozal
and colleagues (13, 15) stated that nNOS inhibition had
minimal effects on peripheral chemoreceptor activity and proposed that
eNOS would provide the major source for NO in the CB, exerting a
downregulatory effect on peripheral chemoreceptor responsiveness. Our
results agree partially with these observations, but we found that TRIM
enhanced the chemosensory responses to high doses of NaCN. More
recently, Kline et al. (27) studied the role played by NO
generated from the eNOS isoform in ventilatory regulation using mutant
mice deficient in the eNOS protein. They found that wild-type mice had
larger ventilatory responses to hypoxia than mutant mice, but the
responses to hyperoxic hypercapnia were similar in both groups of mice.
Thus mutant mice lacking eNOS present a selective blunting of the
ventilatory responses to hypoxia but not to hypercapnia. Mutant mice
showed attenuated ventilatory responses to NaCN and to brief hyperoxic
stimulation (Dejour's test), indicating a reduced peripheral
chemosensory sensitivity. Observations of Kline et al.
(27) contradict our results and those reported by Gozal
and colleagues (13, 15). However, Kline et al.
(27) suggested that the acute pharmacological blockade of
eNOS could be different from the chronic deficiency of the eNOS
protein. In mutant mice, the gene encoding eNOS is defective from
birth, and the animals develop hypertension and glomus cell
hyperplasia. Accordingly, Kline et al. (27) proposed that
the reduced ventilatory responsiveness to hypoxia in mice lacking the
eNOS protein might be secondarily due to the effects of chronic
hypertension in the CB.
Besides the NO-mediated vascular effects, Wang and coworkers (38,
39) proposed that NO produced and released from petrosal nerve
C-fiber terminals produces a retrograde inhibition of glomus cell
activity. They found that electrical stimulation of C fibers in the
carotid sinus nerve evoked a Ca2+-dependent increase in
[3H]citrulline accumulation in the CB, which was blocked
by L-NAME. The electrical stimulation of C fibers in the
carotid sinus nerve elevates cGMP levels in CB vessels and in glomus
cells, an effect that was reversed by L-NAME. Therefore,
they proposed that NO plays a dual role in mediating the chemosensory
inhibition, one via its actions on the CB vasculature and the other
through a direct effect on the glomus cells' excitability, probably
mediated by axon reflex or afferent depolarization of chemosensory
nerve terminals (39). Certainly, NO increases cGMP levels
in glomus cells, but it is difficult to understand how the increased
cGMP could reduce the O2 sensing in glomus cells
(12), because PO2-dependent K+ currents are unaffected by cGMP (17).
Moreover, a direct effect of NO on
PO2-dependent K+ conductance has
not been found. Indeed, Hatton and Peers (17) and Summers
et al. (36) found that the NO donors SNAP and SNP did not
modify the PO2-dependent K+
currents in rat and rabbit glomus cells. However, it is plausible that
NO may affect the function of other ion channels in glomus cells.
Summers et al. (36) found that NO donors SNP and spermine inhibit L-type Ca2+ currents in rabbit glomus cells through
a cGMP-independent mechanism mediated by a direct modification of the
thiol groups of the channel proteins. Accordingly, part of the
inhibitory effect of NO on carotid hypoxic chemoreception could be
attributed to an inhibition of L-type Ca2+ channels in
glomus cells. Petrosal ganglion neurons are another target site for the
action of NO. Because NO is produced in the soma of a population of
petrosal ganglion neurons in the cat (38, 39), it is
possible that NO may modulate the excitability of the sensory
afferents. Because Alcayaga et al. (3) found that the
population of petrosal neurons projecting through the carotid sinus nerve is selectively activated by acetylcholine, we
(1) studied the effects of SNP and L-NAME on the responses
evoked in the carotid sinus nerve by acetylcholine applied to the
isolated petrosal ganglion. SNP partially reduced the sensitivity and
amplitude of the response to acetylcholine, although the maximal
response was less affected, whereas L-NAME slightly
increased the sensitivity of the acetylcholine-induced responses, an
effect that persisted after L-NAME withdrawal.
We found that the injection of the NO donors SNAP and
6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine
to the CB transiently reduced the hypoxia-augmented chemosensory
activity in a dose-dependent manner. However, during normoxia, the same NO donors increased the chemosensory discharge in a dose-dependent manner, showing a dual effect of NO on carotid chemoreception depending
on PO2 levels (22). Accordingly,
we proposed that a high concentration of NO ([NO]) or its metabolite
peroxynitrite might account for the chemosensory excitation, because NO
and peroxynitrite inhibit the electron transport chain and oxidative phosphorylation (4, 6). It is well known that NO and
peroxynitrite reversibly inhibit mitochondrial respiration at different
levels, reducing O2 consumption. In fact, NO at low and
medium concentrations (<5 µM) specifically and reversibly inhibits
cytochrome oxidase in complex IV in competition with
O2 (4). However, higher [NO] may inhibit
other respiratory chain complexes (4). A new and interesting possibility is that mitochondrial NO/O2 ratio
may be crucial to regulate the respiratory rate, playing a
physiological role in O2 sensing. Recently, it has been
found that the mitochondria contains a new NOS isoform
(9), which produces significant amounts of NO, enough for
regulating their own respiration (7), suggesting that NO
may be important for the regulation of energy metabolism. Thus it is
possible that NO inhibition of cytochrome a3
could be involved in the physiological regulation of carotid chemosensory sensing of PO2. Buerk and Lahiri
(5) studied the role of NO in the cat CB, measuring tissue
PO2 in cat CB perfused in vitro before and
after NOS inhibition with L-NAME. They compared the
O2 disappearance curves during stop flow. They found that L-NAME reduced the CB PO2 from
74.5 ± 8.7 to 37.4 ± 6.9 Torr and reduced the maximum rate
of O2 consumption by 18%. As expected, the NO donor SNP
increased the CB PO2 but reduced the rate of O2 disappearance by 15%, indicating that NO inhibits
O2 consumption. Thus they proposed that NO may play a role
in O2 sensing in the CB. Buerk and Lahiri (5)
found an unusually high [NO] in the CB (
300 nM), which suggests
that NO may play a metabolic function in the CB.
Recently, Kline et al. (26) used mutant mice to study the
role played by NO produced by the nNOS isoform in the control of
respiration. Mutant mice lacking the nNOS protein exhibited greater
respiratory responses to hypoxia than wild-type controls. Kline et al.
(26) attributed part of this effect to an enhancement of
peripheral chemosensory responsiveness, because respiratory responses
to brief hyperoxia and to NaCN were more pronounced in mutant mice. The
respiratory response to hypercapnia was similar in both groups of mice,
suggesting that hypoxic chemosensory responses were selectively
augmented in mutant mice deficient in nNOS. However, peripheral as well
as central mechanisms dependent on nNOS integrity may contribute to the
enhanced ventilatory responses to hypoxia observed in mutant mice
(18, 35). Our results suggest that NO produced by the nNOS
isoform in the cat CB may inhibit the chemosensory responses to NaCN.
However, comparison of the magnitude of the effects produced by
L-NAME (inhibition of nNOS and eNOS isoforms) with those
effects produced by TRIM (inhibition of the nNOS) on the responses to
hypoxia and the dose-response curves for nicotine and NaCN points to a
predominant contribution of eNOS to the inhibitory action of NO in the
cat CB (Fig. 12). However, we cannot preclude different contributions
of eNOS and nNOS to the NO production in the CB of different species,
which may explain the contradictory results of Gozal and colleagues
(13, 15) and Kline and colleagues (26, 27).
The marked effect of L-NAME on the duration of chemosensory
responses to nicotine and NaCN supports the idea that NO may contribute to the well-known phenomenon of blood flow autoregulation in the CB
(31). Lahiri et al. (29) recorded
simultaneous cat chemosensory discharges and CB
PO2, measured with an optical method based on the O2-dependent quenching of the phosphorescence of
Pd-meso-tetra-(4-carboxyphenyl)-porphine. The CB
PO2 and chemoreceptor discharges were affected
by hemorrhagic hypotension only when arterial blood pressure fell below
50 Torr, and then CB PO2 decreased producing
chemosensory excitation. The lack of a significant effect of
hypotension indicates that O2 delivery to CB was almost
independent of the systemic blood pressure above 50 Torr, suggesting
that CB microcirculation and PO2 are regulated
by an intrinsic mechanism, which may be partially mediated by NO as is
the case in other vascular beds (23, 24). However, the
mechanisms underlying the blood flow autoregulation in the CB remain to
be investigated.
In summary, our results show that L-NAME enhanced the
amplitude of the response to hypoxia and the duration of chemosensory responses to NaCN and nicotine, resulting in a great increase of
discharges. By contrast, TRIM enhances only the responses to high doses
of NaCN, but not those to hypoxia and nicotine. As expected, NO
produced by the donor SNAP reduces chemosensory responses to nicotine
and NaCN. These results suggest that both NOS isoforms may contribute
to the NO effect in the CB, but the eNOS isoform seems to be the major
source for NO in the cat CB, which maintains a tonic inhibitory effect
on chemoreceptor activity. However, we cannot conclude that all of the
effects of NO produced by eNOS are vascularly mediated. It is possible
that NO produced by endothelial cells may diffuse and reach several
targets in the CB. The proximity of the endothelial cells to the glomus
cells and nerve endings and the high basal levels of NO (300 nM)
reported by Buerk and Lahiri (5) in the cat CB support
this interpretation.
 |
ACKNOWLEDGEMENTS |
We thank Carolina Larraín for assistance in the preparation
of the experiments and of this manuscript.
 |
FOOTNOTES |
Supported by Grant 198-0965 from the National Fund for Scientific and
Technological Development of Chile and by the Office for Research and
Graduate Studies of the Pontificia Catholic University of Chile.
Address for reprint requests and other correspondence: R. Iturriaga, Laboratorio de Neurobiología, Facultad de Ciencias
Biológicas, P. Universidad Católica of Chile, Casilla
114-D, Santiago 1, Chile (E-mail:
riturria{at}bio.puc.cl).
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.
September 6, 2002;10.1152/ajplung.00494.2001
Received 28 December 2001; accepted in final form 26 August 2002.
 |
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