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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Nomega -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 approx  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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Nomega -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 approx  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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (approx 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<UP><SUB>3</SUB><SUP>−</SUP></UP>, 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 (Sigma 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 approx  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 approx 30 µM, IC50 for eNOS approx 1 mM; L-NAME IC50 for eNOS approx 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.

Statistical analysis. Data were expressed as means ± SE. To compare data points from different experiments, we expressed fx and Sigma 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 maxSigma 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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 Sigma 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, Sigma 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 maxSigma 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 Nomega -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: Sigma 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: Sigma fx. * P < 0.01 (Bonferroni test after 2-way ANOVA) statistically different between L-NAME and control dose-response curves.

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 Sigma fx (P > 0.05, by two-way ANOVA), but enhanced the NaCN-induced Sigma 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: Sigma 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: Sigma fx. * P < 0.01 (Bonferroni test after 2-way ANOVA), statistically different between TRIM and control dose-response curves.

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 Sigma 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 (maxSigma 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.

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 approx  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 approx  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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (approx 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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