Departamento de Bioquímica y Biología Molecular y Fisiología, Instituto de Biología y Genética Molecular, Consejo Superior Investigaciones Científicas, Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain
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ABSTRACT |
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The aim of the present work was to elucidate the role of NADPH oxidase in hypoxia sensing and transduction in the carotid body (CB) chemoreceptor cells. We have studied the effects of several inhibitors of NADPH oxidase on the normoxic and hypoxia-induced release of [3H]catecholamines (CA) in an in vitro preparation of intact CB of the rat and rabbit whose CA deposits have been labeled by prior incubation with the natural precursor [3H]tyrosine. It was found that diphenyleneiodonium (DPI; 0.2-25 µM), an inhibitor of NADPH oxidase, caused a dose-dependent release of [3H]CA from normoxic CB chemoreceptor cells. Contrary to hypoxia, DPI-evoked release was only partially Ca2+ dependent. Concentrations of DPI reported to produce full inhibition of NADPH oxidase in the rat CB did not prevent the hypoxic release response in the rat and rabbit CB chemoreceptor cells, as stimulation with hypoxia in the presence of DPI elicited a response equaling the sum of that produced by DPI and hypoxia applied separately. Neopterin (3-300 µM) and phenylarsine oxide (0.5-2 µM), other inhibitors of NADPH oxidase, did not promote release of [3H]CA in normoxic conditions or affect the response elicited by hypoxia. On the basis of effects of neopterin and phenylarsine oxide, it is concluded that NADPH oxidase does not appear to play a role in oxygen sensing or transduction in the rat and rabbit CB chemoreceptor cells in vitro and, in the context of the present study, that DPI effects are not related to NADPH oxidase inhibition.
hypoxia; reactive oxygen species; diphenyleneiodonium; neopterin; phenylarsine oxide; carotid body; partial pressure of oxygen
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INTRODUCTION |
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ALL CELLS IN HIGHER ANIMALS are responsive to hypoxia if intense enough. The response in most cells includes coordinated downregulation of energy demand and energy supply pathways, resulting in a decrease in ATP turnover and a slowing down of ATP-demanding cellular processes such as protein synthesis, maintenance of electrochemical gradients, and, in excitable cells, spiking activity; the goal of these responses is to prolong cell survival (20).
However, in chemoreceptor cells of the carotid body (CB), erythropoietin (EPO)-producing cells, and pulmonary artery smooth muscle cells (PASMC), which are involved in regulation loops aimed to restore O2 availability to the entire organism (14, 32), hypoxia sensing/transduction proceeds in an entirely different manner. First, these cells are activated by hypoxia and become increasingly active with the severity of hypoxia. Second, their hypoxic threshold is a PO2 of ~70 mmHg (31), i.e., they are activated before a significant drop in blood O2 content occurs. Finally, during hypoxia, they maintain high metabolic rates, ATP levels, and ATP turnovers (27, 38).
In these three cell systems, reactive oxygen species (ROS) have been implicated in O2 sensing/signaling. In the CB, it was found that diphenyleneiodonium (DPI), an inhibitor of NAD(P)H oxidase (17), increased the normoxic activity in the carotid sinus nerve (CSN) and within 20-30 min abolished the response to hypoxia (5). Acker and Xue (1) proposed that hypoxia would reduce the activity of NAD(P)H oxidase, leading to a decrease in the production of ROS in chemoreceptor cells. Because transduction of hypoxia in chemoreceptor cells involves changes in the gating of ion channels (15), it was also postulated that the decrease in ROS would alter the opening probability of ion channels to produce cell depolarization, activation of voltage-dependent Ca2+ channels, and Ca2+-dependent release of neurotransmitters (1). Similarly, it was reported that DPI blocks hypoxic vasoconstriction (36). As in the case of the CB, the proposal was that NADPH oxidase was involved in O2 sensing in PASMC (36). However, it has also been reported that hypoxia produces a decrease (2) or a DPI-sensitive increase in ROS levels (26). Additionally, Weir et al. (40) and Wyatt et al. (41) found that DPI is a nonselective blocker of ion currents in PASMC and CB chemoreceptor cells and suggested that the effects of DPI on ionic currents could account for its effects in the response of both cell types to hypoxia. In EPO cells, the key observation leading to involvement of NADPH oxidase in the O2 sensing was that H2O2 inhibited EPO production in response to hypoxia (8); however, DPI failed to induce EPO and inhibited the hypoxic EPO induction (13).
The aim of the present work was to elucidate the role of NADPH oxidase in CB chemoreceptor cell O2 sensing. In in vitro preparations of the rat and rabbit CB, we have studied the effect of the NADPH oxidase inhibitors DPI, neopterin (21), and phenylarsine oxide (PAO; see Ref. 24) on the release of catecholamines (CA) from chemoreceptor cells in normoxia and hypoxia. In normoxia, DPI increased the release of CA from chemoreceptor cells in a dose-dependent manner, but, contrary to hypoxia, the release elicited by DPI was only partially Ca2+ dependent. In addition, DPI- and hypoxia-induced release were additive, indicating parallel transduction pathways. Neither neopterin nor PAO affected normoxic release or the response to hypoxia. We conclude that NADPH oxidase function is not related to O2 sensing and that DPI effects in chemoreceptor cells are not related to NADPH inhibition in isolated rat and rabbit CB.
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MATERIALS AND METHODS |
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Surgery and CB isolation. Adult New Zealand White rabbits (2-2.5 kg) and adult Sprague-Dawley rats (250-300 g) were anesthetized with 40 and 60 mg/kg pentobarbital sodium (Sigma, Madrid, Spain), respectively, dissolved in physiological saline and administered via the lateral vein of the ear (rabbit) and intraperitoneally (rat). The animals were tracheostomized, the area of the carotid bifurcation was dissected, and the block of tissue containing the carotid bifurcation was removed and placed in a Lucite chamber filled with ice-cold Tyrode (in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1.1 MgCl2, 5.5 glucose, and 10 HEPES) adjusted to pH 7.40 with 1 N NaOH. The CBs (6-12/experiment) were identified and cleaned of surrounding connective tissue under a dissecting microscope and were collected in glass vials containing fresh Tyrode. After the removal of the carotid artery bifurcations, the animals were killed by intracardiac injection of 100 mg/kg of pentobarbital sodium.
Labeling of CA deposits of the CB. The
6-12 CBs used for an experiment were incubated in a glass vial
placed in a metabolic shaker at a constant temperature of 37°C. The
incubating solution was Tyrode solution containing 100 mM
6-methyltetrahydropterine (a tyrosine hydroxylase cofactor; Sigma), 1 mM ascorbic acid (a cofactor of dopamine -hydroxylase), and
[3H]tyrosine (Amersham
Ibérica, Madrid, Spain), the natural precursor of CA; in the
experiments with rabbit CB the concentration of [3H]tyrosine was 40 µM and the specific activity was 20 Ci/mmol, and in those with rat CB
the concentration was 30 µM and the specific activity 52 Ci/mmol.
During the 2 h of incubation, each rabbit CB synthesized ~12 pmol of
[3H]dopamine
([3H]DA), equivalent
to ~3 × 105 dpm, and ~1
pmol of
[3H]norepinephrine
([3H]NE), equivalent
to ~2.5 × 104 dpm (9, 28),
and the mean rat CB synthesis amounted to 3.1 ± 0.4 and 0.36 ± 0.06 pmol of [3H]DA
and [3H]NE,
respectively, amounting to 1.78 × 105 and 2.1 × 104 dpm (unpublished observation).
Release of [3H]CA and their analyses. In all of the experiments, after the labeling period, CBs were separated and transferred to new vials (1 CB/vial) containing 4 ml of precursor-free Tyrode bicarbonate (of identical composition to that above except for the replacement of 24 mM NaCl with an equimolar concentration of NaHCO3) continuously bubbled with a gas mixture (21% O2-5% CO2-balance N2) saturated with water vapor. In the experiments with the rabbit CB, the solution was renewed every 30 min during 2 h and was discarded, and in the experiments with the rat CB this washing period lasted 1 h and the solution was renewed every 15 min; in the washing period, most of the precursor, as well as the labile pool of labeled CA, was lost, and afterward the basal release of [3H]CA was stable for several hours. Thereafter, the incubating solutions were renewed every 10 min and were collected for later analysis of their [3H]CA content. The specific pattern of stimuli application to the CB is specified in RESULTS. The analysis of the released [3H]CA included acidification of the collected incubating solutions to pH 3.2 with a mixture of glacial acetic and ascorbic acid to avoid degradation of CA, bulk adsorption of all catechols released in alumina at a pH of 8.6, intense washing of alumina columns with distilled water, and bulk elution of all catechols with 1 ml of 1 N HCl. The alumina eluates were counted in a liquid scintillation spectrometer, and the released [3H]CA was expressed as disintegrations per minute per 10 min (11, 28).
Statistics. Significance of the differences observed between groups was assessed by the use of a two-tailed Student's t-test for unpaired data. Means ± SE are given. ![]() |
RESULTS |
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Time course and dose response of DPI on the release of
[3H]CA in the rat and rabbit CB.
In a first group of experiments, we studied the effect of DPI (10 µM)
on the basal or normoxic release of
[3H]CA from rat CB chemoreceptor
cells. Figure 1 compares the time course of
the basal release of [3H]CA in
control CBs with that elicited by 10 µM DPI applied during four
consecutive 10-min incubating periods. The mean basal release of
[3H]CA obtained from eight
individualized control CBs (1 CB/vial) was monotonic, whereas DPI
produced a time-dependent increase in the release of
[3H]CA. The mean release of
[3H]CA obtained in eight
DPI-treated CBs (1/vial) reached a maximum 30-40 min after DPI
application and remained above basal for >30 min after DPI removal.
It should be noted that the time course of 10 µM DPI action on the
release of [3H]CA is analogous
to that seen on rat CSN chemoreceptor activity (5). Due to the
difficulties of handling of the rat CB by reason of its small size
(average rat CB weight is ~50 µg; see Ref. 10), we have used rabbit
CB to perform a full dose-response study on the effect of DPI on the
normoxic release of [3H]CA.
Figure 2 shows the results. As in the case
of the rat CB, the basal release of
[3H]CA in control CBs (no DPI)
was monotonic, and the lowest concentration of DPI tested (0.2 µM)
did not alter it. The rest of the concentrations of DPI tested (1, 5, 10, and 25 µM) augmented the release of
[3H]CA in a dose-dependent
manner and with time courses (Fig.
2A) comparable to that observed in
the rat CB. In Fig. 2B, we have plotted the DPI-evoked release as a function of DPI concentration (each
data point is the mean ± SE of the evoked release in 8-12 individual CBs). It appears that, within the range of concentrations tested, there are two clear components in the effect of DPI on the
release of [3H]CA from
chemoreceptor cells. Up to 10 µM, the dose-response curve for DPI
followed a Michaelis-Menten-like relationship with calculated apparent
Michaelis constant and maximal velocity of 3.05 µM and 3.25 × 103 dpm/10 min, respectively; at
25 µM the response fell far above the fitting for the rest of the
data, suggesting the participation of a different mechanism in the
response. The difference between the release of
[3H]CA elicited by 10 µM DPI
in the rat CB (30,018 ± 2,818 dpm; n = 8) and in the rabbit CB (25,704 ± 4,345 dpm; n = 12) was not statistically significant.
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Comparative
Ca2+ dependence
of the release of [3H]CA induced by DPI
and hypoxia in the rabbit CB.
The Ca2+ dependence is a
well-established property of the release of
[3H]CA induced by hypoxia in the
rabbit CB chemoreceptor cells (11, 28). Therefore, it was of prime
importance to define if the release response elicited by DPI shared
this property with the natural stimulus. Figure
3A shows
the actual time course of the release of
[3H]CA elicited by 5 µM DPI in
normal Ca2+-containing and in
nominally Ca2+-free solutions. It
is evident that, although the elimination of
Ca2+ retarded the onset of the
response to DPI, a significant part of the response persisted in 0 Ca2+. Figure
3B shows total evoked responses in
Ca2+-containing (21,584 ± 2,732 dpm; n = 8) and in
Ca2+-free (6,613 ± 1,560 dpm;
n = 6;
P < 0.001) solutions, i.e., nearly 30% of the DPI response survived the removal of
Ca2+ from the incubating solution.
Figure 3, C and
D, shows comparable experiments using
hypoxia as stimulus. Hypoxia-induced release decreased from 12,975 ± 1,375 dpm (n =11) in
Ca2+-containing solution to 334 ± 95 dpm (n = 5) in 0 Ca2+ (97.5%;
P < 0.001). The release responses
produced by DPI and hypoxia in 0 Ca2+ were statistically different
(P < 0.005).
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Effects of DPI on the release of [3H]CA
induced by hypoxia in the rat and rabbit CB.
Cross et al. (5) observed that perfusion of the rat CB-CSN preparation
during 27 min with 10 µM DPI abolished the effect of hypoxia on CSN
discharges. On the basis of this observation, it was proposed (1; see
introduction) that, after pharmacological inhibition of NADPH oxidase,
a decrease of the response to hypoxia in chemoreceptor cells, where the
enzyme is located (22, 42), should be expected; full inhibition of the
oxidase should obliterate hypoxic responses. To directly test the
proposal, we compared the release of
[3H]CA from chemoreceptor cells
in three groups of rat CBs. In the first group, the CBs were subjected
to a hypoxic stimulus (10 min incubation with a solution equilibrated
with 2% O2;
PO2 20 mmHg); in the second
group, the CBs were incubated for 40 min with 10 µM DPI; and, in the
third group, the organs were similarly incubated with 10 µM DPI for
40 min but in the last 10 min were stimulated with the same level of
hypoxia as the first group. This timing for hypoxia application was
chosen based in the observations of Cross et al. (5). Figure
4A shows
the mean time course of the release of
[3H]CA in the three groups of
rat CBs (8 CBs/group; 1 CB/vial) and shows evidence that DPI did not
appear to alter the time course of the response to hypoxia, inasmuch as
the sum of the release elicited by DPI and by hypoxia at each time when
applied individually is equivalent to the release produced by the
simultaneous application of hypoxia and DPI. Figure
4B shows the total evoked responses in
the three groups, proving that the response to DPI and to hypoxia applied simultaneously (38,150 ± 2,294 dpm;
n = 8) essentially equals the sum of
the responses to hypoxia (7,722 ± 924 dpm;
n = 8) and to DPI (30,013 ± 2,818 dpm; n = 8) applied
separately. Differences of evoked release in the three experimental
groups were statistically different (P < 0.001, hypoxia vs. DPI or DPI + hypoxia;
P < 0.05, DPI vs. DPI + hypoxia),
but the difference between the sum of the evoked release by hypoxia and
DPI applied separately (37,735 ± 2,294) and the release evoked by
the two stimuli applied simultaneously was not statistically
significant (P > 0.4). Figure 4,
C and
D, shows the results obtained in an identical experiment performed with rabbit CBs. As it was the case in
the experiments in the rat CB, the total evoked responses by hypoxia
(12,975 ± 1,373 dpm; n = 11), DPI
(25,704 ± 4,345 dpm; n = 12), and
hypoxia plus DPI applied simultaneously (44,380 ± 2,749 dpm;
n = 8) exhibited differences that were
statistically significant (P < 0.01, hypoxia vs. DPI and DPI + hypoxia; P < 0.01, DPI vs. DPI + hypoxia). The difference between the sum of the
evoked release by hypoxia and DPI applied separately (38,679 ± 3,241 dpm) and the release evoked by the two stimuli applied simultaneously was not statistically significant
(P > 0.1).
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Effects of DPI on the release of [3H]CA
induced by high external
K+ in the rat
and rabbit CB.
The release of [3H]CA induced by
hypoxia in the rabbit and rat CB is greatly dependent on
Ca2+ entering the cells via
voltage-dependent channels (28, 33) and, on the other hand, it has been
shown that 10 µM DPI inhibits Ca2+ channels in isolated neonatal
rat chemoreceptor cells (41). However, we did not observe any decrease
in the release elicited by hypoxia in the presence of DPI, when
Ca2+ channels should be partially
inhibited. Therefore, it was of great interest to disclose, in the
intact CB, the significance of the observed inhibitory effect of DPI on
the Ca2+ channels of isolated
chemoreceptor cells. Because the release of
[3H]CA elicited by high
extracellular K+ in the intact CB
is inhibited by dihydropyridine antagonists of L-type
Ca2+ channels by 80% in the rat
(19) and fully in the rabbit (28), high external
K+ was the stimulus of choice to
test for the functional significance of DPI inhibition of
Ca2+ channels. Figure
5A
compares the mean time courses of the release responses elicited by 10 µM DPI, 35 mM external K+, and
both stimuli applied simultaneously in the rat CB, and Fig. 5B compares the total evoked release
of [3H]CA in the three groups.
Total evoked release elicited by DPI amounted to 30,013 ± 2,818 dpm
(n = 8), the release produced by 35 mM
external K+ was 7,518 ± 790 (n = 6), and that produced by both
stimuli applied simultaneously was 38,253 ± 1,476 (n = 6). Differences of evoked release
in the three experimental groups were statistically different (P < 0.001, 35 mM
K+ vs. DPI and 35 mM
K+ + DPI applied simultaneously;
P < 0.05, DPI vs. 35 mM
K+ + DPI applied simultaneously),
but the sum of the release evoked by the two stimuli applied separately
(37,531 ± 2,192) was not statistically different
(P > 0.4) from the release produced
by both stimuli applied simultaneously. Comparable results were
obtained in the rabbit CB (Fig. 5, C
and D).
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Effects of neopterin and PAO on the normoxic and hypoxia-induced release of [3H]CA. The ability of DPI to activate the release of [3H]CA and its inability to alter the response to hypoxia would indicate that inhibition of NADPH oxidase activity is not related to sensing or transduction of hypoxia but at the same time leaves open two alternative possibilities: 1) DPI activation of the neurosecretory response in chemoreceptor cells is the result of NADPH oxidase inhibition; or 2) DPI effects on chemoreceptor cells is a side effect unrelated to NADPH inhibition.
To resolve this problem experimentally, new experiments were performed using neopterin and PAO, specific inhibitors of NADPH oxidase inhibitors at the concentrations used and with reportedly different mechanisms of action (21, 24). Figure 6A shows that 100 µM neopterin and 1 µM PAO, which are concentrations capable of producing nearly full NADPH oxidase inhibition in phagocytic cells, do not alter the normoxic release of [3H]CA in the rat CB. The use of neopterin and PAO was extended to the rabbit CB to study the effects of several concentrations of each agent on the normoxic release of [3H]CA and the effects of selected concentrations on the release of [3H]CA induced by hypoxia. Neopterin did not affect basal release of [3H]CA at any of the concentrations tested (3, 10, 30, 100, and 300 µM); Fig. 6B shows the results for 3, 30, and 300 µM neopterin. As shown in Fig. 6C, PAO at concentration of 0.5, 1, and 2 µM was equally ineffective at modifying the normoxic release of [3H]CA. Figure 7, A and B, compares the time course of release of [3H]CA induced by hypoxia in the absence and in the presence of 100 µM neopterin and 2 µM PAO. Neither inhibitor affected the time course or peak release response produced by hypoxia in the rabbit CB.
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DISCUSSION |
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The present study was undertaken to clarify the role of NADPH oxidase and, thereby, the possible significance of NADPH oxidase-derived ROS in the oxygen sensing/signaling in chemoreceptor cells of the CB. We have found that only DPI, one of three inhibitors of NADPH oxidase used, is able to activate chemoreceptor cells in basal or normoxic conditions to produce a dose-dependent increase in the release of [3H]CA. The other two inhibitors, neopterin and PAO, did not activate chemoreceptor cells in basal conditions, and none of the three altered the release response elicited by hypoxia.
It should be remarked that NADPH oxidase has been demonstrated in the chemoreceptor cells of the CB of all species studied, including rat (22, 42), guinea pig, and human (22). Therefore, although NADPH oxidase has not been specifically localized in the rabbit CB, there are no reasons to suspect that rabbit CB chemoreceptor cells do not express the enzyme. As a consequence, and due to the close identity of results obtained in the rat and rabbit CB, we shall discuss the data obtained in both species as a whole.
The parameter selected in our intact preparation of the CB to measure activation of chemoreceptor cells is the release of [3H]CA. In the case of the rabbit CB, it is extensively documented that nearly 96% of the [3H]CA synthesis occurring in the intact CB takes place in chemoreceptor cells (9, 15). Most (~93%) of the [3H]CA synthesized is [3H]DA, which is located in chemoreceptor cells, the remaining being [3H]NE located both in chemoreceptor cells and intraglomic sympathetic endings. The situation in the rat CB is similar. It is well documented that the rat CB is also a CA-rich organ with DA-to-NE ratios varying between 3 and 5 (16 and unpublished observation). In the rat CB, as in the case in the rabbit CB, all DA is stored in chemoreceptor cells, and NE is preferentially stored in sympathetic endings since surgical sympathectomy did not affect CB DA content but reduced by over 50% NE levels (16). Recent studies from our laboratory (unpublished observation) have shown that, in a rat CB preparation identical to that used in the present study, hypoxia and high external K+ released [3H]DA and [3H]NE in ratios of 9.7 to 1 and 8.1 to 1, respectively. Therefore, even when the functional significance of CA in the CB is in dispute (15), it is evident that both in the rat and rabbit CB the release of [3H]CA is a rather specific index of chemoreceptor cell function.
There is no doubt that DPI can activate the CB. Cross et al. (5) found that DPI (10 µM) augmented the action potential frequency in the rat CSN, and here we show that the same concentration of the drug augments the release of [3H]CA from rat CB chemoreceptor cells (Fig. 1); in the rabbit CB, DPI increases the release of [3H]CA from chemoreceptor cells in a dose-dependent manner (Fig. 2). However, in the same article, Cross et al. (5) also reported that DPI (10 µM) was able to abolish the CSN response elicited by hypoxia, whereas we find it does not modify the response of chemoreceptor cells to hypoxia in either species (Fig. 4). Because NADPH oxidase is located in chemoreceptor cells (22, 42), a possible explanation for the discrepancy could be that the abolition of the CSN response to hypoxia produced by DPI is the result of another action of the inhibitor occurring at the level of the nerve endings of the CSN and not at the NADPH oxidase of chemoreceptor cells. It is known that 10 µM DPI fully or nearly fully inhibits NADPH oxidase (5, 17, 26), so that in the presence of this concentration of DPI hypoxia should be unable to inhibit the enzyme further. Therefore, if hypoxia activates chemoreceptor cells via inhibition of NADPH oxidase, in the presence of 10 µM DPI, hypoxia should be ineffective in triggering the release of [3H]CA. The data presented in Fig. 4 showing that the release of [3H]CA elicited by hypoxia is not affected by DPI indicate that hypoxia sensing and transduction is independent of the activity of this enzyme. Consistent with this conclusion are our findings with neopterin and PAO (Fig. 7, A and B); neither inhibitor of NADPH oxidase at concentrations capable of fully inhibiting the enzyme (see below) alters the sensing of hypoxia and its transduction into the neurosecretion of [3H]CA. Finally, if as postulated by Cross et al. (5; see also Ref. 1) the mechanism involved in the activation of the CB by DPI is the same for hypoxia (i.e., the inhibition of NADPH oxidase), it should be expected that the responses elicited by both stimuli share the same properties. However, this is not the case. DPI-induced release of [3H]CA is only partially dependent on the presence of Ca2+ in the extracellular milieu, whereas that induced by hypoxia is totally dependent on extracellular Ca2+ (Fig. 3).
A close inspection of the data of Cross et al. (Fig. 4 in Ref. 5) supports the contention raised in the previous paragraph. DPI (10 µM) produced an increase in the discharges of the CSN equivalent to about one-third of that produced by hypoxia, and yet it abolished the response to hypoxia. As a first alternative to explain this observation, it might be argued that this concentration of DPI did not fully inhibit NADPH oxidase and thereby explain the inability of DPI to produce the same level of activity as the hypoxic stimulus; however, if the enzyme was only partially inhibited, it should be possible to inhibit it further with hypoxia, and then the response to hypoxia should not be fully abolished. As a second alternative, it could be assumed as shown by Cross et al. (5) that 10 µM DPI inhibited NADPH oxidase in full; then, it should produce an activation of the CB equivalent or higher than (see below) that produced by the hypoxic stimulus tested. Therefore, in neither alternative do the data support a causal link between NADPH oxidase inhibition and CSN activation. Interestingly, the intensity of the hypoxic stimulus used by Cross et al. (5), although strong, is submaximal and comparable to the stimulus used in the present experiments (~20 mmHg). In their study, the effect of 10 µM DPI on peak CSN discharges was small (one-third) in comparison with hypoxia, whereas in our experiments the same concentration of DPI produced a peak effect on the release of [3H]CA higher than (rat CB) or comparable to (rabbit CB) the hypoxic stimulus. Because, at least in the rabbit CB, there is a close parallelism between the release of [3H]CA and the activity in the sinus nerve (11), these considerations would also suggest that DPI is inhibiting the firing of the CSN.
A different but related issue is if DPI activation of the CB is related to its ability to inhibit NADPH oxidase. Our data would indicate that DPI activates chemoreceptor cells by a mechanism unrelated to NADPH oxidase inhibition. Thus neither neopterin, at concentrations capable of totally inhibiting NADPH oxidase in peritoneal macrophages (21) and in neural tissue (4), nor PAO, also at supramaximal concentrations to inhibit the enzyme in neutrophils (24), was able to activate chemoreceptor cells to induce release of [3H]CA (Fig. 6, A-C). Therefore, our conclusion would be that NADPH oxidase inhibition is incapable of triggering the activation of chemoreceptor cells and consequently that DPI effects on the CB are, in the present context, nonspecific.
However, the saturation-like kinetics of the release of
[3H]CA elicited by DPI at
concentrations below 10 µM suggest the involvement of an enzymatic or
binding mechanism in the genesis of the response. It is known that DPI
acts by acceptance of electrons at the reduced flavin centers of
flavoenzymes (29), and thereby it can inhibit, probably with different
potency, any enzyme, which being present in chemoreceptor cells uses
flavins as cofactors. It is known, for example, that DPI can inhibit
nitric oxide synthase (6, 34), cytochrome
P-450 reductase (35), xanthine oxidase
(7), and protoporphyrinogen oxidase (3), and it appears to activate guanylate cyclase (6). Although any of these enzymes could be the
target responsible for the effects of DPI in chemoreceptor cells, it
should be pointed out that cytochrome
P-450 has been proposed to be involved
in CB and PASMC hypoxia sensing and transduction (18, 43), and
therefore it might be that the observed effects of DPI on the release
of [3H]CA are the result of
cytochrome P-450 reductase inhibition. At higher concentrations (inhibitory constant 30 µM), DPI can also inhibit mitochondrial complex 1 (12,
25), and this inhibition of mitochondrial function could explain the
great effect of DPI on the release of
[3H]CA at 25 µM, as all
mitochondrial poisons are powerful stimulants of the CB (15).
The effects of DPI on the release of [3H]CA induced by high external K+ observed in the experiments using intact rat and rabbit CB (Fig. 5) contrast with the marked inhibition of Ca2+ currents produced by DPI in isolated chemoreceptor cells of the neonatal rat (41). As the release of [3H]CA induced by high external K+ in the rabbit and rat CB is inhibited by antagonists of voltage-dependent Ca2+ channels (19, 28), inhibition of the release of [3H]CA should be expected with the use of DPI. However, we observe that DPI does not inhibit the release of [3H]CA elicited by high external K+. We do not have a sound explanation for the apparent contradiction existing between both groups of results, but we envision two possibilities. First, it might be argued that DPI does not inhibit Ca2+ channels in intact chemoreceptor cells in the entire organ, the inhibition seen in isolated cells being the result of cell isolation and culture or of cell dialysis during whole cell recording. Second, since DPI also inhibits K+ channels (40, 41), it would be possible that chemoreceptor cells are partially depolarized by DPI in the absence of any other stimulus; this inhibition of K+ channels would in turn determine that hypoxia and high external K+ produce a more intense depolarization in the presence of DPI, and as a consequence more Ca2+ channels would be activated. This extra recruitment of Ca2+ channels would tend to balance their inhibition, with the net result of only minor modifications in the secretory response. DPI inhibition of K+ channels and depolarization might contribute to or be responsible for that part of the release of [3H]CA induced by DPI that is Ca2+ dependent.
The main conclusions of our work are that NADPH oxidase is not involved in O2 sensing or transduction in chemoreceptor cells of the rat and rabbit CB in vitro and that the capacity of DPI to activate the CB is not due to its ability to inhibit the oxidase because other inhibitors of different chemical structure and mechanisms of action do not activate CB chemoreceptors. However, in relation to the proposal implicating ROS in the O2 sensing/signaling, our results do not exclude that ROS of origin different from NADPH oxidase might have a potential significance in the overall response of the chemoreceptor cells (and PASMC and EPO cells) to hypoxia. On the one hand, it is known that ROS, per se, and the redox pairs expected to be altered by changes in the rate of ROS production are able to modify the gating properties of ion channels, albeit not always in the predicted direction (37). On the other hand, it is conceivable that hypoxia, the sensed signal, alters the rate of production of ROS. However, difficulties in the methodologies of ROS measurement (30) yielded conflicting results regarding the direction of the change in the rate of ROS production during hypoxia (see Introduction). Therefore, we think that, at least for the chemoreceptor cells, most of the path (23) to ascribe a role to ROS in the O2 sensing and transduction remains to be worked out.
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ACKNOWLEDGEMENTS |
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We thank María de los Llanos Bravo for technical support in the realization of the experiments.
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FOOTNOTES |
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This work was supported by Spanish Dirección General de Investigación Científica y Técnica Grant PB92/0267.
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. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: C. González, Departamento de Bioquímica y Biología Molecular y Fisiología, IBGM, CSIC, Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain (E-mail: constanc{at}ibgm.uva.es).
Received 3 August 1998; accepted in final form 17 December 1998.
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