Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice

L. He1, J. Chen1, B. Dinger1, K. Sanders2, K. Sundar2, J. Hoidal2, and S. Fidone1

Departments of 1 Physiology and 2 Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah 84108


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Various heme-containing proteins have been proposed as primary molecular O2 sensors for hypoxia-sensitive type I cells in the mammalian carotid body. One set of data in particular supports the involvement of a cytochrome b NADPH oxidase that is commonly found in neutrophils. Subunits of this enzyme have been immunocytochemically localized in type I cells, and diphenyleneiodonium, an inhibitor of the oxidase, increases carotid body chemoreceptor activity. The present study evaluated immunocytochemical and functional properties of carotid bodies from normal mice and from mice with a disrupted gp91 phagocytic oxidase (gp91phox) DNA sequence gene knockout (KO), a gene that codes for a subunit of the neutrophilic form of NADPH oxidase. Immunostaining for tyrosine hydroxylase, a signature marker antigen for type I cells, was found in groups or lobules of cells displaying morphological features typical of the O2-sensitive cells in other species, and the incidence of tyrosine hydroxylase-immunopositive cells was similar in carotid bodies from both strains of mice. Studies of whole cell K+ currents also revealed identical current-voltage relationships and current depression by hypoxia in type I cells dissociated from normal vs. KO animals. Likewise, hypoxia-evoked increases in intracellular Ca2+ concentration were not significantly different for normal and KO type I cells. The whole organ response to hypoxia was evaluated in recordings of carotid sinus nerve activity in vitro. In these experiments, responses elicited by hypoxia and by the classic chemoreceptor stimulant nicotine were also indistinguishable in normal vs. KO preparations. Our data demonstrate that carotid body function remains intact after sequence disruption of the gp91phox gene. These findings are not in accord with the hypothesis that the phagocytic form of NADPH oxidase acts as a primary O2 sensor in arterial chemoreception.

hypoxia; reactive oxygen species; sensory transduction; chemoreceptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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IT IS WIDELY HELD that O2 chemoreception in the mammalian carotid body is initiated by specialized chemosensory type I cells that respond to hypoxia with depolarization and release of multiple neurotransmitter agents (9, 11). The cascade of molecular and cellular events involved in O2 chemotransduction has been the subject of intense scrutiny in recent years. Numerous laboratories (5, 14, 21, 22, 24, 27) have reported that low PO2 inhibits the conductance of a variety of voltage-sensitive and voltage-insensitive K+ channels in type I cells, yet the molecular mechanisms underlying the modulation of these currents remain uncertain and controversial. Various heme proteins have also been proposed as the primary O2 sensors (11), and one set of data in particular suggests the involvement of a multicomponent cytochrome b-containing NADPH oxidase that may be similar, if not identical, to an enzyme commonly found in phagocytic cells (1, 2). According to this hypothesis, the oxidase generates reactive oxygen species (ROS) in proportion to available O2, which then modulates K+ channels via the formation of H2O2 from ROS by the action of superoxide dismutase. Immunoreactivity for multiple subunits of the phagocytic form of the enzyme, including p22phox, gp91phox, p47phox, and p67phox, has been localized to type I cells (17), and an inhibitor of the oxidase, diphenyleneiodonium (DPI), alters carotid body chemoreceptor activity evoked by hypoxia (17).

Neuroepithelial bodies (NEBs) located in lung airways have also been proposed as O2 chemoreceptors, and like the carotid body, they consist of specialized cells that are thought to release neuroactive agents in response to hypoxia (18-20). NEB cells also express O2-sensitive K+ channels, and immunocytochemical studies indicate that these cells contain components of the phagocytic NADPH oxidase (29, 32). A possible functional link between the oxidase and K+ channels in NEB cells was established in gp91phox gene knockout (KO) mice in which the K+ currents were inhibited by H2O2 but not by hypoxia or DPI, suggesting that the ROS-producing enzyme is an essential component of the transduction machinery (10). In contrast, other investigators (3) using gp91phox KO animals reached opposite conclusions in studies of pulmonary arterial smooth muscle cells (PASMCs) that showed that K+ currents displayed normal sensitivity to hypoxia.

In the present study, we have used the gp91phox gene KO strain of animals to examine the hypothesis that the phagocytic form of the NADPH oxidase is the primary O2 sensor in the carotid body. A multidisciplinary research strategy was employed to evaluate multiple functional components of the chemotransduction pathway. Our data suggest that normal vs. KO animals equally express tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, which has been established as a signature marker antigen for chemosensory type I cells. Moreover, functional properties of the chemosensory cells, including hypoxia-evoked Ca2+ responses and depression of K+ currents by low PO2, did not differ after gene KO. Finally, the carotid sinus nerve (CSN) responses evoked by hypoxia in vitro were indistinguishable in the two strains of mice.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Animals. All procedures involving animals were approved by the University of Utah Institutional Animal Care and Use Committee. Experimental mice containing the disrupted gp91phox gene (gp91phox KO) were generated at the Indiana University School of Medicine and kindly provided by M. C. Dinauer. The gp91phox gene is located on the X chromosome, and, consequently, all experimental mice used in this study were males hemizygous for the disrupted gene. A previous study by Pollock et al. (25) showed that neutrophils from these mice lack normal respiratory burst activity. Age-matched male mice of the C57BL/6 strain that possessed normal NADPH oxidase activity served as control animals.

Polymorphonucleocytes obtained from C57BL/6 mice demonstrated a respiratory burst in response to stimulation with phorbol 12-myristate 13-acetate, but polymorphonucleocytes from gp91phox KO mice did not respond to phorbol 12-myristate 13-acetate (Sanders and Hoidal, unpublished observations).

Immunocytochemistry. While the animals were under pentobarbital sodium anesthesia (50-60 mg/kg ip), carotid bodies were surgically removed from eight C57BL/6 (normal) and eight gp91phox KO mice. Tissues were immediately immersed in ice-cold 4% paraformaldehyde in 0.1 M PBS (pH 7.4). After fixation for 1 h, the surface of each carotid body was darkened with a concentrated solution of colloidal india ink. This procedure greatly improved the visibility of these exceedingly small organs during subsequent washing and sectioning. The carotid bodies were equilibrated for 1 h with a solution of cold 20% sucrose in 0.1 M PBS and were then embedded in optimum cutting temperature compound at -20°C. Sectioning (4 µm) each mouse carotid body yielded 17-20 useful specimens containing type I cells. Thaw-mounted sections were first exposed to avidin-biotin preblocking reagents (20 min; Vector) and incubated at 4°C overnight in anti-TH primary antibody (Chemicon) diluted 1:1,000 in PBS containing 0.3% Triton X-100. Sections were then rinsed in PBS at room temperature and incubated for 2 h in biotinylated goat anti-rabbit IgG (diluted 1:200; Vector), rinsed in PBS for 20 min, incubated in avidin-biotinylated horseradish peroxidase complex (2 h; Vector Elite kit), and treated with 3',3'-diaminobenzidine tetrahydrochloride and H2O2.

Dissociation of carotid body cells. In pentobarbital sodium-anesthetized mice (10 C57BL/6, 14 gp91 KO), the carotid arteries, including the bifurcations, were surgically exposed, removed, and placed in ice-cold modified Tyrode solution containing (in mM) 112 NaCl, 4.7 KCl, 2.2 CaCl2, 1.1 MgCl2, 42 sodium glutamate, 5.6 glucose, and 5 HEPES buffer (pH 7.43; 37°C) and equilibrated with 100% O2. The carotid bodies were dissected free of surrounding connective tissue and transferred to Hanks' balanced salt solution (HBSS; Ca2+- and Mg2+-free) containing 0.2% collagenase and 0.2% trypsin. Each carotid body was cut into two pieces and incubated for 20 min in a CO2 incubator (5% CO2-95% air) at 36.5°C. Tissue fragments were centrifuged (1,730 g for 7 min at 4°C) in HBSS (Ca2+- and Mg2+-free) and then transferred to poly-L-lysine-coated glass coverslips where they were triturated in a small volume of Ham's F-12 medium plus 10% fetal calf serum and 5 µg/ml of insulin. The coverslips containing dissociated type I cells were placed in the CO2 incubator for 2-5 h before use.

Whole cell patch-clamp recordings. The coverslips containing the type I cells were placed in a 0.3-ml flow chamber mounted on the stage of a Zeiss phase-contrast, inverted microscope. Cells were bathed in modified Tyrode solution delivered at 0.5 ml/min via a peristaltic pump. Bath temperature was maintained at 35-36.5°C. The bath was grounded via a Ag/AgCl electrode. Patch pipettes were fabricated from borosilicate glass tubing (outer diameter, 1.5 mm; inner diameter, 0.75 mm; Sutter Instrument) in a Flaming/Brown micropipette puller (model P-87; Sutter Instrument). Pipette resistance varied between 2 and 10 MOmega . For K+ current measurements, bath solutions contained 135 mM choline-Cl, 5 mM KCl, 50 µM CdCl2, 1.0 mM CaCl2, 1.0 mM MgCl2, 5.6 mM glucose, and 10 mM HEPES buffer, pH 7.43, at 37°C. The pipette solution contained 145 mM potassium glutamate, 15 mM KCl, 2 mM MgCl, and 20 mM HEPES, pH 7.2, at 37°C. K+ current was evoked by step voltage changes from a holding potential of -70 mV.

Under control conditions, the cells were superfused in solution equilibrated with air (PO2 ~120 Torr). Hypoxic solutions were equilibrated with air and contained 1 mM sodium dithionite, resulting in a bath PO2 of ~32 Torr, an O2 tension similar to that of the internal carotid body during moderate hypoxia (6, 7). Bath PO2 was measured with a Diamond General model 760 needle electrode connected to a Harvard model 102 oxygen electrode amplifier. The system was calibrated in buffer solutions equilibrated with 100% O2 and 100% N2 at 37°C.

Whole cell currents were recorded with an Axopatch 200A patch-clamp amplifier and a CV 201A headstage (Axon Instruments). Records were simultaneously displayed on an oscilloscope and digitized with a DigiData 1200 computer interface for analysis with pCLAMP version 5.0 software (Axon Instruments). The series resistance was typically 40 MOmega for perforated whole cell recordings and was not compensated in these experiments. Junction potentials, which varied from 2 to 4 mV, were canceled at the beginning of the experiment. Data shown in the text were plotted after subtraction of any capacitance and leakage currents.

Measurement of intracellular Ca2+ concentration. Details of these procedures have been published previously (13). Briefly, dissociated type I cells attached to coverslips were incubated in Ham's F-12 medium containing 0.5 µM fura 2-AM for 10-15 min in a CO2 incubator at 36.5°C. Coverslips were placed in a flow chamber where they were superfused with modified Tyrode solution equilibrated with air (PO2 ~120 Torr) at 0.75-1.0 ml/min. The temperature was maintained at 35-36.5°C. Cells were made hypoxic by the addition of 1.0 mM sodium dithionite to the superfusate (PO2 ~31-33 Torr). The chamber was mounted on the stage of a Zeiss inverted microscope incorporated into a Zeiss/Attofluor workstation equipped with an excitation wavelength selector (filter changer) and an intensified charge-coupled device camera system. Fura 2 fluorescence emission was measured at 520 nm in response to alternating excitation wavelengths of 334 and 380 nm. Data were collected and analyzed with Attofluor Ratiovision software (version 6.0). Typically, fluorescence observations were obtained from isolated type I cells. However, some data were also obtained from multiple cells aggregated into clusters. In these instances, data from each cell were analyzed separately. We did not observe any consistent difference in the basal or stimulus-evoked responses from isolated vs. clustered cells. Cells selected for analysis displayed morphology typical of type I cells, and they responded to the low-O2 stimulus with at least a doubling of the basal intracellular Ca2+ concentration ([Ca2+]i).

Electrophysiological recording of CSN activity. While the animals were under pentobarbital sodium anesthesia and with the aid of a dissecting microscope, the carotid bifurcations containing the carotid bodies were located and removed from nine C57BL/6 and seven gp91phox KO mice and placed in a lucite chamber containing 100% O2-equilibrated modified Tyrode solution at 0-4°C. Each carotid body, along with its attached nerve, was carefully removed from the artery and cleaned of surrounding connective tissue, and the preparation was then placed in a conventional superfusion chamber where the carotid body was continuously superfused (up to 4 h) with modified Tyrode solution maintained at 37°C and equilibrated with a selected gas mixture. The CSN was positioned in the tip (inner diameter ~75 µm) of a glass suction electrode for monopolar recording of chemoreceptor activity (12). The bath was grounded with a Ag-AgCl2 wire, and neural activity was led to an alternating current-coupled preamplifier, filtered, and transferred to a window discriminator and a frequency-to-voltage converter. Signals were processed by an analog-to-digital/digital-to-analog converter for display of frequency histograms on a PC computer monitor. Basal (resting) CSN activity was established in solutions equilibrated with 100% O2, which resulted in a bath PO2 ~450 Torr. The superfused preparations were stimulated with solutions equilibrated with air (PO2 ~120 Torr) or 100% N2 (PO2 ~40 Torr) or that contained 100 µM nicotine, a classic chemoreceptor stimulant (9).


    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
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TH-immunoreactive cells in normal vs. KO carotid bodies. Immunocytochemical staining of frozen sections from normal and KO mice carotid bodies revealed immunoreactivity for TH, the rate-limiting enzyme for catecholamine synthesis (Fig. 1). TH has been established as a signature marker antigen for chemosensory type I cells in other mammalian species, including cat and rat (30, 31). In the normal mouse carotid body (Fig. 1A), TH immunostaining was found in groups of cells that formed island-like lobules surrounded by vascular and connective tissue, features typical of carotid bodies from other species (28). Also typical, the immunostained cells appeared to be 8-12 µm in diameter, and they contained large ovoid nuclei. The TH immunoreactivity varied in different cells from light to intensely dark. The incidence of cell lobules displaying comparable morphological and immunostaining characteristics appeared similar in carotid bodies from normal vs. KO mice (Fig. 1, A vs. B).


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Fig. 1.   Tyrosine hydroxylase (TH) immunostaining in normal and gp91 phagocytic oxidase (gp91phox) gene knockout (KO) mouse carotid body. Bar, 20 µm.

K+ currents recorded in dissociated cells. Figure 2A shows outward K+ currents recorded in cells dissociated from normal and KO carotid bodies in response to a voltage step to +60 mV from a holding potential of -70 mV. The three traces show the current recorded in solutions equilibrated with air (control; PO2 ~120 Torr), air-equilibrated solutions containing 1 mM sodium dithionite (hypoxia; PO2 ~31-33 Torr), and a trace recorded 2-3 min after reintroduction of the normoxic solution (recovery). Depression of the outward K+ current by low O2 tension is characteristic of chemosensory type I cells (11). The time course of the evoked outward currents was similar in normal and KO cells, as was the depression of the current by hypoxia. Typical current-voltage relationships established with incremental voltage steps between -60 mV and +60 mV were indistinguishable between the two groups of cells (Fig. 2B). The summary data in Fig. 2C show that currents evoked by a voltage step to +60 mV and the effects of hypoxia on the evoked currents were not statistically different in groups of 15 normal cells vs. 14 cells from KO animals.


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Fig. 2.   Outward K+ current recorded in normal and gp91phox gene KO cells dissociated from mouse carotid bodies. A: current evoked by a voltage step from a holding potential of -70 to +60 mV. Control and recovery currents were recorded in solution at PO2 ~120 Torr; hypoxic current (hypoxia) was recorded at PO2 ~31-33 Torr. B: typical current-voltage relationships in cells from normal and KO animals were indistinguishable. C: summary data from 15 normal and 14 KO cells show that voltage-evoked (voltage step from -70 mV to +60 mV) currents in normoxia and hypoxia are statistically identical (P > 0.19), and depression of currents by hypoxia is likewise not different after gp91phox gene KO (P > 0.9).

[Ca2+]i regulation in normal vs. KO chemosensory cells. [Ca2+]i was assessed in 67 normal and 83 KO cells. In cells from normal mice, basal [Ca2+]i, recorded in solutions equilibrated with air (PO2 ~120), ranged from 3.0 to 24.0 nM, with a mean of 12.30 ± 0.69 (±SE). The basal [Ca2+]i in cells from KO animals displayed a similar range (2.0-27.0 nM), and the mean (11.05 ± 0.056) was not statistically different from the value measured in normal cells (P = 0.187). Figure 3 shows four typical [Ca2+]i responses evoked by a 60-s low O2 stimulus (PO2 ~31-33 Torr) in each of two cells from both normal and KO animals. Peak [Ca2+]i increases in cells from normal animals varied from 33 to 467 nM, with a mean (±SE) in 67 cells of 87.9 ± 13.3 nM. In cells from KO animals, peak responses to hypoxia ranged from 25 to 442 nM, and the mean response (81.1 ± 11.1) in 83 cells was not statistically different from that in normal cells (P > 0.05). The variability in the Ca2+ responses reported here in normal and KO cells is in accord with observations made in normal rat type I cells (4).


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Fig. 3.   Hypoxia-evoked intracellular Ca2+ concentration ([Ca2+]i) responses in type I cells from normal vs. gp91phox KO mice. Two sample responses each from normal (left) and KO (right) type I cells evoked by low O2 solutions (PO2 ~31-33 Torr) are shown at top. Basal [Ca2+]i was established at PO2 ~120 Torr. Summary data from 67 normal and 83 KO type I cells are shown at bottom. P > 0.07 by Mann-Whitney rank sum test.

Resting and evoked CSN activity. CSN activity was successfully recorded in vitro in 13 normal and 13 KO mouse preparations. Resting (basal) neural activity measured at bath PO2 ~450 Torr ranged from 4.8 to 23.8 Hz, and the mean (±SE) basal activity in normal vs. KO preparations was virtually identical (normal, 15.61 ± 1.36 Hz; KO, 15.65 ± 1.44 Hz). The top and middle sections of Fig.4A show that lowering bath PO2 to ~120 or ~40 Torr [moderate and severe hypoxic stimuli, respectively, for rat carotid bodies superfused in vitro (12)] elicited a steep increase in neural activity from both normal and KO mouse preparations. During the ~100 s of moderate or severe hypoxia, CSN activity was maintained at a high level of discharge; neural activity rapidly decreased when the bath PO2 was switched back to 450 Torr. Figure 4B summarizes observations from normal and KO preparations and shows that the nerve discharge, averaged over the 100-s hypoxic episode, was not statistically different between normal and KO preparations for both moderate (PO2 ~120) and severe (PO2 ~40) hypoxic stimulus conditions.


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Fig. 4.   A: effect of hypoxia and nicotine on carotid sinus nerve (CSN) activity in normal (left) and gp91phox gene KO (right) mice. Carotid body/CSN preparations were superfused in vitro, and CSN activity was recorded with a suction electrode. Typical traces show CSN activity evoked by lowering of the bath PO2 (superimposed trace) to ~120 (top) and ~40 Torr (middle) and by superfusion with 100 µM nicotine (bottom). B: summary data from multiple preparations show that responses were statistically identical in normal vs. KO animals. Nos. in parentheses, no. of mice; imp, impulses. P > 0.7.

The introduction of 100 µM nicotine (Fig. 4A, bottom) also elicited a rapid rise in neural activity in both normal and KO preparations, but unlike hypoxic stimulation, the peak response declined to a lower level during the continued presence of the drug. Discharge rates returned slowly to prestimulus levels over a period of 2-3 min after the reintroduction of nicotine-free superfusate. As with hypoxia, the average increase in the nicotine-evoked nerve discharge did not differ between normal and KO carotid bodies (Fig. 4B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The data presented here indicate that multiple features of carotid body function are not affected by disruption of the gp91phox gene. The assessments in our study included functional properties that are exquisitely sensitive to hypoxia, namely, whole cell voltage-gated K+ current, [Ca2+]i, and CSN activity. Moreover, the morphology of the carotid body and the expression of TH in type I cells do not appear to be altered as a result of gp91phox gene KO. Thus our data are not in accord with the hypothesis that the phagocytic form of the NADPH oxidase functions as the primary O2 sensor in carotid body chemoreception (1, 2). Roy et al. (26) reached a similar conclusion in a study that evaluated ventilatory reflexes and type I cell [Ca2+]i responses evoked by hypoxia. However, these findings conflict with a study of resting and hypoxia-evoked changes in ventilation in neonatal mice deficient in gp91phox (16).

A primary postulate of the oxidase hypothesis is that the generation of ROS in type I cells results in increased open probability of O2-sensitive, voltage-gated K+ channels (1). NEB cells in mice express similar hypoxia-sensitive, voltage-gated K+ currents (32). However, unlike the K+ currents recorded in the present experiments, NEB cell K+ currents lose their sensitivity to hypoxia after gp91phox gene KO (10). Recent studies (5, 24) suggest that type I cell depolarization is not initiated by the voltage-sensitive K+ currents recorded in the present study but, instead, suggest that cell activation may involve a separate set of voltage-insensitive K+ channels, which give rise to resting leak currents across the plasma membrane. If hypoxia-sensitive leak currents regulate type I cell membrane potential, then it is possible that the ROS generating oxidase is coupled to sets of K+ channels with divergent properties in type I cells vs. NEB cells. Consequently, disruption of gp91phox would not necessarily alter the expression of voltage-sensitive K+ currents nor their response to hypoxia in type I cells. However, our finding that later steps in the transduction cascade (i.e., hypoxia-evoked [Ca2+]i responses and CSN activity) remain intact in gp91phox KO mice indicates that gene disruption does not affect the overall performance of the chemotransduction machinery, including any alternative sets of hypoxia-sensitive K+ currents in type I cells that may be required for cell activation.

Because sequence disruption is gene specific, the present data do not eliminate the possibility that a different isoform of the gp91phox subunit remains functional in the carotid bodies of KO mice. However, other recent studies further support the notion that NADPH oxidases are unlikely candidates for primary O2 sensors in carotid body type I cells or for other O2-sensitive cells, for that matter. For example, Obeso et al. (23) reported that although the NADPH inhibitor DPI activated type I cells and evoked the release of catecholamines, this effect occurred via a mechanism that differed from hypoxia-elicited cell activation. Furthermore, other specific NADPH oxidase blockers, neopterin and phenylarsine oxide, did not promote the release of catecholamines from carotid bodies superfused in vitro (23). In PC12 cells, which share multiple functional characteristics with carotid body type I cells, Kummer and colleagues (15) have shown that the induction of TH by hypoxia (a response that also occurs in type I cells) does not depend on the generation of ROS. Moreover, Kummer et al. have pointed out that the ubiquitous expression of the gp91phox subunit in sensory neurons of visceral and dorsal root ganglia suggests a general function rather that a specific role for NADPH oxidase in arterial chemoreception (8). In addition, other studies with the gp91phox gene KO model have shown that loss of NADPH oxidase function does not alter hypoxia-induced K+ current depression and vasoconstriction in PASMCs (3), nor does it effect the hypoxia-evoked transcription of the erythropoietin gene in kidney (Sanders and Hoidal, unpublished observations).

Previous studies have shown that gp91phox gene KO mice lack phagocyte superoxide production and manifest a correspondingly increased susceptibility to infection (25). According to the "oxidase hypothesis" of chemoreception, disruption of oxidase-dependent ROS production should substantially compromise hypoxic chemotransduction in type I cells, resulting in altered resting and hypoxia-evoked chemoreceptor activity (1). Our data indicate that chemoreceptor function is normal in gp91phox KO mice and are not in agreement with the hypothesis that the phagocytic form of NADPH oxidase acts as the primary O2 sensor in carotid body type I cells. These results are in accord with the effect of oxidase disruption in PASMCs (3) and erythropoietin-producing cells, but they conflict with data from studies of NEB cells in lung airways (10). Thus O2 sensing may occur via diverse mechanisms that are specific to particular tissues and cells.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants NS-12636, NS-07938, and HL-50153.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Fidone, Dept. of Physiology, Univ. or Utah School of Medicine, 410 Chipeta Way, Salt Lake City, UT 84108-1297 (E-mail: S.J.Fidone{at}m.cc.utah.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 31 May 2001; accepted in final form 23 August 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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