Departments of 1 Physiology and 2 Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah 84108
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 M. 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.
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).
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RESULTS |
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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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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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[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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants NS-12636, NS-07938, and HL-50153.
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FOOTNOTES |
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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.
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