Effect of p47phox gene deletion on ROS production and ... - CiteSeerX

Am J Physiol Lung Cell Mol Physiol 289: L916 –L924, 2005; doi:10.1152/ajplung.00015.2005.

Effect of p47phox gene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells L. He,1 B. Dinger,1 K. Sanders,2 J. Hoidal,2 A. Obeso,3 L. Stensaas,1 S. Fidone,1 and C. Gonzalez3 1

Department of Physiology and 2Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah; and 3Departamento de Bioquimica y Biologia Molecular y Fisiologia/Instituto de Biologıá y Gene´tica Molecular Facultad de Medicina, Universidad de Valladolid y Consejo Superior de Investigaciones Cientı´ficas, Valladolid, Spain Submitted 11 January 2005; accepted in final form 12 July 2005

He, L., B. Dinger, K. Sanders, J. Hoidal, A. Obeso, L. Stensaas, S. Fidone, and C. Gonzalez. Effect of p47phox gene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells. Am J Physiol Lung Cell Mol Physiol 289: L916 –L924, 2005; doi:10.1152/ajplung.00015.2005.—Membrane potential in oxygensensitive type I cells in carotid body is controlled by diverse sets of voltage-dependent and -independent K⫹ channels. Coupling of PO2 to the open-closed state of channels may involve production of reactive oxygen species (ROS) by NADPH oxidase. One hypothesis suggests that ROS are produced in proportion to the prevailing PO2 and a subset of K⫹ channels closes as ROS levels decrease. We evaluated ROS levels in normal and p47phox gene-deleted [NADPH oxidase knockout (KO)] type I cells using the ROS-sensitive dye dihydroethidium (DHE). In normal cells, hypoxia elicited an increase in ROS, which was blocked by the specific NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 3 mM). KO type I cells did not respond to hypoxia, but the mitochondrial uncoupler azide (5 ␮M) elicited increased fluorescence in both normal and KO cells. Hypoxia had no effect on ROS production in sensory and sympathetic neurons. Methodological control experiments showed that stimulation of neutrophils with a cocktail containing the chemotactic peptide N-formylMet-Leu-Phe (1 ␮M), arachidonic acid (10 ␮M), and cytochalasin B (5 ␮g/ml) elicited a rapid increase in DHE fluorescence. This response was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (10 ␮M). KO neutrophils did not respond; however, azide (5 ␮M) elicited a rapid increase in fluorescence. Physiological studies in type I cells demonstrated that hypoxia evoked an enhanced depression of K⫹ current and increased intracellular Ca2⫹ levels in KO vs. normal cells. Moreover, AEBSF potentiated hypoxia-induced increases in intracellular Ca2⫹ and enhanced the depression of K⫹ current in low O2. Our findings suggest that local compartmental increases in oxidase activity and ROS production inhibit the activity of type I cells by facilitating K⫹ channel activity in hypoxia.

EXPOSURE OF CAROTID BODY ARTERIAL chemoreceptors to lowered PO2 elicits increased neural activity in the carotid sinus nerve (CSN) and reflex cardiopulmonary adjustments that mitigate the adverse effects of hypoxemia. Increased carotid body activity occurs at relatively moderate PO2 levels, in contrast to the severe hypoxia required to elicit metabolic and functional adjustments in non-O2-sensing tissues (19, 20, 23). Efforts to understand the molecular physiology of hypoxic chemotransduction in the carotid body have focused primarily on the relationship between PO2 and the activity of K⫹ channels in neuroectoderm-derived type I cells (23, 40). In the rat, multiple

studies have demonstrated that hypoxia inhibits two types of K⫹ channels: 1) a large-conductance, voltage- and calciumdependent channel [maxiK (39, 50)] and 2) a voltage-independent TASK-type channel that mediates a background leak current (5, 7). Available data indicate that TASK-1 channels play a major role in setting the membrane potential (Em) and that closure of these channels initiates cell depolarization (6, 17). In rat type I cells, the current-voltage characteristics of maxiK channels suggest that they do not open until Em is more positive than ⫺20 mV. Multiple studies have reported that specific maxiK blockers fail to activate isolated type I cells, which has supported the notion that these channels modulate cell activity and facilitate repolarization of the membrane (6, 17, 42). However, in thin slices of rat carotid body, Pardal and Lo´pez-Barneo (37) demonstrated that the specific maxiK blocker iberiotoxin evokes catecholamine release from type I cells. There is general agreement that maxiK and TASK-1 channels (also expressed in non-O2-sensing tissues, brain, and elsewhere) are not inherently sensitive to molecular oxygen and must somehow be coupled to cellular components that are modulated by the prevailing PO2 (38). Previous studies have delineated differing points of view regarding O2 sensing such that either an O2-binding heme protein in the plasma membrane (33, 49) or specialized O2-sensitive cytoplasmic components (1, 48) modulate the activity of K⫹ channels. Importantly, a recent review by Prabhakar (43) pointed out that these different mechanisms are not necessarily mutually exclusive and that chemoreceptor cells may integrate signals from multiple O2 sensors, which collectively determine K⫹ channel activity. One hypothesis suggests that a phagocytic-like NADPH oxidase couples PO2 to K⫹ channel activity by generating superoxide anion (O⫺ 2 ) in proportion to available O2. H2O2 derived from O⫺ 2 by the rapid action of superoxide dismutase (SOD) would facilitate K⫹ channel activity (1). Indeed, application of H2O2 to the in vitro rat carotid body/CSN preparation has been shown to depress chemoreceptor activity (2), and an inhibitor of the oxidase, diphenyleneiodonium (DPI), alters nerve activity evoked by hypoxia (14). Furthermore, certain subunits common to the phagocytic and nonphagocytic forms of the enzyme, including p22phox, gp91phox, p47phox, and p67phox (phox: phagocytic oxidase), have been localized to type I cells by the use of immunocytochemical staining techniques (32). Support for the involvement of NADPH oxidase

Address for reprint requests and other correspondence: B. Dinger, Dept. of Physiology, Univ. of Utah School of Medicine, 410 Chipeta Way, Salt Lake City, 84108 Utah (e-mail: [email protected]).

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reactive oxygen species; potassium channels; cellular redox; dihydroethidium; cell calcium

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HYPOXIA AND ROS IN CHEMORECEPTOR CELLS

also has come from studies of p47phox gene-deleted mice, which display increased CSN activity and ventilatory reflexes evoked by hypoxia (45). Opposing these findings are multiple observations that question the validity of the NADPH oxidase hypothesis of chemotransduction. First, recent studies in the rat have shown that the heme-binding molecule carbon monoxide (CO) activates K⫹ channels and blocks the inhibitory effects of hypoxia, suggesting that a heme protein mediates the effects of hypoxia on channel activity (33). Second, although early studies demonstrated that maxiK and TASK-1 channels fail to respond to hypoxia after excision of membrane patches from type I cells (50), more recent studies have obtained opposite results indicating that cytosolic factors (i.e., subunits of NADPH oxidase) are not required for low-O2 inhibition of the voltage-dependent K⫹ current (IK) (44). In addition, studies using genetically modified mice lacking the gp91phox subunit demonstrated normal inhibition of IK by hypoxia in type I cells, as well as normal hypoxia-evoked Ca2⫹ responses and CSN activity (25). Finally, a recent study indicates that O2 sensitivity in maxiK channels is mediated by CO produced by hemeoxygenase-2 (49). An important postulate of the NADPH oxidase hypothesis is that hypoxia depresses ROS levels in O2-sensitive cells (1, 2). ROS production has indeed been shown to decrease in response to hypoxia in a cell line derived from airway chemoreceptors [neuroepithelial body (NEB) cells], in which NADPH oxidase appears to be a central component of the O2-sensing machinery (34). However, the effect of hypoxia on ROS has not been firmly established in type I cells, and the effects of recently available specific oxidase inhibitors have not been tested on K⫹ channel function. In the present experiments, we have used p47phox gene-deleted knockout (KO) mice and normal mice to reexamine the NADPH oxidase hypothesis. Our experiments focused first on establishing proper experimental conditions for the measurement of ROS in phagocytic neutrophils (polymorphonuclear neutrophils, PMN) from normal vs. KO mice. We then used identical methods to examine the effects of hypoxia on NADPH oxidase activity in type I cells. Finally, we have demonstrated the effects of a highly specific inhibitor of NADPH oxidase on hypoxia-evoked depression of the voltage-dependent maxiK current and hypoxia-evoked Ca2⫹ responses in mouse type I cells. Our data suggest that NADPH oxidase is an important component of the transduction machinery in type I cells. However, the findings are not in accord with the notion that decreased oxidase activity initiates cell depolarization.

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Preparation of PMN PMN were prepared from blood obtained by intracardiac puncture in anesthetized mice (10 mg/kg ketamine ⫹ 0.9 mg/kg xylazine, im). Samples enriched in PMN were prepared using a Ficoll-Hypaque technique for human blood (3), modified to accommodate small blood volumes (0.7–1 ml). Giemsa staining and differential counting showed that 60 – 80% of the isolated cells were PMN. Cells were plated on poly-L-lysine-coated coverslips in Hanks’ balanced salt solution and maintained in a CO2 incubator (36.5°C, PO2 120 mmHg) for 60 –120 min before loading with dihydroethidium (DHE; 30 min). For fluorescence measurements, PMN were superfused with an airequilibrated Tyrode solution containing (in mM) 112 NaCl, 4.7 KCl, 2.2 CaCl2, 1.1 MgCl2, 42 Na-glutamate, 5.6 glucose, and 5 HEPES buffer (pH 7.40). Dissociation and Culture of Carotid Body Type I Cells and Ganglionic Neurons Carotid bodies were removed from anesthetized young adult mice (20 –25 g), cleaned of connective tissue, and cut into pieces, which were placed on a coverslip and incubated in 100 ␮l of Ham’s F-12 medium (Ca2⫹ and Mg2⫹ free) containing collagenase and trypsin (0.2%; 30 min) in a CO2 incubator. Peripheral nerve ganglia were similarly minced and incubated (petrosal ganglion, 20 min; nodose ganglion, 15 min, and superior cervical ganglion, 40 min). Tissue fragments were rinsed in F-12 medium (Ca2⫹ and Mg2⫹ free) and transferred to poly-L-lysine-coated glass coverslips, where they were triturated with a polished Pasteur pipette in 100 ␮l of F-12 medium containing 10% fetal calf serum and 5 ␮g/ml insulin. Coverslips containing isolated cells were maintained in a CO2 incubator for later recording (2– 6 h). Perforated Whole Cell Patch-Clamp Recordings Detailed methods have been published previously (26). In brief, coverslips containing carotid body type I cells were positioned in a 0.3-ml flow chamber mounted on the stage of an inverted microscope and perfused (0.5 ml/min) at 35–36.5°C. Bath solution contained (in mM) 141 NaCl, 4.7 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH 7.40) and was routinely air-equilibrated (PO2 120 mmHg). Pipette solution (in mM: 145 K-glutamate, 15 KCl, 2 MgCl2, and 20 HEPES, pH 7.2 and 37°C) also contained nystatin (150 –200 ␮g/ml); pipette resistances varied from 2 to 10 M⍀. Hypoxic solutions have a PO2 of 30 –32 mmHg, similar to that in carotid body tissue during moderate hypoxia (8). IK were evoked by step voltage changes from a holding potential of ⫺70 mV, recorded using an Axopatch 200A patch-clamp amplifier and a CV 201A head stage (Axon Instruments), displayed on an oscilloscope, and digitized with a DigiData 1200 computer interface for analysis using pCLAMP 5.0 software (Axon Instruments). The series resistance (typically 40 M⍀) was not compensated. Junction potentials (2– 4 mV) were canceled at the onset of the experiment. Capacitance and leakage currents were subtracted. Measurement of Intracellular Ca2⫹ Concentration and ROS Production

METHODS

Animals The phagocytic isoform of NADPH oxidase is composed of the catalytic subunit gp91phox and the regulatory subunits p22phox, p47phox, p40phox, p67phox, and the small GTPase Rac. p47phox genedeleted mice (p47phoxKO), originally produced in the laboratory of S. M. Holland (29), were phenotyped for NADPH oxidase before being used in experiments. Control mice were obtained from “backcrossed” heterozygous littermates to ensure that normal vs. NADPH oxidase-deficient animals share a similar genetic background. All protocols were approved by the University of Utah Institutional Animal Care and Use Committee. AJP-Lung Cell Mol Physiol • VOL

Intracellular Ca2⫹. Detailed methods have been published previously (26). Coverslip-attached cells were loaded with 0.5 ␮M fura-2 AM (20 –25 min) in the CO2 incubator. The recording chamber (Tyrode solution, perfusion rate 1.0 –1.2 ml/min, 32–34°C) was mounted into a Zeiss/Attofluor workstation equipped with an excitation wavelength selector and an intensified charge-coupled device camera system. Fura-2 emission (520 nm) was obtained by alternating excitation at 334 and 380 nm, and the 334/380 ratios were used to calculate intracellular Ca2⫹ concentration ([Ca2⫹]i) (26). Fluorescence signals were obtained from isolated and clustered type I cells, with no differences in basal or stimulus-evoked responses (see also

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Ref. 30). Cells selected for analysis displayed morphology typical of type I cells (42) and responded to hypoxia (bath PO2 24 –26 mmHg) with at least a doubling of the basal [Ca2⫹]i. ROS production. Chemoreceptor type I cells attached to coverslips were loaded with 5 ␮M DHE in the CO2 incubator (30 min). Coverslips were superfused in the chamber used for [Ca2⫹]i measurements, and the DHE fluorescence (excitation at 535 nm) was recorded at 645 nm (sampling rate 100 ms/4 s). Data were collected and analyzed using Attofluor Ratiovision software. As in the case of [Ca2⫹]i, recordings from isolated and clustered cells gave identical signals. Much of the fluorescent oxidized form of DHE is cell trapped; fluorescence gradually increases due to basal ROS production, and any stimulus that elevates ROS production increases the signal slope (47). RESULTS

Effect of Cell Activation on DHE Fluorescence in Mouse Neutrophils PMN are suitable biological controls because their physiological function is to rapidly produce ROS generated via NADPH-oxidase (the “respiratory burst”) as part of a “killing” mechanism to destroy invading pathogens. Therefore, normal PMN will generate an unequivocal positive fluorescent signal, whereas PMN from p47phoxKO mice should lack ROS production. Preliminary experiments established optimal conditions for measuring ROS in isolated cells attached to poly-L-lysinecoated coverslips. Background fluorescence was assessed in cell-free areas of the coverslip. Basal fluorescence for each cell was recorded for 10 –12 min, followed by the introduction of a stimulus cocktail consisting of the chemotactic peptide Nformyl-Met-Leu-Phe (fMLP; 1 ␮M), arachidonic acid (AA; 10 ␮M), and cytochalasin B (CB; 5 ␮g/ml). Figure 1A shows that in normal cells, fMLP/AA/CB elicited a marked, rapid increase in the slope of the fluorescence signal. Within 30 – 60 s after application of the stimulus, the slope receded but the signal continued to increase gradually. The introduction of DPI, a commonly used inhibitor of NADPH oxidase, substantially decreased the signal, consistent with decreased ROS production. After DPI washout, the fluorescence continued to increase gradually. Finally, the introduction of azide (5 ␮M; an agent known to increase ROS via inhibition of cytochrome oxidase and catalase) elicited a second rapid increase in the signal, which declined after washout. The lower trace in Fig. 1A shows results from a p47phoxKO PMN. This cell failed to respond to fMLP/AA/CB, and DPI elicited only a slight decrease in the signal, findings consistent with the absence of functioning NADPH oxidase. However, 5 ␮M azide markedly increased the fluorescence, demonstrating the viability of the cell. Summarized in Fig. 1B are data from 98 normal and 185 p47phoxKO PMN, in which slopes of fluorescence signals were analyzed for each experimental condition over a 100-s interval. The data demonstrate the deleterious effect of gene deletion on the response to the stimulus cocktail and the blocking effect of DPI. However, the effects of azide on ROS production were similar in normal and NADPH oxidase-deficient PMN. Effect of Hypoxia and Azide on ROS in Normal and p47phox Gene-Deleted Type I Cells Assessments of ROS production in mouse type I cells are shown in Fig. 2. In normal cells, basal fluorescence was flat or AJP-Lung Cell Mol Physiol • VOL

Fig. 1. A: a cocktail of neutrophil-stimulating agents [N-formyl-Met-Leu-Phe (fMLP peptide; 1 ␮M), arachidonic acid (AA; 10 ␮M), and cytochalasin B (CytB; 5 ␮g/ml)] produced a sharp increase in dihydroethidium (DHE) fluorescence in a single attached polymorphonuclear neutrophil (PMN) from a normal mouse and no response in a p47phox knockout (KO) PMN. Diphenyleneiodonium (DPI; 10 ␮M) decreased the fluorescence signal in the normal mouse and had no effect on the p47phoxKO PMN. Azide (5 ␮M) caused a similar increase in fluorescence in the normal and p47phoxKO PMN. B and C: summaries of DHE fluorescence for 98 normal (B) and 185 p47phoxKO PMN (C). Data are expressed as slopes of fluorescence intensity change (⌬I/10 s) during the initial 100 s of the experimental conditions designated in A. ***P ⬍ 0.001 vs. drug-free conditions.

increased only slightly in media equilibrated with air (PO2 ⬃120 Torr), not unlike the behavior of PMN (Fig. 2A). The introduction of hypoxic media equilibrated at PO2 ⬃24 –26 Torr elicited an increase in fluorescence. However, unlike the activated PMN, which produced a sharp and rapid elevation in ROS, hypoxia evoked a noticeably slower increase in the fluorescence signal, consistent with less robust ROS production. ROS levels increased substantially as hypoxia continued for several minutes. Moreover, superfusion with hypoxic media containing the specific NADPH oxidase inhibitor 4-(2aminoethyl)-benzenesulfonyl fluoride (AEBSF; 3 mM) (16) elicited a marked decrease in the fluorescence emission, and after washout of the drug, the signal again indicated increased ROS production that subsided upon reintroduction of normoxic

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Fig. 2. A: effect of hypoxia on DHE fluorescence in carotid body type I cells isolated from normal and p47phoxKO mice. In normoxia, fluorescence gradually increases and is markedly elevated by hypoxia in the normal but not in the p47phoxKO cell. 4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF; 3 mM) depresses hypoxic increase of reactive oxygen species (ROS) production in the normal type I cell. Azide (5 ␮M) increased the fluorescence in the normal and p47phoxKO cells. B and C: summaries of DHE fluorescence for 32 normal (B) and 44 p47phoxKO type I cells (C) harvested from 4 normal and 4 KO animals. Slopes (⌬I/10 s) were quantified as described in Fig. 1. ***P ⬍ 0.001 vs. control, normoxic conditions.

media. Subsequently, exposure to azide (5 ␮M) elicited a large increase in the production of ROS. The lower trace in Fig. 2A demonstrates that hypoxia fails to elevate ROS levels in p47phoxKO type I cells. However, the presence of azide evoked a marked increase in the fluorescence signal, consistent with increased ROS production in mitochondria. Summary data in Fig. 2B show that the moderate hypoxia used in these experiments elicited significant increases in ROS in normal type I cells but failed to alter ROS production in p47phoxKO type I cells. However, azide evoked similar increases in ROS in both normal and oxidase-deficient cells. In hypoxia, AEBSF evoked a significant decrease in fluorescence signal in normal but not p47phoxKO type I cells. Using immunocytochemical techniques, Dvorakova et al. (18) reported the presence of NADPH oxidase subunits in sensory neurons. The function of enzyme components in these cells is unknown, but their presence suggests that oxidase function may not be specific to type I cells. To test this possibility, we examined the effects of hypoxia on ROS production in cultured sensory neurons from petrosal ganglion (which innervates the carotid body) and nodose ganglion, as AJP-Lung Cell Mol Physiol • VOL

well as from postganglionic sympathetic neurons in the superior cervical ganglion. Figure 3A shows that hypoxia had no effect on ROS production in these cells but that azide uniformly evoked a large increase in the fluorescence signal. These findings confirm that hypoxia-evoked activation of NADPH oxidase is a specialized function of type I cells. Previous reports have documented the artifactual production of ROS via the interaction of short-wavelength light with assembled NADPH oxidase (28). Our standard protocol utilized 100-ms excitatory light pulses separated by 4-s dark intervals. In separate control experiments, we compared the rate (slope of fluorescence signal) of ROS production during hypoxia in 17 mouse type I cells exposed to 100-ms pulses in three experimental trials in which the dark intervals were 4, 8, and 16 s, respectively (Fig. 3B). The data show that the slopes of the responses in hypoxia were not decreased by longer intervals between light pulses, suggesting that increased ROS production is not an artifact of exposure to light. These findings are not unexpected, given that light-induced ROS production in mammalian cells is due to exposure to intense violet-blue light below 450 nm (28), whereas in our experiments, DHE

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this drug evoked a small inhibition of the K⫹ current, but it substantially enhanced current depression during hypoxia. In p47phoxKO type I cells (Fig. 4G), AEBSF had no effect on the hypoxic depression of K⫹ current, consistent with the absence of enzyme activity. Figure 4H shows that in normal mouse type I cells, the introduction of 100 ␮M H2O2 tended to reverse the depressive effect of hypoxia on the voltage-dependent outward current, confirming channel sensitivity to the redox state of cytosolic constituents. Dampening of the hypoxia-evoked current depression by H2O2 was similar in both normal and p47phox gene-deleted cells (data not shown). Hypoxia-Evoked Ca2⫹ Responses in Type I Cells and Effect of p47phox Gene Deletion

Fig. 3. A: sample records showing the lack of effect of hypoxia on DHE fluorescence in petrosal (PG), nodose (NG), and superior cervical ganglion (SCG) neurons. B: effect of sampling rate on slope of hypoxia-evoked DHE fluorescence. Left, records of fluorescence in a type I cell at 3 sampling rates with dark intervals of 4, 8, and 16 s. Right, summary data of fluorescence slope (⌬I/min) from 17 cells.

is excited by light filtered between 500 and 560 nm (peak ⬃535 nm). Voltage-Dependent K⫹ Currents in Mouse Type I Cells Under normoxic conditions (PO2 ⬃120 Torr), depolarizing steps from a holding potential of ⫺70 mV evoked outward currents at test potentials above ⫺30 mV in nearly all cultured cells of 8 –10 ␮m in diameter from normal and p47phoxKO preparations (Fig. 4, A and B). The amplitude of the current did not noticeably inactivate during 200-ms pulses. In addition, the current-voltage relationship increased steadily between 0 and ⫹40 mV (Fig. 4, C and D). Thus, in the nystatin patch configuration, these voltage-dependent currents did not display the inflexion of Ca2⫹-dependent K⫹ currents typically recorded in the whole cell configuration (39). However, the highly selective maxiK channel blocker iberiotoxin (10 nM) (22) inhibited ⬃70% of the hypoxia-sensitive current in these cells (data not shown), consistent with the presence of a set of maxiK or BK channels previously reported in rat type I cells (39). The introduction of hypoxic media (PO2 ⬃24 –26 Torr) depressed outward current by ⬃50% in normal cells at test potentials of 0 and ⫹40 mV. This effect of hypoxia was significantly enhanced in p47phoxKO cells, where the reduction of the current was some 15% greater (Fig. 4E). Involvement of NADPH oxidase in K⫹ channel function was tested via the application of the specific inhibitor AEBSF (3 mM). Figure 4F demonstrates that in normoxic conditions, AJP-Lung Cell Mol Physiol • VOL

Figure 5A presents summary data from assessment of [Ca2⫹]i in 87 normal and 323 p47phoxKO type I cells. In cells from normal mice, basal [Ca2⫹]i, recorded in solution equilibrated with air (PO2 ⬃120 Torr), ranged from 1 to 41 nM, with a mean of 5.0 ⫾ 0.7698 nM (⫾SE). The basal [Ca2⫹]i in cells from gene-deleted animals displayed a wider range (1.0 –180 nM), and the mean (22.11 ⫾ 1.53 nM) was some fourfold higher than normal (P ⬍ 0.0001). Peak increases in [Ca2⫹]i evoked by PO2 ⬃24 –26 Torr in cells from normal animals varied from 5 to 718 nM, with a mean of 124.43 ⫾ 15.77 nM. In cells from oxidase-deficient animals, peak responses to hypoxia ranged from 16 to 1,145 nM, and the mean (276.49 ⫾ 12.12 nM) response was more than twice the value found in normal cells (P ⬍ 0.0001). The variability in the Ca2⫹ responses presently reported in normal and gene-deleted cells is in accord with observations made in normal rat type I cells (4). In 34 normal and 39 p47phoxKO type I cells, we tested the effect of AEBSF on the hypoxic [Ca2⫹]i. In normal cells, AEBSF more than doubled (reversibly) the hypoxic response, raising [Ca2⫹]i levels to those seen in p47phoxKO type I cells (Fig. 5B). However, AEBSF had no effect on hypoxia-evoked Ca2⫹ responses in p47phoxKO type I cells (Fig. 5C). In normal type I cells, increasing concentrations of H2O2 (1, 10, and 100 ␮M) significantly depressed hypoxia-evoked Ca2⫹-responses in a dose-related manner, further indicating an influence of cellular redox potential on cell excitability (Fig. 5D). DISCUSSION

A role for ROS in O2 sensing has been proposed in multiple cells and tissues including cardiomyocytes, vascular smooth muscle, erythropoietin-producing cells in kidney and liver, and the morphologically similar cells that comprise chemoreceptors in NEB and carotid body (1, 9, 48, 51). Various methods have been applied to assess the relationship between ROS production and PO2, resulting in diverse sets of data and conflicting views. Numerous studies have used the fluorescent dye dichlorofluorescein (DCF) as a specific indicator of H2O2. However, recent studies indicate that DCF is the substrate for multiple oxidative enzymes and that fluorescence signals may be artifactual (47). In the current study, we established a DHE-based method, combined with assessments of normal and gene-deleted PMN as positive and negative controls, respectively, for increased NADPH oxidase-dependent ROS production. Our data using PMN attached to coverslips are consistent with the known function of the oxidase in these highly specialized cells, and consequently they can be viewed as addi-

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Fig. 4. A and C: sample records (A) and current-voltage curves (C) obtained in a normal type I cell in normoxia, hypoxia, and again in normoxia (recovery). B and D: sample records (B) and current-voltage curves (D) obtained in a cell from a p47phoxKO mouse. E: mean hypoxic depression of the normoxic voltage-dependent K⫹ current (IK) at membrane potentials (Em) of 0 and ⫹40 mV obtained in 31 normal and 32 p47phoxKO cells. *P ⬍ 0.05; ***P ⬍ 0.001 vs. normal cells. F and G: effects of AEBSF (3 mM) in normal (n ⫽ 10; F) and p47phoxKO type I cells (n ⫽ 12; G). ***P ⬍ 0.001 vs. AEBSF. ⫹⫹⫹P ⬍ 0.001 vs. hypoxia. H: H2O2 (100 ␮M) decreased hypoxic inhibition of IK in normal cells. Values are means ⫾ SE; n ⫽ 12 cells. ***P ⬍ 0.001 vs. normoxia (100%). ⫹⫹⫹P ⬍ 0.001 vs. hypoxia.

tional confirmation of appropriate conditions for the detection of biologically important ROS in isolated cells. Given that the oxidation products of DHE bind to DNA, it was somewhat surprising that inhibitors of NADPH oxidase decreased the fluorescence signal in normal PMN and type I cells during stimulation. A previous study of ROS production evoked by ischemic preconditioning in heart muscle reported similar results with DHE (31). These results suggest that some products are not irreversibly oxidized or that leakage of precursor and/or products is a continuous process. In any case, our data suggest that DHE can detect decreasing as well as increasing ROS levels in viable cells. The prevailing hypothesis of NADPH oxidase involvement in type I cell O2 sensing proposes that ROS, including H2O2, are produced in direct proportion to local PO2 (1, 2). In opposition to this view, our data clearly indicate that ROS production in normal mouse type I cells is increased by moderate hypoxia. Moreover, the effects of the highly specific inhibitor AEBSF support the notion that hypoxia-dependent ROS production is a consequence of increased NADPH oxidase activity. This conclusion is substantiated by data demonstrating that hypoxia does not alter ROS levels in p47phox-genedeleted type I cells. The absence of hypoxia-evoked ROS production in type I cells lacking NADPH oxidase activity strongly suggests that mitochondria are not a source of ROS in AJP-Lung Cell Mol Physiol • VOL

moderate hypoxia. However, our data do not rule out a role for mitochondria in ROS production in severe hypoxia, where electron transport may be compromised (11, 12). Likewise, given that O2 is a substrate for NADPH oxidase, it is conceivable that production of ROS may be depressed in more severe conditions (2). Our findings call for an explanation of increased oxidase activity in an environment where an essential substrate, namely, oxygen, is decreasing. Because the reported mean Km for O2 of phagocytic NADPH-oxidase is 17 ␮M (PO2 12.7 Torr) (15), the hypoxia used in our experiments should not markedly limit enzyme activity. In this regard, it is important to note that NADPH is an equally important substrate for oxidase activation. This enzyme cofactor, which is widely utilized in the enzymatic transfer of hydrogen, is synthetically formed in the hexose monophosphate shunt (HMS) pathway of glucose metabolism. Previous studies in our laboratory have shown that glucose uptake in type I cells is elevated by hypoxia and that this response is absent in the presence of the Na⫹/ K⫹-ATPase inhibitor ouabain (36). Thus it appears that glucose metabolism is stimulated in type I cells by membrane depolarization during hypoxia. Given these facts, it is conceivable that hypoxia stimulates ROS production consequent to an elevation in the cellular pool of NADPH. Indeed, in preliminary experiments we found that HMS inhibitors depress

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Fig. 5. A: normoxic (basal) and hypoxic intracellular Ca2⫹ concentration ([Ca2⫹]i) measured with fura-2 in type I cells from normal and p47phoxKO mice. Both normoxic and peak hypoxic [Ca2⫹]i levels were significantly higher in the KO cells (n ⫽ 87 normal and 323 KO cells). ***P ⬍ 0.001 vs. normal cells. B: in normal cells, AEBSF (3 mM) more than doubled (in a reversible manner) the peak [Ca2⫹]i response elicited by hypoxia, bringing it to levels nearly identical to those seen in KO cells. Values are means ⫾ SE; n ⫽ 34 cells. ***P ⬍ 0.001 vs. hypoxia. C: AEBSF did not affect the hypoxic [Ca2⫹]i responses of p47phoxKO cells. Values are means ⫾ SE; n ⫽ 39 cells. D: increasing concentrations of H2O2 caused a progressively greater inhibition of [Ca2⫹]i responses evoked by hypoxia in type I cells. Values are means ⫾ SE; n ⫽ 31 cells. ***P ⬍ 0.001 vs. control hypoxic response.

hypoxia-evoked ROS production in type I cells. It is noteworthy that type I cells likely contain an isoform of gp91phox, because deletion of this gene does not alter carotid body function (25). Importantly, an isoform of the enzyme may have regulatory properties that differ from the well-known phagocytic prototype, including altered affinities for O2 and/or NADPH. Our studies using the perforated patch-clamp technique showed that mouse type I cells contain an iberiotoxin-sensitive K⫹ current that is similar, if not identical, to maxiK currents previously described in rat type I cells (39). The mouse data indicate that depression of this current by hypoxia is opposed by exposure to H2O2. Moreover, AEBSF or deletion of p47phox

enhances the effects of hypoxia on the depression of the voltage-dependent K⫹ current, as well as elevation of [Ca2⫹]i. Combined with the finding that hypoxia increases ROS, these data strongly suggest that hypoxia-triggered NADPH oxidase activity facilitates the open state of the voltage-dependent K⫹ channels. The current-voltage relationship of these channels shows that they open at voltages above ⫺30 mV, suggesting that in a resting (i.e., normoxic) cell, they contribute minimally to the EM. It thus seems likely that these channels facilitate cell repolarization and that although low PO2 per se promotes channel closure, the consequent increase in ROS tends to enhance channel activity. Riesco-Fagundo et al. (44) recently demonstrated that maxiK channels in rat type I cells are closed

Fig. 6. Redox modulation of O2 sensing in carotid body type I cells. See text for detailed explanation. Hem Ox2, hemeoxygenase-2; CO, carbon monoxide; HMP, hexose monophosphate shunt pathway; Po, channel open probability.

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by hypoxia in isolated membrane patches and that CO can substitute for O2, suggesting that hypoxic depression of the current occurs via a heme-binding protein and is independent of cytosolic factors such as ROS. These findings are in accord with the involvement of CO production by hemeoxygenase-2 in the regulation of maxiK channels (49). Previous studies have indicated that hypoxia depresses ROS production in a cell line (H-146) of small-cell carcinoma, which is thought to be derived from NEB airway chemoreceptors (34). Like type I cells, NEB and H-146 cells express O2-sensitive K⫹ channels. However, in genetically modified mice deficient in gp91phox, hypoxia fails to inhibit the K⫹ current in the airway chemoreceptor cells (21). Likewise, in normal NEB cells, DPI occludes channel sensitivity to low O2, further supporting the hypothesis that NADPH oxidase is an important component of the O2-sensing machinery that facilitates cell depolarization during hypoxia (21). In contrast, the current study of type I cells demonstrated enhanced sensitivity to hypoxia following oxidase inhibition or gene deletion of a key NADPH oxidase subunit. In a recent study of carotid sinus nerve activity, our laboratory group also demonstrated enhanced basal and hypoxia-evoked responses in p47phox KO preparations (45). Our data strongly support the hypothesis that the production of ROS modulates the excitatory effects of hypoxia in type I cells. Thus isoforms of the oxidase appear to perform fundamentally different roles in airway vs. arterial chemoreceptor cells. Recent studies have shown that a nonspecific inhibitor of NADPH oxidase, DPI, promotes the dose-dependent release of radiolabeled catecholamines from rat and rabbit carotid bodies in normoxia and enhancement of catecholamine release evoked by hypoxia (35). Although the DPI-evoked release is only partially Ca2⫹ dependent, these findings are in general agreement with the hypothesis that ROS produced by NADPH oxidase subserve an inhibitory role with respect to type I cell activity. However, in the same study, other inhibitors of the oxidase did not stimulate release of catecholamine. These latter observations may indicate the presence of a novel isoform of the oxidase that is not sensitive to conventional inhibitors of the phagocytic enzyme (13). Our data imply that type I cell activity evoked by hypoxia is in part determined by a change in the redox state of cytosolic constituents. Recently, one of our group (C. Gonzalez) coauthored a study showing that hypoxia does not alter redox conditions in rabbit and calf carotid body tissue (46). In addition, artificially elevating the ratio of reduced-to-oxidized glutathione with N-acetylcysteine (NAC) neither excited nor inhibited the release of catecholamine from type I cells. One possible explanation of these seemingly conflicting data is that increased ROS production due to NADPH oxidase activity is balanced by increased NADPH synthesis in the HMS pathway after hypoxia-induced glucose uptake. Thus hypoxia may elicit an overall increase in reducing equivalents, which maintains a redox balance in the face of increased ROS production. Moreover, hypoxia-induced ROS generation could occur locally and contribute minimally to the overall redox status. A highly compartmentalized production of ROS adjacent to target molecules may, in addition, be relatively protected from immediate scavenging, thereby obscuring effects of external manipulations. Nonetheless, oral administration of NAC enhances the hypoxic ventilatory response in humans, suggesting an AJP-Lung Cell Mol Physiol • VOL

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important role for cellular redox status within at least one of the multiple components comprising the chemoreflex pathway (27). Figure 6 presents a summary of current concepts of O2 chemoreception in carotid body chemoreceptor type I cells, indicating potential targets for ROS. The diagram schematizes the main transduction cascade that connects decreased PO2 with the activation of type I cells and the release of neurotransmitters (23). This scheme postulates a membrane-delimited O2 sensor (44) capable of transmitting the decrease in PO2 via regulatory ␤-subunits to K⫹ channels, decreasing their opening probability (Po) (41), and thereby promoting cell depolarization, activation of Na⫹ and Ca2⫹ channels, and Ca2⫹-dependent exocytotic release of neurotransmitters. Low PO2 activates an adenylate cyclase and increases cAMP levels (10); cAMP acts like hypoxia, decreasing K⫹ currents in type I cells. Hypoxia also decreases the activity of hemeoxygenase-2, leading to a decrease in CO production and decreased Po of O2-sensitive maxiK channels in rat type I cells (49). Hypoxia produces an increase in the glucose consumption in type I cells that is secondary to Na⫹ and Ca2⫹ entry, as well as activation of the plasma membrane ATPases (36). Unpublished data from our laboratory indicate that a part of this glucose enters the hexose monophosphate pathway, leading to an increase of NADPH levels, which facilitates activation of NADPH oxidase. Alternatively, this enzyme might be directly activated by hypoxia or indirectly via direct interaction with the membranebound O2 sensor; in addition, a hypoxia-driven increase in intracellular Ca2⫹ may activate NADPH oxidase (24). Superoxide anion generated by NADPH oxidase, and/or the resulting H2O2, acts on K⫹ channels, either directly or via regulatory ␤-subunits, opposing the effects of hypoxia. Thus ROS negatively modulate the hypoxic transduction cascade and control type I cell excitability. ACKNOWLEDGMENTS We thank Dr. Caroline Owen for expert instruction in the preparation of PMNL. GRANTS This work was supported by United States Public Health Service Grants NS-12636, NS-07938, and NL-50153 and Spanish Direccioń General de Investigacioń Cientı´fica y Tećnica Grant BFU2004-06394 and Red Respira Grant RTIC C03/11. REFERENCES 1. Acker H. Mechanisms and meaning of cellular oxygen sensing in the organism. Respir Physiol 95: 1–10, 1994. 2. Acker H, Bolling B, Delpiano MA, Dafau E, Gorlach A, and Holtermann G. The meaning of H2O2 generation in carotid body cells for PO2 chemoreception. J Auton Nerv Syst 41: 41–51, 1992. 3. Boyum A. Separation of white blood cells. Nature 204: 793–794, 1964. 4. Bright GR, Agani FH, Haque U, Overhold JL, and Prabhakar NR. Heterogeneity in cytosolic calcium responses to hypoxia in carotid body cells. Brain Res 706: 297–302, 1996. 5. Buckler KJ. A novel oxygen-sensitive potassium current in rat carotid body type I cells. J Physiol 498: 649 – 666, 1997. 6. Buckler KJ. Background leak K⫹-currents and oxygen sensing in carotid body type 1 cells. Respir Physiol 115: 179 –187, 1999. 7. Buckler KJ, Williams BA, and Honore E. An oxygen-, acid- and anaesthetic-sensitive TASK-like background potassium channel in rat arterial chemoreceptor cells. J Physiol 525: 135–142, 2000. 8. Buerk DG, Nair PK, and Whalen WJ. Evidence for second metabolic pathway for O2 from PtiO2 measurements in denervated cat carotid body. J Appl Physiol 67: 1578 –1584, 1989.

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29. Jackson SH, Gallin JI, and Holland SM. The p47phox mouse knock-out model of chronic granulomatous disease. J Exp Med 182: 751–758, 1995. 30. Jiang RG and Eyzaguirre C. Effects of hypoxia and putative transmitters on [Ca2⫹]i of rat glomus cells. Brain Res 995: 285–296, 2004. 31. Kevin LG, Camara AK, Riess ML, Novalija E, and Stowe DF. Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion. Am J Physiol Heart Circ Physiol 284: H566 –H574, 2003. 32. Kummer W and Acker H. Immunohistochemical demonstration of four subunits of neutrophil NAD(P)H oxidase in type I cells of carotid body. J Appl Physiol 78: 1904 –1909, 1995. 33. Lo´pez-Lo´pez JR and Gonzalez C. Time course of K⫹ current inhibition by low oxygen in chemoreceptor cells of adult rabbit carotid body: effects of carbon monoxide. FEBS Lett 299: 251–254, 1992. 34. O’Kelly I, Lewis A, Peers C, and Kemp PJ. O2 sensing by airway chemoreceptor-derived cells: protein kinase C activation reveals functional evidence for involvement of NADPH oxidase. J Biol Chem 275: 7684 –7692, 2000. 35. Obeso A, Gomez-Nino A, and Gonzalez C. NADPH oxidase inhibition does not interfere with low PO2 transduction in rat and rabbit CB chemoreceptor cells. Am J Physiol Cell Physiol 276: C593–C601, 1999. 36. Obeso A, Gonzalez C, Rigual R, Dinger B, and Fidone S. Effect of low O2 on glucose uptake in rabbit carotid body. J Appl Physiol 74: 2387– 2393, 1993. 37. Pardal R and Lo´pez-Barneo J. Carotid body thin slices: responses of glomus cells to hypoxia and K⫹-channel blockers. Respir Physiol Neurobiol 132: 69 –79, 2002. 38. Patel AJ and Honore´ E. Molecular physiology of oxygen-sensitive potassium channels. Eur Respir J 18: 221–227, 2001. 39. Peers C. Hypoxic suppression of K⫹ currents in type I carotid body cells: selective effect on the Ca2⫹-activated K⫹ current. Neurosci Lett 119: 253–256, 1990. 40. Peers C and Buckler KJ. Transduction of chemostimuli by the type I carotid body cell. J Membr Biol 144: 1–9, 1995. 41. Pe´rez-Garcia MT, Lo´pez-Lo´pez JR, and Gonzalez C. Kv␤1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to Kv4.2 but not to Shaker channels. J Gen Physiol 113: 897–907, 1999. 42. Pe´rez-Garcia MT, Obeso A, Lo´pez-Lo´pez JR, Herreros B, and Gonzalez C. Characterization of cultured chemoreceptor cells dissociated from adult rabbit carotid body. Am J Physiol Cell Physiol 263: C1152–C1159, 1992. 43. Prabhakar NR. Oxygen sensing by the carotid body chemoreceptors. J Appl Physiol 88: 2287–2295, 2000. 44. Riesco-Fagundo AM, Pe´rez-Garcıá MT, Gonzalez C, and Lo´pezLo´pez JR. O2 modulates large-conductance Ca2⫹-dependent K⫹ channels of rat chemoreceptor cells by a membrane-restricted and CO-sensitive mechanism. Circ Res 89: 430 – 436, 2001. 45. Sanders KA, Sundar KM, He L, Dinger B, Fidone S, and Hoidal JR. Role of components of the phagocytic NADPH oxidase in oxygen sensing. J Appl Physiol 93: 1357–1364, 2002. 46. Sanz-Alfayate G, Obeso A, Agapito MT, and Gonzalez C. Reduced to oxidized glutathione ratios and oxygen sensing in calf and rabbit carotid body chemoreceptor cells. J Physiol 537: 209 –220, 2001. 47. Tarpey MM and Fridovich I. Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide and peroxynitrite. Circ Res 89: 224 –236, 2001. 48. Weir EK and Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB J 9: 183–189, 1995. 49. Williams SE, Wootton P, Mason HS, Bould J, Iles DE, Riccardi D, Peers C, and Kemp PJ. Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel. Science 306: 2093–2097, 2004. 50. Wyatt CN and Peers C. Ca2⫹-activated K⫹ channels in isolated type I cells of the neonatal rat carotid body. J Physiol 483: 559 –565, 1995. 51. Youngson C, Nurse C, Yeger H, and Cutz E. Oxygen sensing in airway chemoreceptors. Nature 365: 153–155, 1993.

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