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J Biol Chem, Vol. 275, Issue 11, 7684-7692, March 17, 2000

O₂ Sensing by Airway Chemoreceptor-derived Cells
PROTEIN KINASE C ACTIVATION REVEALS FUNCTIONAL EVIDENCE FOR INVOLVEMENT OF NADPH OXIDASE^*

Ita O'Kelly^§, Anthony Lewis, Chris Peers^§, and Paul J. Kemp^¶

From the  School of Biomedical Sciences and the ^§ Institute for Cardiovascular Research, University of Leeds, Leeds LS2 9JT, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Accumulating evidence suggests that neuroepithelial bodies are airway O₂ sensors. Recently, we have established the H-146 small cell lung carcinoma line as a suitable model to study the biochemical basis of neuroepithelial body cell chemotransduction. Here we explore the possibility that hypoxic modulation of K⁺ channels is intimately linked to activity of NADPH oxidase. Graded hypoxia caused graded inhibition of whole cell K⁺ currents, which correlated well with membrane depolarization. Pretreatment with the phorbol ester, 12-O-tetradecanoyl (TPA), inhibited K⁺ currents at all potentials. Although 4-phorbol 12,13-didecanoate and TPA in the presence of bisindolylmaleimide were also able to depress K⁺ currents, only TPA could significantly ameliorate hypoxic depression of these currents. Thus, protein kinase C (PKC) activation modulates the sensitivity of these cells to changes in pO₂. Furthermore, because the addition of H₂O₂, a downstream product of NADPH oxidase, could only activate K⁺ currents during hypoxia (when endogenous H₂O₂ production is suppressed), it appears likely that PKC modulates the affinity of NADPH oxidase for O₂ potentially via phosphorylation of the p47^phox subunit, which is present in these cells. These data show that PKC is an important regulator of the O₂-transduction pathway and suggests that NADPH oxidase represents a significant component of the airway O₂ sensor.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ventilation-perfusion matching is essential for efficient oxygenation of pulmonary blood in the face of varying demands of the body's tissues. Numerous physiological processes have long been known to ensure optimization of ventilation-perfusion matching, but the cellular mechanisms underlying such processes are only now beginning to be understood. One such important mechanism is that of O₂ chemoreception, i.e. an active cellular response to an acute general and/or regional reduction of pO₂. Most studies to date have focused on the carotid body arterial chemoreceptors. Type I cells of these sensors express O₂-sensitive K⁺ channels, and exposure to hypoxia causes rapid channel inhibition. This leads to cell depolarization, Ca²⁺ entry via voltage-gated Ca²⁺ channels, and subsequent triggering of neurosecretion, an essential step in the initiation of afferent information, which leads to reflex increases of ventilation (1, 2). In vascular smooth muscle, O₂-sensitive K⁺ channels (3) and Ca²⁺ channels (4) have more recently been reported, and their rapid modulation by hypoxia evokes appropriate vasodilatation or vasoconstriction (depending on the location of the blood vessel (5)).

Compelling evidence is emerging that neuroepithelial bodies (NEBs),¹ located in clusters at bronchial bifurcations throughout the lung, serve as airway chemoreceptors (6, 7). Like their vascular counterparts in the carotid body, NEB cells possess numerous transmitters, which appear to be released in hypoxia (8). These transmitters (particularly serotonin) may act to initiate afferent nerve activity to medullary respiratory centers and may also act to control local vasomotor tone, hence directly influencing local ventilation-perfusion matching (9). Compared with type I cells of the carotid body, our understanding of O₂ chemoreception by NEB cells is in its infancy, due largely to the technically demanding nature of their isolation/primary culture or to the difficulty in recording currents in situ using the lung slice. However, it has been established that they are electrically excitable and, most importantly, possess O₂-sensitive K⁺ channels (7, 10), suggesting that they may respond, at least superficially, like carotid body type I cells. In recent studies, we have built on the suggestions of others to establish that small cell lung carcinoma (SCLC) cells, which are believed to be derived from NEB cells (11) and with which they share numerous similarities (12), represent immortal airway chemoreceptors. Thus, we have recently identified, in the SCLC cell line H-146, a specific component of the whole cell K⁺ current, which is sensitive to hypoxia and influences membrane potential (13, 14). This current is 4-aminopyridine- (4-AP) and Ca²⁺-insensitive, quinidine-sensitive, and may be a novel member of the tandem P-domain family of K⁺ channels. The ease of use of cell lines as compared with isolated NEB cells or lung slices suggests that they may be exploited to understand the molecular and biochemical mechanisms underlying rapid effects of hypoxia on chemoreceptor cells.

The mechanism(s) which couple a fall of pO₂ to K⁺ channel inhibition remain elusive, but recent work has implicated the involvement of NADPH oxidase as an O₂ sensor in both NEB cells and SCLC cells (15). The NADPH oxidase model for O₂ chemoreception suggests that, under normoxic conditions, the oxidase tonically generates superoxide from O₂, which is rapidly converted to H₂O₂ by a number of enzymes including superoxide dismutase and catalase. This H₂O₂ is believed to promote channel activity. It follows, therefore, that H₂O₂ levels will decline in hypoxia, causing a concomitant reduction in channel activity. Several independent lines of evidence support this idea: (i) NEB and SCLC cells express the mRNA encoding K⁺ channels known to be modulated by H₂O₂; (ii) in situ hybridization and immunohistochemistry have indicated that specific subunits of NADPH oxidase (gp91^phox and p22^phox) are present in both cell types; (iii) H₂O₂ generation in NEB cell clusters is significantly greater than in surrounding tissue, can be enhanced by protein kinase C (PKC) activation using phorbol ester (which specifically phosphorylates another subunit, p47^phox, of the oxidase complex), and can be suppressed by diphenyleneiodonium, a known NADPH oxidase inhibitor and; iv), K⁺ currents can be enhanced (albeit transiently) by application of H₂O₂ (10, 15). Collectively, these observations support the idea that hypoxic inhibition of K⁺ channels in airway chemoreceptor cells and SCLC cells involves reduced levels of H₂O₂ derived from NADPH oxidase activity, but a direct link between the observations is still lacking. Indeed, it has, to date, never been demonstrated directly that hypoxia causes a fall of H₂O₂ levels in these cells.

In the present study, we demonstrate for the first time that H₂O₂ production declines during hypoxia and have exploited the fact that NADPH oxidase activity can be regulated by PKC-dependent phosphorylation (16) to test further this model for O₂ chemoreception in SCLC cells. Our results provide compelling functional evidence to support a central role for NADPH oxidase in this important process, and we propose that PKC may modulate chemoreception by altering the affinity of the oxidase for O₂.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture

The small cell lung carcinoma cell line, H-146, was purchased from American Tissue Type Cell Collection (Rockville, MD) and was of unknown passage number. Cells were grown in suspension culture in RPMI 1640 medium (containing L-glutamine) supplemented with 10% fetal calf serum, 2% sodium pyruvate, and 2% penicillin/streptomycin (all from Life Technologies, Inc.) in a humidified atmosphere of 5% CO₂/95% air at 37 °C. Medium was changed every 2 days and cells were passaged every 6-7 days by splitting in the ratio 1:5. Cells were used between nominal passage numbers 1 and 6.

Electrophysiology

Solutions and Chemicals-- Unless stated otherwise, all chemicals were of the highest grade available and were purchased from Sigma. Standard pipette solution was K⁺-rich and contained: 10 mM NaCl, 117 mM KCl, 2 mM MgSO₄, 10 mM HEPES, 11 mM EGTA, 1 mM CaCl₂, 2 mM Na₂ATP, pH 7.2, with KOH; free [Ca²⁺] = 27 nM. Standard bath solution was Na⁺-rich and contained: 135 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 5 mM HEPES, 2.5 mM CaCl₂, 10 mM D-glucose, pH 7.4, with NaOH. 4-AP was added to the bath solution where indicated, and osmolarity was maintained by isoosmotic substitution of NaCl. Where indicated, cells were preincubated with 100 nM of the phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and 4-phorbol 12, 13-didecanoate (4-PDD) and in some cases with the PKC inhibitor, bisindolylmaleimide (BIM, 1 µM), for 10 min at room temperature. Where used, 1 µM BIM was also included in the pipette solution.

All tubing was gas-impermeant (Tygon tubing BDH, Atherstone, Berkshire, UK). Normoxic solutions were equilibrated with room air. Solutions were made hypoxic, where appropriate, by bubbling with N_2(g) for at least 30 min prior to perfusion of cells. This procedure produced no shift in pH. Solution flow rate was ~5 ml·min¹. Graded hypoxia was achieved by splicing variable lengths of gas-permeant tubing into the perfusion lines. pO₂ was measured (at the cell) using a polarized (800 mV), calibrated carbon fiber electrode (16); for the experiments reported herein, the pO₂ values were 150 (normoxia), 85, 45, 25, and 15 (hypoxia) mm Hg.

Whole Cell Recording-- Following trituration (10 passes through a 1-ml automatic pipette tip), cells were allowed to adhere at 37 °C for at least 1 h to poly-L-lysine-coated glass coverslips before being placed in a temperature-controlled perfusion chamber (Brook Industries, Lake Villa, IL) mounted on the stage of a Nikon TMS inverted microscope. All experiments were carried out at 21 ± 1 °C. Patch pipettes were manufactured from standard walled borosilicate glass capillary tubing on a two-stage Narishige PP-83 pipette puller (Narishige Scientific Instrument Lab, Kasuya, Tokyo, Japan), were heat-polished on a Narishige microforge, and had measured tip resistances of 3-8 M (when filled with K⁺-rich pipette solution).

Resistive feedback voltage-clamp was achieved using an Axopatch 200A amplifier (Axon Instruments, Forster City, CA). Voltage protocols were generated, and currents were recorded using pClamp 6.0.3 software employing a Digidata 1200 A/D converter (Axon Instruments). Data were filtered (4-pole Bessel) at 2 kHz and digitized at 5 kHz. Following successful transition to the whole-cell recording mode (12), capacitance transients were compensated for and measured. Where necessary, series resistance compensation was used at 100%.

To evoke ionic currents in H-146 cells, two voltage protocols were used: (a) ramp protocol: holding potential = 70 mV, 100 to +60 mV, ramp duration = 1 s, frequency = 0.1 Hz; (b) time series: holding potential = 70 mV, single increment to 0 mV, step duration = 50 ms, frequency = 0.1 Hz.

Fast current clamp was achieved using the same amplifier, and the solutions were the same as those used in the voltage-clamp experiments. Cells were clamped at I = 0 pA and the recorded voltage was filtered at 1 kHz and digitized at 2 kHz.

Single Cell Fluorescence

H-146 cells were loaded for 30 min at 37 °C with 10 µM (in 0.01% dimethyl sulfoxide) H₂O₂ fluorescent indicator 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA, Molecular Probes, Leiden, Netherlands) during adherence to poly-L-lysine-coated coverslips (17). Coverslips were mounted on the stage of Nikon Diaphot 300 and perfused with the standard normoxic (150 mm Hg) bath solution (see above for composition). H₂DCFDA was excited with light of wavelength 488 nm using a monochromator and emitted fluorescence (>510 nm) collected by a CCD camera (Hamamatsu, C4880-80). Cells were imaged using Openlab software at 2-s intervals, and pixel intensity was calculated off-line. Following a control period of 20 s, cells were perfused with standard bath solution for 2 min, which was either air-equilibrated (150 mm Hg) or hypoxic (15 mm Hg). Recovery was followed for a further 3 min.

Data Handling and Calculations

Electrophysiology-- The magnitude of the steady-state outward currents (time-series protocol) was measured as the mean current between 46 and 49 ms of the voltage-pulse. In the time-series plots, the currents from each separate experiment were normalized by dividing the current at each point by the mean of the first five currents in that series; in the figures, the numbers represent the mean (± S.E.); n refers to the number of cells. Current amplitudes were not corrected for cell size, because this did not vary between the four groups of cells studied, as determined by membrane capacitance measurements; control cells, 5.0 ± 0.13 picoFarad (n = 93); TPA-treated cells, 4.8 ± 0.23 picoFarad (n = 57); 4-PDD treated cells, 4.7 ± 0.21 picoFarad (n = 23); TPA- and BIM-treated cells, 5.7 ± 0.50 picoFarad (n = 16). Statistical comparisons were made using the paired or unpaired Student's t test, as appropriate, with p < 0.05 being considered significant.

Fluorescence-- H₂DCFDA is effectively trapped within cells and, in common with most other flourescence indicators, undergoes photobleaching. Because it is not a dual excitation or emission dye, this artifact cannot be removed by signal ratioing. Thus, during the experimental period, fluorescence intensity declines exponentially with time. Nevertheless, a change in the rate of decay is indicative of a change in cellular H₂O₂ content/production. Mean pixel intensity was calculated from each cell area at intervals of 2 s, background fluorescence was subtracted, and the subtracted intensity was plotted against time. The rate of change of fluorescence was calculated as a rolling average of slope of five consecutive frames for the entire duration of the experiment. To account for any differential dye loading, slope data from each cell were normalized such that the first average from each protocol was unity. In Fig. 2, the mean data plot shows this rolling average versus time. A downward deflection (indicative of an increase in normalized slope value) represents a decrease in H₂O₂ production/content. Exemplar, nonadjusted data are shown in the insets of Fig. 2.

RT-PCR-- Total RNA was extracted from pelleted H-146 cells using the RNeasy Micro Kit (Qiagen, Crawley, W. Sussex, UK). The extracted RNA was then divided; 50% was treated (cleaned) with RQ-1 RNase-free DNase (1 unit·µg¹ RNA; Promega, Southampton, Hampshire, UK) to remove genomic DNA contamination, before re-extraction using the RNeasy Micro Kit. The remaining 50% was kept in an untreated (uncleaned) state. The yield, purity, and integrity of the RNA was verified by spectrophotometry at 260/280 nm, followed by electrophoresis on 1% agarose, and was then stored in aqueous solution at 80 °C. RT was performed on 1-µg aliquots of both cleaned and uncleaned RNA using the reverse transcription system A3500 (Promega), comprising Avian Myeloblastosis Virus reverse transcriptase and oligo(dT) (15) primers (42 °C, 15 min). The resulting cDNA was amplified by PCR and screened for p47^phox RNA expression using oligonucleotide primers (22-mers) designed against the published sequence of human neutrophil cytosolic factor 1 (GenBank^TM accession number M55067 (18)). These primers (upstream: 5'-atgcaggtgagccatacgtcgc-3', downstream: 5'-tctttcctgatgacccaccagc-3') were chosen because they were predicted to amplify a region within a single exon. Therefore, products from both reverse transcribed mRNA and genomic DNA (the positive control used in this study) would be of identical length. PCR reactions using the cleaned and uncleaned (as positive control) cDNA were run in parallel. Amplification of 1-µl cDNA (equivalent to 160 ng of reverse-transcribed RNA) was performed using a Hybaid (Ashford, Middlesex, UK) express thermal cycler, in a volume of 50 µl, containing 2.5 units of Taq DNA polymerase (Promega), under optimized conditions with 2.0 mM MgCl₂: hot start = 94 °C/1 min; denaturing = 94 °C/1 min, annealing = 64 °C/1 min, extension = 72 °C/1 min for 30 cycles followed by a final cycle, which had a 5-min extension. Products were separated on 2% agarose gels and visualized with ethidium bromide/UV transillumination. Because signal was relatively low from cleaned cDNA, a "turbo" PCR was performed on 5 µl of the primary PCR products. Turbo PCR conditions were identical to primary PCR. Sequencing was carried out by dye terminator PCR with an ABI PRISM automated sequencer (School of Biology, University of Leeds, UK).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Response to Graded Hypoxia-- In H-146 cells, we have recently shown that acute hypoxia (pO₂ ~15 mm Hg) causes rapid and reversible inhibition of whole cell K⁺ currents and membrane depolarization (13). Fig. 1 extends our earlier observations to show that reducing perfusate pO₂ in a graded manner (between 150 and 15 mm Hg) results in correspondingly graded inhibition of these K⁺ currents and also causes graded membrane depolarization (Fig. 1A). In this series of experiments, a pO₂ of 15 mm Hg significantly reduced mean outward K⁺ current amplitudes (measured at 0 mV) from 223.2 ± 28.9 pA to 149.6 ± 41.2 pA (p < 0.005, n = 7), a reduction of ~33%. The same degree of hypoxia caused a significant depolarization from -47.9 ± 1.3 mV to 38.4 ± 3.8 mV (p < 0.05, n = 7), a reduction of 9.5 mV. These values are similar to our previously published data (13). Fig. 1B shows a plot of the mean current amplitude versus membrane potential at the five grades of hypoxia examined. Hypoxic reduction in whole cell K⁺ current correlates well with membrane potential (r = 0.96) and strongly suggests, therefore, that the O₂-sensitive current contributes to the resting membrane potential in this airway chemoreceptor cell model.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The response of H-146 cells to graded hypoxia. A, mean outward K+ currents (left axis, open circles) and membrane potentials (right axis, closed circles) recorded during graded depression in pO2 from 150 to 15 mm Hg. Currents were recorded during 50-ms step depolarizations from a holding potential of 70-0 mV; n  7 cells. Membrane potential was measured in current clamp with I = 0 pA; n  7 cells. B, mean current amplitude versus mean membrane potential at the five separate pO2 values. r = 0.96; n  7.

Effects of Hypoxia on H₂O₂ Production in H-146 Cells-- Central to the hypothesis that NADPH oxidase acts as the O₂ sensor in airway chemoreceptors is the notion that H₂O₂ levels are decreased during hypoxia. Fig. 2 demonstrates that this is indeed the case in H-146 cells. During normoxia, fluorescence intensity gradually declined because of photobleaching (Fig. 2A and inset). However, perfusion with the hypoxic solution (15 mm Hg) caused a far greater decline in fluorescence intensity, approaching twice the base-line bleaching rate (observed under normoxic conditions (Fig. 2B and inset)). This effect was maximal around 30 s after exchange to the hypoxic solution and was reversible on reperfusion with normoxic solution. The time-course of this effect was similar to that of hypoxic inhibition of K⁺ currents (see Ref. 13 and also Figs. 5 and 6). These data show clearly that H₂O₂ levels in H-146 cells are regulated by environmental pO₂ and suggest that K⁺ channels are under tonic control by a H₂O₂-generating system.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Fluorescence measurement of H2O2 production. A, normalized mean rate of change of H2DCFDA fluorescence (± S.E.) during normoxic conditions (n = 6). B, normalized mean rate of change of H2DCFDA fluorescence (± S.E.) before, during, and after perfusion of cells with hypoxic (15 mm Hg) bath solution (n = 9). The period of hypoxia is indicated by the horizontal bar. For both A and B, the rate of fluorescence change was calculated as described under "Data Handling and Calculations" from raw data exemplified in the two insets.

Effects of Phorbol Esters on H-146 K⁺ Currents-- Preincubation of H-146 cells for 10 min with the membrane-permeable PKC activator, TPA (100 nM), caused a significant (p < 0.05, n = 11 control cells and 8 TPA-treated cells; unpaired Student's t test) reduction in ramp currents at all potentials (Figs. 3, A and B). To determine the contribution which PKC activation made to this reduction in current, we employed: (a) a biologically inactive phorbol ester, 4-PDD (100 nM); and (b) TPA together with the PKC inhibitor, BIM (1 µM bath and pipette). These maneuvers resulted in smaller, nonsignificant (p > 0.29, n = 5; p > 0.52, n = 10, respectively) reductions in currents (Fig. 3, C-E), than were seen with TPA. This suggests that the inhibition caused by TPA was attributable to both nonspecific actions of phorbol esters and activation of PKC.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   The effects of phorbol esters in normoxic conditions. A-D, mean current-voltage relationships (± S.E.; dotted lines) recorded using the ramp protocol in normoxia after no pretreatment (A, n = 11), 10-min pretreatment with 100 nM TPA (B, n = 8), 100 nM 4-PDD (C, n = 5), and 100 nM TPA in the presence of 1 µM BIM (D, n = 10). E, mean current amplitudes recorded in normoxia at 0 mV with no bath additions (Control) and following pretreatment with the agents shown beneath each bar. Number of observations in each condition is as stated in A-D.

Effects of PKC Activation on Hypoxic Inhibition of K⁺ Currents in H-146 Cells-- Fig. 4A shows currents evoked by ramp depolarizations from a typical H-146 cell before, during, and after exposure to two levels of acute hypoxia. These currents exemplify the reversible, graded inhibition caused by hypoxia, which is summarized in Fig. 1A. In contrast to these findings, currents recorded in cells pretreated with 100 nM TPA, although reduced in amplitude (see above), were virtually unaffected by hypoxia (e.g. Fig. 4B). Average current amplitudes (measured at 0 mV) at all levels of hypoxia examined are plotted in Fig. 4C. Thus, TPA markedly and significantly (p < 0.01, n = 6 control cells; n = 4 TPA-treated cells) suppressed hypoxic inhibition of K⁺ currents in H-146 cells. The results shown in Fig. 4D (determined from currents recorded at 0 mV during normoxia and hypoxia at 15 mm Hg) indicate that this was attributable to PKC activation, because neither 4-PDD (p = 0.18, n = 4) nor TPA in the presence of BIM (p = 0.87, n = 5) could significantly suppress the level of hypoxic inhibition of currents.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   The effects of phorbol esters on hypoxic depression of K+ currents. A and B, exemplar I-V relationships during normoxia and two grades of hypoxia (45 and 15 mm Hg) in untreated (A) and TPA-pretreated (B) cells. Currents were recorded during 1-s ramp depolarizations from a holding potential of 0 mV. C, mean outward K+ current recorded from TPA-treated cells during graded depression in pO2 from 150 to 15 mm Hg. Currents were recorded at 0 mV during 1-s ramp depolarizations from a holding potential of 70 to 60 mV, n = 4 cells. D, mean current amplitudes recorded in hypoxia (15 mm Hg) at 0 mV with no bath additions (Control) and following pretreatment with the agents shown beneath each bar. Number of observations in each condition is as stated under "Results."

We have previously shown that the O₂-sensitive K⁺ current is resistant to 4-AP (14). Following treatment with TPA, the proportion of the whole cell current, which was 4-AP-insensitive, remained essentially unchanged compared with control (Fig. 5, A and D). However, in the presence of a maximally effective concentration of 4-AP (10 mM), hypoxia (15 mm Hg) caused over 50% inhibition of the residual current (e.g. Fig. 5, A, C, and D). In contrast, hypoxic inhibition of the 4-AP-resistant current was significantly (p < 0.005, n = 8) reduced to around 25% following TPA treatment (e.g. Fig. 5, B, C, and E). Fig. 5C shows the mean time course of these effects. This observation shows that although TPA caused a generalized reduction in current (see also Fig. 3), the 4-AP-resistant component was less O₂-sensitive in these cells when PKC was activated.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of PKC activation on hypoxic depression of 4-AP-insensitive K+ currents. A and B, exemplar currents under control conditions and then before (4-AP), during (4-AP + Hypoxia), and after (4-AP + Recovery) hypoxia (15 mm Hg) in the presence of 10 mM 4-AP. Traces were recorded from untreated (A) and TPA-pretreated (B) cells. Currents were recorded during 50-ms step depolarizations from a holding potential of 70 to 0 mV. C, mean time series plots of current amplitudes (with vertical S.E. bars, averaged for eight control cells (open circles) and eight TPA-treated cells (closed circles)) evoked by repeated step depolarizations from 70 to 0 mV (50-ms duration, 0.2 Hz). 4-AP and hypoxia were applied for the periods indicted by the horizontal bars. n = 8 cells in both conditions. D and E, bar graphs (with vertical S.E. bars) showing effect of TPA treatment on proportion of current inhibited by 10 mM 4-AP (D) and proportion of 4-AP-resistant current, which is inhibited by hypoxia (E, 15 mm Hg). Adapted from the same data as shown in C.

Although there are many PKC-dependent mechanisms that could account for these data, one potential site of action for PKC might be the NADPH-oxidase complex, which has been recently implicated in NEB cell O₂ sensing (15). If this is the case in our NEB cell model, we would predict that products downstream of NADPH oxidase activity would regulate the O₂-sensitive K⁺ channel. We addressed this possibility by applying one such product, H₂O₂, in the presence of 4-AP. Fig. 6A shows that under normoxic conditions, H₂O₂ was without effect. However, during hypoxic inhibition of the K⁺ current, H₂O₂ caused a dramatic and transient reactivation of the current. The time course of this effect was similar to that previously reported in NEB cells (15).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of H2O2 on 4-AP-insensitive K+ current. A, typical currents evoked from an example cell before (Normoxia), during (Normoxia + H2O2), and after (Normoxia) bath application of 2 mM H2O2. B, typical currents evoked from a representative cell before (Normoxia) and during hypoxia (Hypoxia) and during continued exposure to hypoxia but in the additional presence of 2 mM H2O2 (Hypoxia + H2O2). Currents were evoked by step depolarizations from 70 to 0 mV, and scale bars apply to both A and B. 10 mM 4-AP was present throughout in both cases. C, mean time series plot of currents evoked from five cells as in A but normalized to the first five currents evoked after the effects of 10 mM 4-AP had stabilized. H2O2 was applied for the period indicated by the horizontal bar. D, as C, except cells (n = 7) were exposed to hypoxia before and during H2O2 application, as indicated by horizontal bars.

A probable site of PKC-dependent phosphorylation of the heteromeric enzyme, NADPH oxidase, is p47^phox (16). However, although there is good evidence for the presence in both native NEB cells and H-146 cells of some of the components of the enzyme (15), evidence for the presence of p47^phox in H-146 cells is still lacking. Fig. 7 shows the use of RT-PCR to address this question directly. Amplification of a product compatible with the presence of p47^phox (130 base pairs) in genomic DNA (uncleaned lanes) shows that the PCR protocol that we employed can detect p47^phox (Fig. 7A). Using this protocol, we could detect only a small signal from cDNA reverse transcribed from DNase-treated H-146 RNA (cleaned lanes). However, a turbo PCR reaction produced a strong signal (130 base pairs) from the cleaned sample (Fig. 7A), indicating that p47^phox was indeed being transcribed by these cells (confirmed by sequencing, Fig. 7B) but that its expression was low because of either rapid degradation or low copy number of p47^phox mRNA; PCR cannot distinguish between these two possibilities. Regardless, p47^phox is present and is, therefore, a possible site of PKC-dependent regulation of the oxidase in our model airway chemoreceptor cells.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   RT-PCR of H-146 cell RNA employing p47phox primers. A, 2% agarose gel of PCR reaction products. Lane 1, water control. Lanes 2 and 3, cDNA reverse transcribed from cleaned RNA following primary (lane 2) and turbo (lane 3) PCR. Lanes 4 and 5, cDNA reverse-transcribed from uncleaned RNA following primary (lane 4) and turbo (lane 5) PCR. Lane 6, DNA ladder with marker sizes as indicated. 10 µl of reaction product was loaded in each lane, and DNA was visualized using ethidium bromide. B, the aligned sequences of p47phox and the PCR product from cleaned cDNA show 99.2% identity. Underlined sequences show the primer sites that were employed for the PCR reaction in A.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Electrophysiological studies have demonstrated that acute reductions in pO₂ suppress K⁺ currents in airway chemoreceptor NEBs (7) in a manner comparable to their vascular counterparts, the type I cells of the carotid body (1, 19). Such an effect underlies membrane depolarization, which presumably leads to activation of voltage-gated Ca²⁺ channels, thereby permitting Ca²⁺ influx to trigger neurotransmitter release in both cell types (8, 20). Previous studies, including our own, have demonstrated that SCLC cell lines (such as H-146 used here), which are derived from the same precursor pool as NEBs (11), provide an excellent model for studying mechanisms underlying hypoxic inhibition of NEB cell K⁺ channel inhibition (13, 14). This is an important advance given the present limitations of acutely isolated, primary cultured NEB cells.

An important aim of the present study was to use PKC activation as a method by which to investigate the transduction pathway responsible for hypoxic suppression of K⁺ currents in our recently established model of NEB O₂ sensing. In NEB cells, it has been proposed, but not directly substantiated, that NADPH oxidase is an important upstream component of the chemoreception pathway (6, 15). NADPH oxidase is a heteromultimeric enzyme that contains, importantly, a PKC-activable subunit, p47^phox (16). Fig. 7 shows, for the first time, that mRNA for this subunit is present in H-146 cells. This observation has allowed us to exploit both PKC-regulation of NADPH oxidase and the fact that the candidate K⁺ channel is not selectively regulated by PKC per se (21) to test the hypothesis that O₂ transduction relies, at least in part, on the sensing of pO₂ by NADPH oxidase. Clearly, there are many targets for PKC-dependent phosphorylation. However, there is accumulating evidence in NEB and NEB-derived cells that, in terms of O₂ signal transduction, NADPH oxidase activity, and its regulation by PKC, are of paramount importance. First, H₂O₂, a product of the sequential actions of NADPH oxidase and catalase/superoxide dismutase, appears to provide the required oxidized environment needed for tonic K⁺ channel activity in normoxia (15); Fig. 2 shows clearly that production of H₂O₂ by H-146 cells is suppressed during hypoxia with a time course similar to that of hypoxic K⁺ current inhibition (13). Second, phorbol 12-myristate 13-acetate treatment (another PKC-stimulating phorbol ester) causes a dramatic increase in intracellular H₂O₂ concentration; presumably via activation of p47^phox (15). Based upon these observations, we would predict that any agent that increases the affinity of NADPH oxidase for substrate will ameliorate the inhibitory action of hypoxia.

To investigate the potential involvement of PKC in O₂ sensing by H-146 cells, it was important first to characterize more completely the O₂ sensitivity of hypoxic inhibition of K⁺ currents in these cells. Fig. 1A shows that the inhibition of K⁺ currents (measured at a test potential of 0 mV) by hypoxia was clearly graded, with significant inhibition only observed at a pO₂ of 45 mm Hg or lower. This is in accordance with studies of the [Ca²⁺]_i response of arterial chemoreceptor cells (20) and further validates our own model system of airway chemoreceptor cells. Most importantly, we found an excellent correlation between hypoxia-induced K⁺ current suppression and hypoxia-evoked membrane depolarization (Fig. 1B). This finding justifies our use of measuring current amplitude at 0 mV (which provides an acceptable compromise between signal to noise ratio and resolution of the physiological action of hypoxia) as a reliable indicator of events occurring at resting membrane potential, as we have earlier suggested (14).

Phorbol ester activation of PKC often requires several minutes to become maximal, and so to investigate the effects of PKC activation on K⁺ currents in H-146 cells, we pretreated cells for 10 min with such agents. Nonspecific inhibitory effects of phorbol esters on ion channels have been documented previously (22), and the data shown in Fig. 3 agree with this finding; phorbol ester treatment suppressed mean current amplitude but this can be divided into both PKC-dependent and -independent components. Because the effects of TPA were partially reversed by inhibiting PKC with BIM, it appears that the PKC-dependent component is significant. However, 4-PDD, a phorbol ester that is inactive with respect to PKC activation, also caused some current depression. Although these data contrast with recent studies in type I carotid body cells, which indicate that TPA selectively inhibits O₂-sensitive Ca²⁺-dependent K⁺ channels solely via PKC activation (23), the O₂-sensitive K⁺ current in H-146 cells is clearly not Ca²⁺-dependent (13). Indeed, our most recent study (14) has shown that the O₂-sensitive K⁺ current in H-146 cells is likely to be a member of the recently characterized tandem-P domain family of K⁺ channels (TWIK (24), TREK (25) TASK-1 (21), TASK-2 (26), and TRAAK (27)) and shows structural similarities to TASK. Furthermore, our data in Fig. 5D show very clearly that proportionally the 4-AP-insensitive current (of which the O₂-sensitive current is a large component) is unaffected by phorbol ester treatment. Therefore, although TPA causes a generalized depression of K⁺ currents, it does not affect the O₂-sensitive component per se, but, instead, it specifically ameliorates the hypoxia sensitivity of that component.

Indeed, the most important observation of the present study is that TPA modulates the O₂ sensitivity of K⁺ currents in H-146 cells (Figs. 4 and 5), and such a finding has important implications for the frequently proposed involvement of NADPH oxidase in O₂ chemoreception in various tissues, including airway chemoreceptors (15), arterial chemoreceptors (28), and pulmonary vascular smooth muscle (29). This model proposes that O₂ sensing is dependent on the generation of H₂O₂ by NADPH oxidase under normoxic conditions. This enzyme has been shown to produce H₂O₂ tonically, and both native and recombinant subtypes of K⁺ channels (including those found in NEB cells and SCLC cells (15)) have been shown to be enhanced by H₂O₂, raising the attractive concept that hypoxia inhibits K⁺ currents by reducing H₂O₂ production by NADPH oxidase simply by limiting the enzyme's substrate, O₂. We have now provided direct evidence that acute hypoxia reduces H₂O₂ production (Fig. 2). The ability of NADPH oxidase to generate H₂O₂ can be enhanced by the phorbol ester phorbol 12-myristate 13-acetate, which is another known activator of PKC. Our findings indicate that the PKC-activating phorbol ester, TPA, suppressed the ability of hypoxia to inhibit K⁺ currents in H-146 cells. This specific action of TPA can be attributed to PKC activation, because it was not mimicked by another phorbol ester, 4-PDD (which does not activate PKC) and was prevented by the PKC inhibitor, BIM. Because the nonspecific suppressing effects of TPA on K⁺ currents measured under normoxic conditions were largely because of a PKC-independent action of TPA (see above), the PKC-dependent suppression of hypoxic inhibition is likely to occur at a site upstream of the channel itself. Furthermore, the channel which is likely to underlie the hypoxia-sensitive K⁺ current in H-146 cells is possibly TASK-related, and TASK is insensitive to the actions of phorbol esters (21). The cytosolic protein p47^phox is one of several subunits comprising the NADPH oxidase complex and is a known substrate for PKC (16). Its stimulation by phorbol 12-myristate 13-acetate (presumed to be mediated by PKC phosphorylation) has been suggested to account for the enhanced H₂O₂ production under normoxic conditions in NEB cells, as determined by rhodamine 123 fluorescence (15). In the context of this finding, our results provide compelling evidence that supports the idea that NADPH oxidase is the O₂ sensor in the hypoxic signal transduction pathway. In this model, the tonic generation of H₂O₂ in H-146 cells provides the appropriate environment for normoxic channel activity. That this activity is maximal in normoxia is evidenced by the ability of exogenously applied H₂O₂ to enhance the O₂-sensitive current amplitude only during hypoxia (Fig. 6B) and not during normoxia (Fig. 6A). This is in contrast to results reported for native NEB cells where H₂O₂ transiently activates K⁺ currents in normoxia (15). This may be because of differences between NEB and H-146 cell NADPH oxidase turnover or specific class of K⁺ channel targeted. Although this may suggest limitations of our model, it does not change the picture of the basic transduction pathway per se. Thus, following PKC activation, H₂O₂ levels are likely to be enhanced in H-146 cells as they are in NEB cells (15) due possibly to increased affinity of NADPH oxidase for O₂. Evidence for a PKC-dependent increase in substrate affinity is shown by the kinetic analyses of depression of K⁺ currents during graded hypoxia. These analyses, which are summarized in Fig. 8, show that PKC increases O₂ affinity ~6-fold and provide an explanation for how H₂O₂, and therefore, channel open state probability is maintained in the face of reduced pO₂.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Kinetic analyses of hypoxic inhibition of K+ currents. The main figure shows data from Figs. 1A and 3C normalized and fitted to the Michaelis-Menton equation by iterative fitting using the method of least squares. Open circles, control cells; filled circles, TPA-pretreated cells. The inset shows the same data transformed as a double reciprocal plot as described by the axes. Both analyses predict that preincubation of cells with TPA decreases the calculated Km of the system for O2 approximately 6-fold.

In summary, PKC activation reduces the sensitivity of H-146 cells to changes in pO₂. Furthermore, because the addition of H₂O₂, a downstream product of NADPH oxidase, only activates K⁺ currents during hypoxia (when H₂O₂ production is suppressed), it appears likely that PKC-dependent phosphorylation modulates the affinity of NADPH oxidase for O₂, and the PKC substrate p47^phox (a component of the functional NADPH oxidase) is clearly present in these cells. Taken together, these data show that PKC is an important regulator of the O₂-transduction pathway and provide functional evidence that suggests that NADPH oxidase is a major airway O₂ sensor.

	FOOTNOTES

^* 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.

^¶ To whom correspondence should be addressed: School of Biomedical Sciences, Worsley Medical and Dental Bldg., University of Leeds, Leeds LS2 9JT, UK. Tel.: 44 113 233 4236; Fax: 44 113 233 4228; E-mail: p.z.kemp@leeds.ac.uk.

	ABBREVIATIONS

The abbreviations used are: NEB, neuroepithelial body; SCLC, small cell lung carcinoma; 4-AP, 4-aminopyridine; PKC, protein kinase C; TPA, 12-O-tetradecanoyl; 4-PDD, 4-phorbol 12,13-didecanoate; BIM, bisindolylmaleimide; H₂DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; pO₂, partial pressure of oxygen (mm Hg); RT, reverse transcription; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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