Oxidative stress decreases pHi and Na+/H+ exchange and increases excitability of solitary complex neurons from rat brain slices

Daniel K. Mulkey,1 Richard A. Henderson, III,1,2 Nick A. Ritucci,1 Robert W. Putnam,1 and Jay B. Dean1

1Department of Anatomy and Physiology, Environmental and Hyperbaric Cell Biology Facility, and 2Department of Community Health, Wright State University School of Medicine, Dayton, Ohio 45435

Submitted 28 July 2003 ; accepted in final form 5 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pHi). pHi was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pHi in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H2O2 decreased pHi by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pHi by 0.26 ± 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pHi, causing a 0.42 ± 0.03 (n = 21) pH unit acidification. CT slowed pHi recovery mediated by Na+/H+ exchange (NHE) from NH4Cl-induced acidification by 53% (n = 20) in -buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pHi. These results indicate that oxidative stress 1) decreases pHi in some SC neurons, 2) together with hypercapnia has an additive effect on pHi, 3) partially inhibits NHE, and 4) directly affects excitability of CO2/H+-chemosensitive SC neurons independently of pHi changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction.

hyperoxic hyperventilation; O2 toxicity; pH regulation; brain stem; reactive oxygen species


CO2/H+-CHEMOSENSITIVE NEURONS, which are believed to function as CO2 chemoreceptors for the cardiorespiratory control system, are highly sensitive to oxidative stress. For example, we found that CO2/H+-chemosensitive, but not CO2/H+-insensitive, SC neurons are stimulated by hyperoxia (38). We went on to show that the responses to CO2 and hyperoxia are mediated by separate mechanisms; the CO2 signal involves decreased intracellular pH (pHi) (21) whereas hyperoxia depends on oxidation (38). However, when SC neurons were exposed to CO2 and hyperoxia in combination, the firing rate response was larger than to either stimulus alone (38), thus suggesting that oxidative stress and CO2 signaling mechanisms are at least additive in SC neurons.

A possible point of interaction between the oxidative stress and CO2 signaling pathways is pHi; that is, oxidative stress may cause an intracellular acidosis. There is evidence for an oxidant-induced acidification in several nonneuronal preparations. For example, hydrogen peroxide (H2O2) causes an intracellular acidosis in rat cerebellar astrocytes (63), C6 glioma cells (63), HL60 cells (27), aortic endothelial cells (28), renal epithelial cells (32), and cardiac myocytes (9, 65). Likewise, superoxide () decreases pHi in Xenopus oocytes (10) and in activated neutrophils (25). In addition, preliminary results from our laboratory showed that the chemical oxidants chloramine-T and N-chlorosuccinimide cause an intracellular acidification in SC neurons from rat brain slices (39). Therefore, the current study had four goals. The first goal was to determine whether a variety of oxidants, including reactive oxygen species (ROS) and chemical oxidants, could acidify SC neurons. The second goal was to determine whether oxidative stress affected pHi regulation and Na+/H+ exchange (NHE) activity in SC neurons. The third goal was to determine whether hypercapnia and oxidative stress caused a greater acidification than either stimulus alone, i.e., to investigate whether or not there was an interaction between the oxidative stress and CO2 signaling pathways. The fourth goal was to determine whether chloramine-T could stimulate firing rate independently of its effect on pHi. Preliminary results of these data were presented previously (37, 40).


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Brain slice. Details regarding the preparation of brain slices have been described elsewhere (14). Briefly, a brain stem from a rat pup ranging in postnatal (P) age from P1 to P15 days was isolated, and the medulla oblongata was cut into 300-µm-thick transverse slices by using the Vibratome (series 1000) sectioning system. Slices were taken beginning at obex and moving rostrally for ~900 µm. Animal use procedures are in agreement with the Wright State University Institutional Animal Care and Use Committee guidelines and were approved by the committee (AALAC no. A3632-01). Brain stem slices were incubated at room temperature in artificial cerebral spinal fluid (aCSF) of the following composition (in mM): 124 NaCl, 5.0 KCl, 1.3 MgSO4, 26 NaHCO3, 1.24 KH2PO4, 2.4 CaCl2, and 10 glucose, equilibrated with 95% O2-5% CO2 (PO2 ~720 Torr, PCO2 ~40 Torr) (36). Under these conditions, slices remained viable for electrophysiological experiments for ~8 h.

pHi measurements. We measured pHi by using ratiometric fluorescence imaging microscopy, utilizing two different pH-sensitive fluorescent dyes. The first pH-sensitive dye used was 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) because it can be passively loaded into cells, thus causing minimal cellular disruption. In addition, BCECF is in a state that is not likely to be further oxidized by the level of oxidative stress used in this study (54). Therefore, we assumed that oxidative stress in the form of either chemical oxidants or free radicals would not have any direct effect on the fluorescence of the dye. Details regarding the use of BCECF for pH imaging in brain slices have been previously described (49, 50). Slices were loaded in the dark with 20 µM BCECF (in the membrane-permeable acetoxymethyl ester form) for 30–60 min at 37°C and washed at room temperature in control aCSF (50). Experiments were performed on individual slices transferred to a superfusion chamber positioned on the stage of an inverted Nikon Diaphot microscope while perfused with aCSF, heated to 37°C (pH ~7.45), at 2 ml/min. The dye was excited every 60 s by brief (~1 s) alternating pulses of light with wavelengths of 500 nm (pH sensitive) and 440 nm (pH insensitive). pHi is proportional to the ratio of emitted fluorescence (535 nm) at these two excitation wavelengths (F500/F440). This fluorescence ratio was normalized to the fluorescence ratio value at pH 7.2, determined by using the high-K+/nigericin calibration technique (62), and normalized fluorescence was converted to pHi by using the equation of Ritucci et al. (49, 50). To minimize any effects from nigericin contamination, we used a separate calibration line (distinct from the perfusion lines), and the tissue chamber was extensively washed following each experiment.

In a second series of experiments, we measured pHi by using the pH-sensitive fluorescent dye pyranine while simultaneously measuring membrane potential (Vm) with the use of whole cell recording (WCR) techniques. Pyranine was used in these experiments because 1) it is not membrane permeable and so can only be loaded into neurons by WCR where it tends not to leak out; 2) it is resistant to photobleaching; 3) it has a high quantum yield; and 4) it is less damaging to the cell than BCECF during excitation (23). Pyranine (300 µM) was dissolved in the whole cell solution, which diffused into the cell body only after the patch was ruptured and a WCR established. The dye was excited every 60 s by brief (~2 s) alternating pulses of light with wavelengths of 450 nm (pH sensitive) and 415 nm (pH insensitive). pHi is proportional to the ratio of emitted fluorescence (515 nm) at these two excitation wavelengths (F450/F415). This fluorescence ratio was recorded by using MetaFluor software, normalized to the fluorescence ratio value at pH 7.2, and then normalized fluorescence was converted to pHi- by using the equation of Ritucci et al. (51).

Electrophysiology. Loose extracellular patches and WCR were made by using the Axopatch-1D (CV-4 head-stage gain = 1.0; Axon Instruments), as described previously (30, 42). Briefly, the recording electrode was made from borosilicate glass (TW150-3; World Precision Instruments) by using a two-stage Narishige pipette puller (PP-830). The electrode was filled with a solution containing (in mM) 130 K-gluconate, 0.4 EGTA, 1.0 MgCl2, 0.3 Na2-GTP, 2.0 Na2-ATP, and 10 HEPES, pH 7.45 at room temperature. Compared with standard filling solution, this solution had reduced EGTA and no CaCl2 to reduce washout (22). The recording electrode was connected to the head stage by a Ag-AgCl wire (Medwire), and an AgCl reference was placed in the tissue bath to complete the circuit. Recording electrodes had a tip resistance of ~3 M{Omega}; electrodes with smaller tips (tip resistance > 5 M{Omega}) were not used because they are not conducive to loose patch recording (42). The recording pipette and SC neurons were visualized (x720 magnification) with an Optiphot-2 Nikon microscope with a x40 water-immersion Hoffman contrast objective (NA 0.55). A slight positive pressure was constantly applied to the pipette while it was advanced toward the tissue to prevent the tip from being obstructed and to blow debris from the surface of the target cell. After contact with the cell was established, a slight negative pressure was applied to form a gigaseal. In this configuration loose patch extracellular measurements of firing patterns of individual cells could be made. The gigaseal patch can be ruptured by applying an additional negative pressure or by delivery of a voltage pulse, thereby establishing a WCR. A healthy WCR was assumed when a neuron had a Vm more negative than –40 mV and action potential amplitude of at least 50 mV. All electrophysiological recordings were made in currentclamp mode. Firing rate was integrated in 10-s bins by using a window discriminator (Fredric Haer) and was recorded with the use of a DigiData 1320A acquisition system (Axon Instruments) and stored on videotape (Vetter PCM recorder model 400).

Test conditions. Slices were exposed to oxidative stress by addition of either chemical oxidants or ROS to the aCSF. The chemical oxidants chloramine-T (CT; Sigma-Aldrich) and N-chlorosuccinimide (NCS; Sigma-Aldrich), both of which are water soluble and fairly specific oxidizers of the amino acids cysteine and methionine (55), were used at concentrations previously shown to stimulate firing rate in 67% of SC neurons (500 µM for CT and 1.0 mM for NCS) (38). Slices were also exposed to ROS by application of the superoxide () generator dihydroxyfumaric acid (DHF; 3 mM) or the reactive nonradical derivative H2O2 (4–5 mM). pHi responses to oxidative stress were compared with the response to hypercapnic acidosis (15% CO2), which is also known to stimulate the firing rate of SC neurons (38, 53). Dithiothreitol (DTT; 0.5–1 mM), a hydrophilic and cellpermeable cysteine-specific reducing agent (20), was used to differentiate the effects of CT- and NCS-induced oxidation of cysteine vs. methionine. All test solutions had a pH of 7.45 after equilibration with 5% CO2-balance O2 at 37°C. Finally, to determine the relative effect of oxidative stress in the absence of a change in pHi, we blocked CT-induced acidification by either increasing aCSF bicarbonate () concentration to 52 mM (osmolarity was maintained by decreasing NaCl to 98 mM) or lowering CO2 from 5 to 1.4%.

pHi regulation. NHE activity was determined by measuring pHi recovery from an ammonium chloride (NH4Cl; 20 mM)-induced intracellular acidification (osmolarity was maintained by decreasing NaCl to 104 mM) under control conditions and during oxidative stress imposed by CT. To determine whether our control conditions ( buffering system) unexpectedly contributed to the effects of oxidative stress on NHE activity, we also measured the effects of CT on pHi recovery in HEPES-buffered medium of the following composition (in mM): 124 NaCl, 5.0 KCl, 1.3 MgSO4, 26 HEPES, 1.24 KH2PO4, 2.4 CaCl2, and 10 glucose, pH ~7.4 at 37°C, equilibrated with 100% O2. Amiloride (1.6 mM) was used to block NHE.

Analysis and data presentation. Data were analyzed by using AxoScope 8.1, SigmaPlot, Origin 5.0, and SigmaStat software packages. The CorelDraw 8.0 software package was used for data presentation. To determine whether a test condition caused a change of pHi, a change of >0.05 pH unit was defined as significant. Paired-sample t-tests (P < 0.05 unless otherwise stated) were used to determine when the average change in the pHi, firing rate, or rate of pHi recovery differed significantly from zero. Significant differences among the effects of each test condition on pHi or firing rate were determined by using a one-way ANOVA and Newman-Keuls multiple comparison test (P < 0.05) when appropriate. All results were presented as means ± SE.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The experiments in this study were conducted in two parts. First, we measured pHi to determine whether oxidative stress affects pHi or pH regulation. In the second part of this study, we combined WCR of neuronal excitability with pHi measured with pyranine to determine whether oxidative stress stimulates firing rate independently of changes in pHi.

Effects of oxidative stress on pHi. pHi measured with BCECF in slices incubated in control aCSF was 7.40 ± 0.005 (n = 95) and was similar to values previously reported for SC neurons (49). Exposure to oxidative stress significantly decreased pHi in many SC neurons (Fig. 1). In fact, oxidative stress resulted in acidification of 62% (270 of 436) of SC neurons tested. The chemical oxidants CT and NCS decreased pHi by 0.19 ± 0.007 (n = 111) and 0.20 ± 0.015 (n = 30), respectively (Fig. 1, A and D). Likewise, the free radical , produced by DHF, as well as the reactive nonradical H2O2, decreased pHi by 0.15 ± 0.013 (n = 17) and 0.08 ± 0.002 (n = 112), respectively (Fig. 1, B and D). Acidifications induced by 4 and 5 mM H2O2 were not significantly different and were therefore pooled. Note that both and H2O2 are considered ROS. CT- and NCS-induced acidifications were of a larger magnitude than those induced by either DHF (P < 0.05) or H2O2 (P < 0.001), and H2O2 had the smallest effect on pHi (Fig. 1D). Although both forms of oxidative stress (i.e., chemical oxidants and ROS) decreased pHi to a stable plateau rather than causing a continued acidification during oxidative stress, the acidifications induced by CT and NCS were less reversible than those induced by DHF or H2O2 (Fig. 1, compare A and B). This finding suggests that CT and NCS had a stronger effect on pHi than did DHF or H2O2. This possibility is further supported by the finding that a greater proportion of neurons responded with a change in pHi to CT and NCS (80%) than responded to DHF or H2O2 (50%).



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Fig. 1. Exposure to oxidative stress decreased intracellular pH (pHi) in 62% of solitary complex (SC) neurons. A: pHi traces show that exposure (5–15 min) to the chemical oxidants chloramine-T (CT; 500 µM) and N-chlorosuccinimide (NCS; 1 mM) decreased pHi. In some cells, the effects of CT on pHi were fully reversible (i); however, more typically, chemical oxidants induced an acidification that was only partially reversible (ii, CT; NCS). B: pHi traces show that exposure (5–15 min) to superoxide (), produced from dihydroxyfumaric acid (DHF; 3 mM), or hydrogen peroxide (H2O2; 4–5 mM) reversibly decreased pHi. C: pHi traces show that exposure to CT (500 µM) or NCS (1 mM) caused a sustained acidification and that application of DTT (1 mM) either during (not shown) or ~10 min after exposure to CT or NCS had no effect on pHi recovery. D: bar graph showing the magnitude of the acidification ({Delta}pHi) caused by each oxidant used. All {Delta}pHi were significantly different from zero (P < 0.001). *{Delta}pHi differed significantly from H2O2; **{Delta}pHi differed significantly from DHF and H2O2. DTT did not alter the acidification caused by CT or NCS (P > 0.05).

 

To determine whether the effects of CT or NCS are mediated by the differential oxidation of cysteine or methionine, we attempted to reverse the effects of CT- and NCS-induced acidification with the cysteine-specific reducing agent DTT. If DTT were to reverse the effects of CT or NCS, it would suggest that cysteine-specific oxidation is critical for CT- and NCS-induced acidification. Conversely, if DTT did not reverse the effects of these oxidants, it would suggest that methionine oxidation is critical for CT- and NCS-induced acidification. As shown in Fig. 1C, at a 1:1 (n = 20) or 2:1 (n = 11) ratio of oxidant to reducing agent, DTT did not reverse the acidification caused by CT (n = 28) or NCS (n = 3). That is, the change in pHi caused by CT (Fig. 1Aii) or NCS (Fig. 1A) remained significantly different from control during 10-min recovery in control medium ({Delta}pHi = 0.23 ± 0.02, n = 30) and after 10-min exposure to DTT ({Delta}pHi = 0.21 ± 0.02, n = 30) (Fig. 1C). Exposure to DTT alone had no effect on pHi (data not shown). These results suggest that oxidation of the amino acid methionine plays an important role in CT- and NCS-induced acidification.

The remaining 38% (166 of 436) of SC neurons showed no significant change in pHi (i.e., <=0.05 pH unit) during exposure to chemical oxidants or ROS (Fig. 2). The population of insensitive neurons included neurons whose pHi did not respond to NCS (n = 23) (Fig. 2Aa), CT (n = 12) (Fig. 2Ab), DHF (n = 31), and H2O2 (n = 100) (Fig. 2B). The neurons that did not respond with a change in pHi during oxidative stress appeared to be healthy (strong fluorescence ratio) and were qualitatively similar in size and morphology to cells that were acidified by oxidative stress. In addition, these insensitive neurons exhibited a normal pHi response to hypercapnic acidosis (Fig. 2). Together, these results indicate that the levels and exposure times of chemical oxidants and ROS used decrease the pHi of many, but not all, SC neurons.



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Fig. 2. Exposure to oxidative stress did not significantly decrease pHi in 38% of SC neurons. Aa: 15% CO2 caused a typical acidosis. Subsequent exposure to NCS (1 mM) did not significantly affect pHi. Ab: typical response to 15% CO2 followed by no pHi response to CT (500 µM). B: pHi trace of an SC neuron shows that the neuron responded normally to 15% CO2 but did not acidify in response to DHF (3 mM) or H2O2 (4–5 mM).

 

Oxidative stress slows pHi recovery from acidification. To determine whether NHE activity is slowed by oxidative stress, we measured pHi recovery from an NH4Cl-induced acidification under control conditions and in the presence of CT. We have previously shown (48) that SC neurons regulate pHi from acidification by the activity of NHE only. Therefore, the rate of pHi recovery from an intracellular acidification is assumed to be directly proportional to the activity of NHE.

We found that NHE activity was slowed by oxidative stress. In -buffered medium, pHi recovery from an ~0.3 pH unit acidification, to a minimum pHi of 7.11 ± 0.02 (Fig. 3A, first NH4Cl prepulse), occurred at a rate of 0.017 ± 0.002 pH unit/min. pHi recovery of the same neurons to a second ~0.3 pH unit acidification, to a minimum pHi of 6.83 ± 0.12, was slowed 47% by CT (Fig. 3A, second NH4Cl prepulse). Note that in these experiments, NH4Cl-induced acidification during oxidative stress decreased pHi to a more acidic minimum value. Activity of NHE is known to be inversely proportional to pHi; high pHi values inhibit NHE activity, whereas lower pHi values increase NHE activity (43). Therefore, on the basis of the more acidic minimum pHi value, we would expect higher NHE activity in the presence of CT than in its absence. We observed just the opposite, suggesting that CT inhibits NHE and that we are likely underestimating the inhibitory effect of oxidative stress on NHE activity at the lower initial pHi values.



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Fig. 3. Na+/H+ exchange (NHE) activity is slowed by the chemical oxidant CT. A: representative pHi trace of an nucleus tractus solitarii neuron shows that in artificial cerebral spinal fluid (aCSF; 5% CO, 26 mM ) an 8-min NH4Cl (20 mM) pulse decreased pHi by ~0.3 pH units; pHi recovery from this acidification took ~25 min. After pHi recovery, exposure to CT (500 µM) caused a ~0.15 pH unit acidification. In the presence of CT, a second 8-min NH4Cl pulse decreased pHi ~0.3 pH unit, or ~0.45 pH units from control, and pHi recovery from this acidification took ~55 min. Inset: the pHi recovery phase of an NH4Cl pulse in control (con) and during CT was superimposed to better illustrate the inhibitory effect of CT on NHE activity. Note that during oxidative stress, NH4Cl decreased pHi to a more acidic pHi than during control. NHE activity is pHi dependent and thus more active at lower pHi (43). Therefore, in this experiment, we likely underestimated the inhibitory effect of oxidative stress on NHE. B: representative pHi trace shows that in HEPESbuffered medium, a 10-min pulse of NH4Cl followed by 20-min amiloride (amil; 20 mM), an inhibitor of NHE, decreased pHi by ~0.5 pH units. Amiloride was used to obtain a stable minimum pHi to better match minimum pHi in the absence and presence of CT. Upon removal of amiloride, pHi recovered back to its initial control value. Exposure to CT (500 µM) decreased pHi by ~0.1 pH unit. A second pulse of NH4Cl during CT, followed by amiloride, decreased pHi by ~0.35 pH units for a total fall in pHi of ~0.45 pH units. Upon removal of amiloride, pHi recovered back to its initial value except at a slower rate in the presence of CT compared with control, thus indicating that CT slowed NHE activity. Inset: the pHi recovery phase of an NH4Cl pulse in control and during CT was superimposed to better illustrate the inhibitory effect of CT on NHE activity. C: bar graph summarizing the effects of CT on pHi recovery in -buffered medium (n = 20) and HEPES-buffered medium (n = 10). CT decreased the rate of pHi recovery by 47% in -buffered aCSF and by 43% in HEPES-buffered medium. Significant differences (CT vs. control) were determined by t-test (**P < 0.001).

 

To better determine the effects of oxidative stress on NHE, we measured pHi recovery in the presence and absence of CT under conditions of similar acidification and with minimal buffering power (nominal absence of ). In HEPES-buffered medium, recovery from acidification to a similar minimum pHi value (6.83 ± 0.12; n = 10) was slowed by 43% by CT (Fig. 3B). The effects of CT on pHi recovery and NHE activity are summarized in Fig. 3C. These results clearly demonstrate that NHE activity was significantly slowed by the chemical oxidant CT.

Combined effects of hypercapnia and chemical oxidants on pHi. Oxidative stress and hypercapnia both cause a fall of pHi (Figs. 1,2,3), raising the possibility that the effects of hypercapnia and oxidative stress on pHi are additive. Therefore, we wanted to determine whether neurons could respond to hypercapnia during oxidative stress. We found that exposure to 15% CO2 decreased pHi from a control level of 7.40 ± 0.005 by 0.26 ± 0.006 pH units (n = 95) (Fig. 4). Exposure to the chemical oxidants CT or NCS, as previously mentioned, decreased pHi from 7.32 ± 0.01 by 0.19 ± 0.007 pH units (n = 111) or 0.20 ± 0.015 pH units (n = 30) (Fig. 4). In the presence of CT or NCS, exposure to 15% CO2 decreased pHi by 0.26 ± 0.014 pH units (n = 21), which resulted in a total change in pHi of 0.42 ± 0.003 pH unit from control level (Fig. 4, A and B). Therefore, exposure to 15% CO2 plus oxidant caused a significantly greater fall of pHi than exposure to either 15% CO2 or oxidant alone (Fig. 4C), indicating that the effects of oxidative stress and hypercapnia on pHi are additive.



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Fig. 4. Hypercapnia plus oxidative stress decreases pHi by more than either stimulus alone in SC neurons. A: pHi trace shows that 15% CO2 caused a ~0.3 pH unit acidification. Exposure to CT (500 µM) decreased pHi by ~0.25 pH units. During exposure to CT, the addition of hypercapnia decreased pHi by ~0.2 pH units. B: pHi trace shows that hypercapnia caused a ~0.4 pH unit acidification. Exposure to NCS (1 mM) decreased pHi by ~0.25 pH units. During exposure to NCS, the addition of 15% CO2 decreased pHi by ~0.25 pH units. C: bar graph summarizing the effects of hypercapnia, CT or NCS (CT/NCS), and 15% CO2 plus CT/NCS on pHi. Exposure to CO2 and chemical oxidant in combination decreased pHi significantly (**P < 0.001) more than either stimulus alone.

 

Oxidative stress without a change of pHi. Recently, we showed (38) that oxidative stress, including CT, stimulates firing rate of CO2/H+-chemosensitive SC neurons. These same putative CO2/H+ chemoreceptors also respond to hypercapnia via a mechanism that involves decreased pHi (53). We have shown here that CT decreases pHi (Figs. 1, 3, and 4). Together, these results suggest that oxidant-induced acidification could contribute to the activation of CO2/H+-chemosensitive neurons by oxidative stress. Therefore, to determine whether the firing rate response to CT is mediated by direct ion channel oxidation or indirectly via an oxidant-induced acidification, we attempted to separate the oxidant and pHi effects by blocking the acidification caused by CT with a comparable and simultaneous alkalinization. Two approaches were employed. We made a series of pHi measurements using BCECF while increasing concentration or decreasing PCO2 of the aCSF to determine what level of or CO2 causes an ~0.2 pH unit alkalinization. Increasing the concentration of from 26 to 52 mM increased pHi by ~0.2 pH units (Fig. 5Aa) (48). The time course of this alkalinization was similar to the time course of the acidification caused by CT (Figs. 1, 3, and 4); i.e., the initial change in pHi occurred within ~3 min and reached a maximum in ~5 min (Fig. 5Aa). Therefore, we attempted to block CT-induced acidification by exposing SC neurons to CT and 52 mM at the same time. Simultaneous exposure to CT plus 52 mM resulted in no significant change of pHi (0.019 ± 0.012 pH units; n = 17) (Fig. 5, B and D), indicating that we were able to block the CT induced acidification using high .



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Fig. 5. CT-induced acidification can be blocked by increasing or decreasing CO2 levels. A: representative pHi traces show that increasing from 26 to 52 mM (Aa) or decreasing CO2 from 5 to 1.4% (Ab) caused ~0.15–0.2 pH unit alkalinization. B: pHi trace shows that exposure to CT plus high (52 mM) blocked the CT-induced acidification. C: pHi trace shows that exposure to hypocapnia (1.4% CO2) ~3 min into a CT exposure blocked the CT-induced acidification. D: bar graph summarizing the effects of CT (see Fig. 1D) and CT in either high or low CO2. CT-induced acidification measured with BCECF-AM or pyranine was significantly different from zero (**P < 0.001) but not from each other (P > 0.05). Exposure to CT with high or low CO2 did not decrease pHi significantly. WCR, whole cell recording.

 

Alternatively, CT-induced acidification was blocked by keeping constant at 26 mM, and the CO2 level was decreased from 5 to 1.4%. This approach was used so that could be held constant, because a previous study showed that varying the bicarbonate concentration can affect neuronal excitability (5). Lowering CO2 from 5 to 1.4% caused a 0.19 ± 0.024 (n = 10) pH unit alkalinization (Fig. 5Ab). This hypocapnic alkalinization was of a similar magnitude to the acidification caused by CT (Figs. 1, 3, and 4); however, changing CO2 affected pHi much more rapidly than CT did (Fig. 5Ab). Therefore, to minimize any change in pHi with the use of this paradigm, exposure to hypocapnic solution was initiated ~3 min after initiation of exposure to CT. This procedure resulted in no significant CT-induced change in pHi (0.027 ± 0.09 pH units; n = 36) (Fig. 5, C and D). Thus we were able to block CT-induced acidification by using hypocapnic solutions. Furthermore, either or hypocapnic solutions were equally effective at blocking the acidification caused by CT (ANOVA, P > 0.05) (Fig. 5D). These experiments indicate that we are able to expose SC neurons to oxidative stress imposed by CT in the absence of any change in pHi.

Oxidative stress and decreased pHi stimulate SC neurons. The next goal of this study was to measure the effects of hypercapnia and oxidative stress on firing rate and pHi, simultaneously, of SC neurons. To make this determination, WCRs of Vm were made to measure firing rate while pHi was simultaneously measured by using ratiometric fluorescence imaging microscopy with the pH-sensitive dye pyranine. As shown in Fig. 6A, when the cell membrane was ruptured to establish an intracellular recording, pyranine in the pipette filling solution rapidly diffused into the cell. The time constant for loading an SC neuron with pyranine with a WCR pipette was determined by fitting fluorescence intensity over time to the curve

where Flmax is maximum fluorescence intensity obtained at the pH-insensitive wavelength 415 nm, x is time, and k is one-half Flmax. The time required to reach k, defined as the time constant (tk) for pyranine loading, was 214 ± 63 s (n = 5) (Fig. 6B). Therefore, using the whole cell configuration, we were able to rapidly load individual neurons with pyranine. However, diffusion of pipette solution into the cell also causes the dilution or washout of soluble intracellular material (28), including potentially vital components of the CO2/H+-chemosensitive signaling mechanism. For example, previous studies have shown that CO2/H+ chemosensitivity is highly susceptible to washout during WCR (15, 47).



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Fig. 6. Pyranine rapidly diffuses from the recording pipette into the cytoplasm when the cell membrane is ruptured for WCR. A: images of a pyranine-loaded pipette and neuron. In the loose patch configuration, the cell membrane has not been ruptured and no dye has leaked into the cell. When the patch is ruptured for WCR, dye diffuses into the cytoplasm until fluorescence intensity plateaus. B: fluorescence intensity of pyranine in the neuron at the pH-insensitive wavelength (415 nm) (which is proportional to dye concentration) measured after the loose patch cell membrane was ruptured for WCR was plotted vs. time in seconds. The time constant for pyranine loading was 214 ± 63 s (n = 5).

 

CT sensitivity of SC neurons. In the whole cell configuration, exposure to CT increased firing rate on average by 3.13 ± 0.8 impulses/s from an initial firing rate of 0.71 ± 0.38 impulses/s with a corresponding decrease of pHi of ~0.2 pH units in 15 of 18 (83%) SC neurons. Two of three SC neurons that did not respond to CT with a change in firing rate also did not show a change of pHi during exposure to CT (not shown), thus suggesting that oxidant-induced acidification contributes to the effect of CT on neuronal excitability.

To determine whether SC neurons can respond to CT despite washout of CO2/H+ chemosensitivity, we exposed a cell to 15% CO2 while recording single-cell activity extracellularly in the loose patch configuration. The loose patch recording in Fig. 7A shows that exposure to 15% CO2 increased the firing rate of the neuron. After the patch was ruptured for intracellular recording and ~10 min were allowed for pyranine to fully diffuse into the cell (Fig. 6, A and B), a second exposure to 15% CO2 did not increase the firing rate of the neuron. One concern was that this neuron's spontaneous firing rate ceased during WCR because of a slow hyperpolarizing drift, a phenomenon that has been previously observed (22). To assure that this hyperpolarization was not the basis for loss of CO2/H+ chemosensitivity, we injected small subthreshold depolarizing pulses to bring Vm close to spike threshold. Regardless, hypercapnia was still not able to stimulate firing rate. This washout of the firing rate response to hypercapnia cannot be due to clamping of pHi by the whole cell solution because hypercapnia resulted in a large and maintained acidification of the neuron (Fig. 7B). These results indicate that the firing rate response, but not the pHi response, to hypercapnia had been washed out during WCR. In the absence of a functional CO2/H+-chemosensing mechanism, exposure to CT decreased pHi by ~0.13 pH units and increased the firing rate of the same SC neuron (Fig. 7B). These results indicate that chemosensitive SC neurons can respond to CT with an increase in firing rate in the whole cell configuration. In addition, the CT-induced acidification measured by pyranine, which averaged 0.21 ± 0.012 pH units (n = 11), was similar to that measured by BCECF, 0.19 ± 0.007 (n = 111) (Fig. 5D), thus further indicating that pHi is not being clamped during WCR by the HEPES-buffered pipette solution. Because the neuron response to hypercapnia, but not oxidative stress, is washed out, these results support our hypothesis that the oxidant and pHi signaling mechanisms are functionally distinct.



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Fig. 7. Sensitivity of SC neurons to CT does not washout in CO2/H+-chemosensitive SC neurons. A: in the loose patch configuration, traces of integrated firing rate ({int}FR) and Vm show an SC neuron that is stimulated by 15% CO2. B: traces of {int}FR, pHi (measured by pyranine), and voltage trajectories (3 Vm traces superimposed) show that after the cell membrane was ruptured for WCR, spontaneous activity was lost. A depolarizing current pulse (0.3 nA, 50 ms) was used to bring Vm near action potential threshold. In this configuration, exposure to 15% CO2 decreased pHi but did not result in increased firing rate. These results indicate that CO2/H+ chemosensitivity had been lost due to washout during WCR. In contrast, as shown by the traces of {int}FR and Vm, exposure to CT did increase neuronal firing rate.

 

Oxidant stimulation of firing rate occurs independently of changes of pHi. Exposure to CT increased firing rate with a corresponding decrease in pHi (Figs. 7B and 8A). When the CT-induced acidification was blocked with either the or hypocapnic CT exposure paradigms described above, the oxidant still stimulated firing rate in 14 of 16 (88%) SC neurons tested. For example, exposure of an SC neuron to CT in solution increased firing rate without a change of pHi (Fig. 8B). Likewise, exposure of an SC neuron to CT in hypocapnic solution resulted in an increase of firing rate with no change in pHi (Fig. 8C). These results indicate that the oxidative stress caused by CT can stimulate firing rate independently of changes of pHi.



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Fig. 8. CT stimulates firing rate of SC neurons even in the presence of high or low CO2. A: traces of {int}FR and pHi measured simultaneously by WCR show that exposure to CT increases firing rate and decreases pHi. B: traces of {int}FR and pHi show that when the CT-induced acidification was blocked by increasing concentration from 26 to 52 mM, CT still increased FR. C: traces of {int}FR and pHi show that when CT-induced acidification was blocked by decreasing CO2 from 5 to 1.4%, CT still increased FR. These results indicate that SC neurons can respond to CT independently of changes in pHi.

 

Contribution of pHi to the oxidant-induced stimulation of SC neurons. CO2/H+-chemosensitive neurons, like the ones that we are studying, are stimulated by decreased pHi (21, 53). Therefore, we wanted to quantify the contribution of the oxidant-induced acidification to the CT-induced increase in firing rate of SC neurons. To make this determination, we compared the firing rate response of neurons exposed to CT with a corresponding acidosis (e.g., Figs. 7B and 8A) to the firing rate response evoked by CT in the presence of or hypocapnic solutions (e.g., Fig. 8, B and C). As shown in Fig. 9A, all three conditions, CT and CT with either high or low CO2, were able to stimulate firing rate in the same neuron. However, neurons were typically not exposed to multiple bouts of CT (with or without a corresponding acidosis) because the effects of CT on firing rate tended to be poorly reversible and WCRs frequently became unstable after exposure to CT. Therefore, comparisons were made between populations of neurons exposed to CT alone, CT in the presence of high , and CT in hypocapnic solution. The firing rate response to CT and corresponding acidification was not significantly greater than the firing rate response to CT when either 52 mM or 1.4% CO2 was used to block the CT-induced acidification (Fig. 9B). These results suggest that the effects of CT on pHi do not contribute significantly to the response of SC neurons to CT and, thus, that CT must have direct effects on SC neuron excitability independent of changes of pHi.



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Fig. 9. CT-induced acidification is not a major component of the oxidant signaling mechanism. A: trace of {int}FR shows that CT in the presence of high and CT in the presence of a hypocapnic solution have similar effects on the excitability of an SC neuron. The effects of CT were poorly reversible; therefore, before exposure of an SC neuron to CT in hypocapnic solution, Vm was current-clamped to better match FR to the control level (asterisk). B: bar graph summarizing the effects of CT, CT plus high , and CT plus low CO2 on FR of SC neurons. All 3 conditions significantly increased FR (P < 0.05), but there were no differences in FR among the 3 conditions (P > 0.05).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have described for the first time the effects of oxidative stress on pHi and NHE activity of neurons in a region of CO2/H+ chemoreception for the respiratory control system (i.e., the solitary complex). Our results show that oxidative stress decreases pHi in the majority of SC neurons tested and slows NHE activity. We went on to show that oxidation could activate SC neurons in a manner that is independent of changes of pHi. This conclusion is supported by our findings that the chemical oxidant CT can stimulate firing rate in the absence of a change in pHi as well as when CO2/H+ chemosensitivity has been washed out during WCR. These results suggest that the activity of CO2/H+-chemosensitive neurons can be modulated by redox signaling, possibly by the oxidation of an as-yet unidentified K+ channel (38). Our results offer a possible mechanism by which oxidative stress stimulates CO2/H+-chemosensitive neurons (38) and possibly leads to oxidative stress-induced respiratory dysfunction (2, 3, 11, 31, 46).

Oxidant-induced intracellular acidosis. Our results show that the chemical oxidants CT and NCS, produced by DHF, and H2O2 decrease pHi in 62% of SC neurons. There were, however, differences in the magnitude of the acidification caused by each oxidant. CT and NCS caused a larger and more sustained acidification than did DHF or H2O2, whereas H2O2 evoked the smallest change in pHi and constituted the largest proportion of oxidant-insensitive responses. The remaining 38% of SC neurons tested did not show a significant change in pHi during exposure to oxidative stress.

The concentrations of chemical oxidants used in this study were the same as those previously shown to increase firing rate and input resistance of SC neurons, many of which were CO2/H+ chemosensitive (38). The effects of CT and NCS on pHi were remarkably similar to their effects on excitability of SC neurons as measured by sharp electrode recording (38). Just as with changes of pHi, CT and NCS increased firing rate in about two-thirds of SC neurons tested, and their effects were not fully reversible (38). CT and NCS are thought to specifically oxidize cysteine and methionine amino acids (55), hence CT- and NCS-induced acidification likely involves the oxidation of critical methionine and/or cysteine amino acids, of which NHE has several (64). Furthermore, the acidification caused by CT and NCS was not reversed by the cysteine-specific reducing agent DTT, thus further narrowing the oxidant target to methionine. In addition, comparable to the mild effects of DHF and H2O2 on pHi, hyperoxia (~1,600–2,500 Torr), an apparently milder and probably more general form of oxidative stress, increased firing rate in only 38% of SC neurons and in a reversible fashion (38). These results suggested that the effect of oxidative stress on pHi contributes in part to the effects of oxidative stress on neuronal excitability of SC neurons. Therefore, we went on to test this possibility in the second part of this study by measuring Vm with WCR while simultaneously measuring pHi with pyranine.

Previous studies in nonneuronal preparations have shown that oxidative stress has variable effects on pHi. For example, H2O2 caused an intracellular acidosis in rat cerebellar astrocytes (63), C6 glioma cells (63), human promyelocytic leukemia cell line HL60 (27), human aortic endothelial cells (29), OK and BSC-1 renal epithelial cells (33), and rat and human cardiac myocytes (9, 65). Likewise, produced by the tert-butylhydroxyperoxide (t-BHP) decreased pHi in Xenopus oocytes (10). Furthermore, the H2O2-induced acidification of myocytes was dose dependent at concentrations ranging from 30 µM to 3 mM (34). Conversely, oxidative stress has also been reported to increase pHi in certain cell types. For example, oxidative stress imposed by hyperoxia or by or hydroxyl radicals increased pHi of U937 phagocytes (56), hepatic stellate cells (60), and canine kidney epithelial cells (17).

Together, these observations indicate that oxidative stress can affect pHi. However, similar oxidants have opposite effects on different cell types, whereas similar cell types (e.g., renal epithelial cells) responded differently to different oxidants. These disparate findings suggest that the effects of oxidative stress on pHi depend on both the cell type and the oxidant used. These observations also suggest that the effects of oxidative stress on pHi involve different mechanisms and that different cells likely have different targets of oxidative stress. Therefore, our results provide important insight into the effects of oxidative stress on the central nervous system (CNS). Whether oxidative stress has similar effects on neurons from different regions, including nonchemosensitive areas of the brain stem or cortical regions known to be sensitive to oxidative stress (e.g., substantia nigra), remains to be determined.

Mechanism of oxidant-induced intracellular acidosis. Our results show that oxidant-induced acidification of SC neurons occurred in conjunction with a decrease in amiloride-sensitive NHE activity. This observation is consistent with previous studies conducted in nonneuronal preparations that reported NHE activity is affected by oxidative stress. For example, H2O2, hyperoxia, and the chemical oxidant t-BHP decreased NHE activity and acidified arterial endothelial cells (12, 13, 29), atrial myocardial cells (9), and renal epithelial cells (33). Furthermore, in those preparations where oxidative stress has the opposite effect on pHi, it also had the opposite effect on NHE activity; i.e., ROS produced by hyperoxia or ferric nitrilotriacetate increased NHE activity and alkalinized kidney epithelial cells (17) and hepatic stellate cells (60). These results suggest that the effects of oxidative stress on NHE activity contribute to their effects on pHi. However, Chambers-Kersh et al. (6) showed that inhibition of NHE under control conditions with amiloride did not acidify neurons in the nucleus tractus solitarius (i.e., dorsal SC). Therefore, other factors in addition to decreased NHE activity likely contribute to the oxidant-induced acidification observed in this study.

Possible factors in addition to NHE inhibition that may contribute to the acidification caused by oxidative stress include 1) metabolic acidosis (8); 2) ATP hydrolysis (63); 3) H+ redistribution (33, 54); 4) decreased pH buffer capacity (29, 33); or 5) with regard to CTand NCS-induced acidification, the formation of HCl as a by-product of CTor NCS-mediated protein oxidation. Regarding the first possibility, there is evidence that oxidative stress disrupts components of the electron transport chain (8), thereby slowing or halting oxidative phosphorylation and resulting in increased lactic acid production. In support of this possibility, oxidative stress has been shown to increase lactate production in the CNS (7), and hyperoxia has been shown to decrease metabolism in brain slices (36). The buffering power of cells in this region is high at 45 mM/pH unit (6). Therefore, to change pHi by 0.20 pH units would require ~9 mM lactic acid (1 H+ per lactic acid) to be produced during oxidative stress. This value is similar to the transient increases in lactate levels reported to occur during focal brain activation, ~6 mM (24). Regarding the second possibility, H2O2 has been shown to increase ATP hydrolysis, which caused a pronounced decreased pHi (63). However, for ATP breakdown to decrease pHi by 0.20 pH units would require the hydrolysis of ~9 mM ATP (1 H+ per ATP). This value exceeds total intracellular ATP levels in the brain, which range from ~4.6 to 6.4 mM (4, 58). These first two possibilities would both lead to depleted levels of intracellular ATP, which itself can cause a third possibility, i.e., redistribution of H+ from acidified organelles (e.g., lysosomes) to the cytoplasm by inhibition of the H+ATPase (33). It is also important to note that ATP depletion can also decrease sensitivity of NHE to intracellular H+ (16) and could be part of the mechanism by which oxidative stress slows NHE activity. Evaluation of the possible contribution of these mechanisms must await measurements of lactate, phosphocreatine, ATP, or other metabolic by-products during oxidative stress in SC neurons.

Regarding the fourth possibility, previous studies showed that concentrations of H2O2 that decreased pHi and inhibited NHE activity did not affect pH buffer capacity (29, 33, 54). These results suggest that oxidative stress does not affect pHi buffer capacity. Regarding the final possibility, although it is theoretically possible that CT and NCS produce acid (e.g., HCl) as a by-product of protein oxidation, we are unable to find any experimental evidence to support this possibility. These possibilities are not mutually exclusive but, rather, likely occur in concert such that the effect of oxidative stress on pHi involves a combination of these effects.

Effects of CT on neuronal excitability. The SC contains CO2/H+-chemosensitive neurons that respond to changes in CO2 by a mechanism that involves decreased pHi (53). Recently, it was shown that CO2/H+ chemoreceptors are highly sensitive to hyperoxia by a mechanism that appears to involve ROS (38) and the redox signaling pathway (18). We have shown here that chemical oxidants and ROS decrease pHi. Together, these results beg the question, does oxidative stress stimulate SC neurons directly by redox modulation of ion channels or indirectly by decreased pHi? To test this possibility, we made WCR of Vm while measuring pHi in SC neurons during exposure to CT.

Use of WCR technique to study oxidant sensitivity of SC neurons. The main advantage of measuring Vm by using the whole cell configuration over other patch-clamp recording methods is that pharmacological agents or fluorescent dyes in the pipette can be rapidly introduced into the cell (28). For example, in this study, using the whole cell configuration, we were able to rapidly load SC neurons with the membraneimpermeable fluorescent dye pyranine to perform simultaneous measures of Vm and pHi. However, this advantage also results in the major disadvantage of the WCR technique, that being washout. The rapid exchange between the pipette solution and cytoplasmic material results in dilution or washout of soluble intracellular molecules (28), including important components of the CO2/H+-chemosensitive signaling mechanism (15, 22, 47). To some extent we were able to ameliorate the effects of washout on chemosensitivity by lowering EGTA-Ca2+ concentration in the whole cell filling solution as described previously (22). However, low-EGTA-Ca2+ whole cell solution did not prevent washout in all cells (e.g., Fig. 7). Therefore, low-EGTA-Ca2+ whole cell solution appears to lessen but not block the effects of washout on CO2/H+ chemosensitivity in SC neurons.

Oxidative stress stimulates SC neurons independently of changes of pHi. CT in the presence of either high or low CO2 stimulated firing rate in 83% of SC neurons. Furthermore, there was no difference in the firing rate response evoked by CT in either high or low CO2, indicating that SC neurons are responding to CT and not high , as previously shown to occur in hippocampal neurons (5). Our finding that CT stimulates firing rate in the absence of a change in pHi supports the possibility that oxidative stress stimulates SC neurons by a direct mechanism, possibly involving the redox modulation of cysteine and/or methionine on an as-yet unidentified K+ channel.

There is increasing evidence that redox modulation of ion channels is an important signaling mechanism for controlling neuronal excitability (18). Our results are consistent with previous studies that showed oxidative stress to decrease K+ channel conductance. For example, CT or ROS decreased conductance of delayed rectifier (19, 59) and A-type K+ channels (1) and increased or decreased Ca2+-dependent K+ channel conductance, depending on whether a cysteine or methionine was oxidized (61). Together, these results support the possibility that transmembrane proteins such as NHE or K+ channels can be redox modulated in ways that affect neuronal activity.

Contribution of pHi to the CT response. Oxidative stress and CO2 both increase activity of SC neurons, by increased ROS production (38) and decreased pHi (21, 53), respectively. Our results show that the effects of CT or NCS and CO2 on pHi are additive. Furthermore, we recently showed that the firing rate response to CO2 plus hyperoxia was equal to or greater than the sum change in firing rate evoked by each stimulus alone (38). Therefore, we hypothesized that the CT-induced acidification would contribute to the CT response of SC neurons. Our results did show that CT increased firing rate somewhat more than CT in the presence of high or low CO2, but this difference was not significant. However, this result should be interpreted with caution because our sample size was relatively small. We anticipate that with a larger sample size the difference between CT and CT in high or low CO2 might become significant. Regardless, our results indicate that the CT-induced acidification is not required for the oxidant-induced firing rate response of SC neurons.

It is unclear why the CT-induced acidification does not appear to result in further significant stimulation of the firing rate of SC neurons. It is possible that factors in addition to decreased pHi are required for neuronal stimulation. For example, Filosa and Putnam (22) showed that propionate decreased pHi by ~0.2 pH unit but did not increase firing rate of locus coeruleus neurons. This possibility is further supported by a phenomenon known as the hypoxia paradox; i.e., hypoxiainduced intracellular acidification does not increase activity of CO2/H+ chemoreceptors to stimulate ventilation (41). These results suggest that decreased pHi by itself does not necessarily stimulate activity of CO2/H+ chemoreceptors. However, more experiments are required to confirm this possibility.

Model of CO2/H+ - and oxidative stress-induced signaling pathways in SC neurons. CO2 provides the primary stimulus for breathing, in part, by stimulating CO2/H+-chemosensitive neurons located in various brain stem regions (53). CO2/H+-chemosensitive neurons in the SC are also sensitive to various forms of oxidative stress, including hyperoxia and chemical oxidants (38). Figure 10 presents our current working model summarizing the effects of CO2 and oxidative stress on CO2/H+-chemosensitive neurons. The effect of CO2 and oxidative stress on the excitability of SC neurons appears to be mediated by separate mechanisms; CO2 involves changes in pHi (21), and oxidative stress in the form of CT, NCS, or hyperoxia involves oxidation (38). We have shown here that oxidative stress can decrease pHi and NHE activity; therefore, decreases in pHi (Fig. 10, purple box) are an area of intersection between the two signaling mechanisms. The importance of the oxidantinduced acidification and the potential for an interaction between CO2 and oxidative stress signaling mechanisms remain to be determined.



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Fig. 10. Working model of the pathways by which SC neurons respond to oxidative stress and hypercapnia. Exposure to hypercapnia decreases extracellular pH (pHo; arrow 1) and decreases NHE activity (48), which results in decreased pHi. Hydration of CO2 and dissociation of carbonic acid also directly decreases pHi (arrow 2). It was shown previously that decreased pHi increases firing rate of CO2/H+-chemosensitive neurons (arrow 3) (21) and alters ventilation (reviewed in Refs. 41 and 53), possibly by decreased K+ conductance (22). In addition, we previously showed that oxidative stress in the form of hyperoxia or CT and NCS increases firing rate of CO2/H+-chemosensitive SC neurons, possibly by an oxidant-induced decrease in K+ conductance (38). In this study, we have shown that CT and NCS decrease NHE activity (arrow 4) and pHi (arrow 5). Likewise, ROS produced by DHF or H2O2 (arrow 6) also decrease pHi (arrow 7). However, it is not known whether the amount of ROS produced by hyperoxia (arrow 8) is sufficient to decrease pHi (arrow 9). Furthermore, CT was able to stimulate firing rate independently of changes of pHi, presumably by the redox modulation of an unidentified K+ channel (arrow 10). It remains to be determined whether ROS can also stimulate excitability by direct oxidation of a K+ channel (arrow 11) or whether oxidant-induced acidification can stimulate firing rate (arrow 12). Finally, it remains to be determined whether decreased pHi itself can lead to altered ROS production in SC neurons (arrow 13).

 

Our results suggest that oxidant-induced acidification is not required for the oxidant signaling mechanism. Chemosensitive neurons are sensitive to hypercapnic acidosis (30, 53). It is still conceivable, however, that the oxidant-induced acidification contributes to the effects of oxidative stress on SC neurons, if not through the CO2/H+ signaling mechanism, then possibly by amplifying the oxidant signal. There are several ways in which intracellular acidosis might increase oxidative stress (Fig. 10, arrow 13). These possibilities include 1) acidification-induced dissociation of iron from transferrin (26) and possibly ferritin (44) to facilitate iron catalysis of and H2O2 to the very reactive OH radical (26); 2) acidification-induced increases of the formation of reactive nitrogen species (32); 3) acidification-induced alteration of redox reactions, many of which are pH dependent (52); and 4) acidification-induced decreases in enzymatic antioxidant activity (66). These possibilities are supported by previous studies that have demonstrated that production and/or stability of ROS including , H2O2, OH radical, and/or peroxynitrite are increased at more acidic conditions (pH ranging from 7.0 to 6.1) (35, 45, 57, 66). In addition, decreasing bath pH from 7.2 to 6.2 decreased activity of the enzymatic antioxidants glutathione peroxidase, glutathione S-transferase, and glutathione reductase (66). These findings suggest that the acidification caused by oxidative stress may amplify the oxidant signal by promoting formation of ROS, reactive nitrogen species, or reduction of endogenous antioxidants.

Physiological significance. Increased ventilation in response to high CO2 levels will facilitate CO2 removal in the exhaled breath, thereby maintaining acid/base homeostasis. Paradoxically, oxidative stress in the form of hyperoxia also causes hyperventilation (2, 3, 11). Breathing more during hyperoxia will only further increase oxidative stress and cause a loss of respiratory control (38). Therefore, breathing more during oxidative stress is not thought to have any adaptive value but, rather, may be an early sign of oxygen toxicity resulting from the disruption of CO2/H+ chemoreceptors of the respiratory control system. The mechanism by which oxidative stress affects respiratory control is unknown. Hyperoxia has been shown to destabilize breathing in some infants presenting with recurrent apnea and cyanosis (3). Furthermore, chronic oxidative stress in the form of high iron or alterations in CNS antioxidant levels are thought to contribute to sudden infant death syndrome (31, 46). Our observations that oxidative stress decreases pHi and NHE activity and increases excitability of neurons in a respiratory control brain stem region suggests a possible mechanism by which oxidative stress affects and possibly disrupts respiratory control.


    ACKNOWLEDGMENTS
 
We thank Phyllis Douglas and Raghu Vongole for technical assistance.

GRANTS

This research was supported by National Heart, Lung, and Blood Institute Grant R01-HL-56683, Office of Naval Research Grant N000140110179, and Department of Defense/Office of Naval Research-Defense University Research Instrumentation Program Grant N000140210643.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. B. Dean, Dept. of Anatomy and Physiology, Rm. 235C Bio. Sci Bldg., 3640 Col. Glenn Hwy., Wright State Univ., Dayton, OH 45435 (E-mail: jay.dean{at}wright.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.


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