Systemic hypoxia in mammals is sensed and transduced by the carotid body into increased action potential (AP) frequency on the sinus nerve, resulting in increased ventilation. The mechanism of hypoxia transduction is not resolved, but previous work suggested that fast Na+ channels play an important role in determining the rate and timing of APs (Donnelly, DF, Panisello JM, and Boggs D. J Physiol. 511: 301–311, 1998). We speculated that Na+ channel activity between APs, termed persistent Na+ current (INaP), is responsible for AP generation that and riluzole and phenytoin, which inhibit this current, would impair organ function. Using whole cell patch clamp recording of intact petrosal neurons with projections to the carotid body, we demonstrated that INaP is present in chemoreceptor afferent neurons and is inhibited by riluzole. Furthermore, discharge frequencies of single-unit, chemoreceptor activity, in vitro, during normoxia (Po2 150 Torr) and during acute hypoxia (Po2 90 Torr) were significantly reduced by riluzole concentrations at or above 5 μM, and by phenytoin at 100 μM, without significant affect on nerve conduction time, AP magnitude (inferred from extracellular field), and AP duration. The effect of both drugs appeared solely postsynaptic because hypoxia-induced catecholamine release in the carotid body was not altered by either drug. The respiratory response of unanesthetized, unrestrained 2-wk-old rats to acute hypoxia (12% inspired O2 fraction), which was measured with whole body plethysmography, was significantly reduced after treatment with riluzole (2 mg/kg ip) and phenytoin (20 mg/kg ip). We conclude that INaP is present in chemoreceptor afferent neurons and serves an important role in peripheral chemoreceptor function and, hence, in the ventilatory response to hypoxia.
- hypoxic ventilatory response
- petrosal neuron
hypoxia initiates a prompt increase in ventilation due to stimulation of carotid body chemoreceptors and enhanced action potential (AP) frequency on sinus nerve afferent fibers that terminate in the brain stem (14). The mechanism by which hypoxia leads to increased AP frequency is not well resolved. It is generally accepted that hypoxia is transduced by the glomus cell, a secretory cell that is presynaptic to nerve endings and that responds to acute hypoxia with an increase in intracellular Ca2+ (5, 9) and enhanced secretion of dense-cored storage granules (30). This is speculated to give rise to depolarization events in the nerve terminals, which lead to the generation of afferent APs (34).
However, recent work in our laboratory suggested that the spike generation is a more complicated process. Reductions in excitability produced by isosmotic reductions in extracellular Na+ concentration and low doses of tetrodotoxin (TTX; a blocker of some isoforms of the fast Na+ channel) caused a large decrease in spontaneous discharge frequency but comparatively small changes in nerve conduction velocity or nerve terminal excitability (25). Because rat glomus cells lack fast Na+ channels (31), the experimental result suggested that Na+ channel activity in the nerve terminals was a significant determinant of when APs were initiated. Because this contribution must occur near the resting membrane potential and be relatively constant over time, it suggested a persistent Na+ conductance. In other model systems, this Na+ influx can be due to episodic, low-probability transitions of the fast Na+ channel from the inactive state to the open state and can be reduced by drugs that stabilize the inactive state such as riluzole and phenytoin (41, 62). Accordingly, in this study, we demonstrate the presence of a riluzole-sensitive, TTX-sensitive inward current in intact petrosal chemoreceptor neurons, and we demonstrate an impaired ability to respond to acute hypoxia in terms of discharge activity of isolated peripheral chemoreceptors and ventilatory response. These results may have important implications for patients treated with therapeutic agents targeting Na+ channels, as well as our understanding of the mechanism of chemoreceptor transduction.
The experiments were performed on 2-wk-old Sprague-Dawley rats of both sexes obtained from a commercial breeder. Rats were housed in an animal room with conventional light-dark cycle. Food and water were available ad libitum. The experimental protocols were approved by the Institutional Animal Care and Use Committee.
Whole cell patch-clamp recordings were obtained from cells on the surface of the petrosal ganglia of an in vitro peripheral chemoreceptor complex described below (Fig. 1A). To identify chemoreceptor cells and place them at the surface, an extracellular recording electrode was used to initially record the surface cells and remove them using suction if they did not project to the carotid body. Once candidate petrosal neurons were identified, the extracellular pipette was withdrawn, and a conventional whole cell configuration was obtained with pipette filled with an intracellular solution designed to minimize K+ currents (in mM: 130 CsF, 10 tetraethylammonium chloride, 10 HEPES, 5 EGTA, 1 CaCl2, and 1 Mg-ATP adjusted to pH 7.2). Whole cell patch-clamp recordings were conducted at 36–37°C using pCLAMP 9 (Axon Instruments, Foster City, CA). Electrodes were fabricated from 1.2-mm Drummond capillary glass using a Sutter P-97 puller (Sutter Instruments, Novato, CA), fire polished, and used without any coatings. In voltage-clamp mode, spontaneous APs, presumably originating at the nerve endings in the carotid body, were observed as sharp negative-going current spikes due to the current sink in the initial segment of the axon, and they responded rapidly to changes in chamber oxygen level (Fig. 1, B and C).
Experimental protocol for patch clamp recording.
Persistent Na+ current (INaP) was evoked using a ramp protocol: a hyperpolarization to −120 mV followed by a slow ramp (26 mV/s) to +20 mV. To identify the ionic current's contribution to the ramp current, the ramp protocol was repeated before and after application of TTX (Alomone Labs, Jerusalem, Israel) or riluzole (Sigma-Aldrich, St. Louis, MO). To evoke the transient Na+ current, cells were hyperpolarized to −120 mV for 500 ms followed by a step depolarization over the range of −80 to +20 mV in 5-mV increments.
Single-unit chemoreceptor recording.
Spiking activity of rat chemoreceptors, in vitro, was recorded from the soma of petrosal neurons with projections to the carotid body (26) (Fig. 1A). Rat pups were euthanized using 100% CO2 and decapitated. The peripheral chemoreceptor complex consisting of the carotid body, sinus nerve, part of the glossopharyngeal nerve, and the petrosal ganglion was harvested in toto. The complex was exposed to a dilute mixture of collagenase (Boehringer type P, 1 mg/ml; Boehringer-Mannheim, Mannheim, Germany) and protease (Sigma type IX, 0.2 mg/ml; Sigma-Aldrich) for 30 min at 36°C to aid in the removal of surrounding connective tissue. The complex was transferred to a recording chamber (model RC21C, Warner Instruments, Hamden, CT) mounted on the stage of an inverted microscope and superfused with Ringer solution (in mM: 120 NaCl, 3 KCl, 2 CaCl2, 1 Na2HPO4, 24 NaHCO3, and 10 glucose) oxygenated with 21% O2-5% CO2-balance N2 gas mixture at a rate of 3 ml/min. The temperature in the recording chamber was kept at 36–37°C by an in-line heater (model TC-344B, Warner Instruments).
Single-unit activity was recorded using a glass suction electrode advanced into the petrosal ganglion (Fig. 1A). To facilitate unit identification and for measurement of nerve conduction time, a glass pipette filled with 1 N NaCl (1-MΩ impedance) was placed in the center of the carotid body, and a cathodal electrical stimulus (100 μA, 0.1-ms duration, 1/s) was delivered through the pipette to evoke orthodromic APs (Fig. 1, B and C). Once an evoked potential was detected, stimulus was removed and observed for spontaneous activity. Discharge activity was continuously acquired, and then it was digitized using Digidata 1200 (Axon Instruments) at a sampling rate of 10 kHz. Traces were recorded with Axoscope (Axon Instruments).
Experimental protocol for chemoreceptor recordings.
At the start of each recording period, several orthodromically evoked APs were recorded for measurement of spike conduction time and AP properties (Fig. 1, B and C). Discharge activity during normoxia (Po2 ≈ 150 Torr) was recorded for 5 min. Response to hypoxia was then tested by superfusing the preparation in Ringer solution equilibrated with 12% O2-5% CO2-balance N2 gas for 5 min, resulting in a chamber Po2 ≈ 90 Torr, followed by a return to normoxia.
To determine the effect of riluzole, phenytoin, or vehicle (dimethylsulfoxide; DMSO) on the discharge activity, riluzole, phenytoin (Sigma-Aldrich), or vehicle (DMSO; Sigma-Aldrich) was added in different concentrations to the normoxia and hypoxia reservoirs. After addition of drug or vehicle, a 15-min equilibration time was allowed followed by a hypoxia stimulus period of 5 min. The procedure was repeated with increasing doses of riluzole (2, 5, 10, and 20 μM), phenytoin (100 μM), or equivalent vehicle concentrations. The doses used were based on previous studies (19, 60, 65).
The amount of catecholamine secreted from the carotid body was measured using the same peripheral chemoreceptor complex described above (Fig. 1A). After preparation of the complex, the carotid body was cut from the sinus nerve, transferred to the recording chamber mounted on the stage of an inverted microscope, and then perfused with Ringer solution. A catecholamine electrode was then advanced into the center of the carotid body for measurement. Prefabricated catecholamine electrodes (Axon Instruments) were used in the experiment. The electrodes contained a single 5-μm carbon fiber, which was dip coated with Nafion (Sigma-Aldrich), an ion-exchange resin that discriminates in favor of positively charged ions (23). Carbon-fiber electrode current was monitored in the amperometric mode at +200 mV referenced to an Ag-AgCl-indifferent electrode, which is slightly above the dopamine oxidation (23). Current changes were acquired then digitized using Digidata 1200 (Axon Instruments) at a sampling rate of 10 kHz. Traces were recorded with Axoscope (Axon Instruments).
Experimental protocol for recording of catecholamine secretion.
At the start of each run, the electrode was visually advanced into the carotid body and allowed to settle until the recording was back to baseline. The carotid body was subsequently superfused with Ringer solution equilibrated with 21% O2-5% CO2-balance N2 gas for ∼30 s followed by a hypoxic solution containing 0% O2 glucose oxidase. Hypoxia was continued for 2 min, at which time peak secretion has been achieved (Fig. 1D). The carotid body was then allowed to recover in normoxic Ringer solution. To determine the effect of riluzole and phenytoin on carotid body secretion, the preparation was exposed to Ringer solution containing riluzole, phenytoin, or similar amount of vehicle for 15 min. The hypoxic challenge was repeated after the incubation period.
Breathing was measured in unanesthetized, unrestrained rats using whole body plethysmography. Rat pups were placed in a 0.5-liter cylindrical Plexiglas chamber (Buxco Electronics, Troy, NY) with a bias flow of 1 l/min. Air or test gases entered the chamber though one port at a rate of 1.5 l/min. Pressure changes within the chamber were measured with the accompanying transducer (model TRD5700, Buxco Electronics). Chamber temperature and relative humidity were measured using a flow-through probe (Humidity and temperature voltage output system model HTM 2500, Humirel, Phoenix, AZ) while carbon dioxide tension in the chamber was analyzed via Capstar-100 carbon dioxide analyzer (CWE, Ardmore, PA). Calibrations of all instruments were done daily before the start of the experiment. Data were continuously acquired at a sampling rate of 200 Hz, and they were digitized using Digidata 1320A and Axoscope acquisition program (Axon Instruments). The ventilation waveform was corrected for the high-pass-filtering characteristics of the open or leaky chamber, and the amplitude of the pressure signal during inspiration was used for calculation of tidal volume according to the method of Drorbaugh and Fenn (27).
Experimental protocol for recording respiration.
On the morning of the day of study, animals were weighed and randomly assigned to treatment or control groups. Rats in the former group were administered riluzole dissolved in DMSO (2 mg/kg ip). Although circulating drug concentrations were not measured, we estimated the level to be ∼5 μM. In previous studies in adult humans (33, 43, 44), intravenous infusion of 50 mg (∼1 mg/kg) of riluzole produced a maximum serum concentration of 2.5 μM. In the absence of any significant interspecies variability in the volume of distribution and clearance of the drug, 2 mg/kg of riluzole given intraperitoneally is expected to result to a serum concentration of 5 μM, which is within the range of the in vitro doses used. Based on previous dose-ranging experiments done in our laboratory, phenytoin (administered at 20 mg/kg ip) results in a circulating concentration of ∼100 μM, which is the in vitro dose used. The in vivo dosages are within the therapeutic ranges for humans (33, 43, 68) and were previously used for animal studies (49, 55). Control groups received similar volumes of the drug vehicle, DMSO (8 ml/kg).
After drug administration, the rat was allowed to acclimate to the plethysmograph for ∼20 min or until the rat ceased exploratory behavior. A 5-min recording was taken during room air breathing followed by 10 min of hypoxia (12% O2). Based on the flow rate and chamber size, steady-state inspired O2 fraction levels were achieved in ∼40 s (2 time constants). After the hypoxia period, rats were allowed to recover in room air for 5 min and were then challenged with hypercapnia, 5% CO2 in air, for 10 min.
Current traces evoked by the ramp protocol were leak subtracted, and INaP was quantified as the difference between the electrode current at −40 mV and −80 mV. The effect of application of a drug that may alter INaP was measured by subtracting the raw current trace before and in the presence of the drug or agent and measuring the current difference between −80 mV and −40 mV.
Extracellular AP occurrence times were detected and recorded using FETCHAN 6 (Axon Instruments), which also recorded the peak (extracellular) voltage excursion during an AP. Single-unit recording was confirmed by the unimodal histogram of AP amplitude events. AP occurrence times were converted to frequency (Hz) and plotted using Origin 6.0 (Microcal Software, Northampton, MA). The average frequencies of the discharge activity over 1 min during normoxia and peak of hypoxia were obtained and used for comparison (Fig. 1B). Nerve conduction time was measured on orthodromically evoked spikes after an electrical stimulus delivered to an electrode placed in the carotid body, whereas the AP amplitude and duration were recorded from spontaneous APs during normoxia (Fig. 1C). The conduction time was based on the time lapse from the stimulus artifact to arrival of the somal AP. All were calculated as an average of five trials. Values were expressed at means ± SE. Spike frequencies between vehicle- and riluzole-treated groups were compared using Student's t-test, whereas AP conduction time, amplitude, and duration were compared using one-way ANOVA with Bonferroni-corrected, t-tests for post hoc analysis. A level of 0.05 was considered statistically significant.
Tissue catecholamine concentration was calculated based on the carbon-fiber electrode current (Fig. 1D). Postdrug treatment levels were expressed as percentage of predrug treatment values compared with control for varying initial peak secretion measurements. Values were expressed as means ± SE. Normalized secretion values after vehicle and riluzole administration were compared with Student's t-test. A level of 0.05 was considered significant.
Ventilatory pressure traces were analyzed using Minianalysis (Synaptosoft, Decatur, GA), which was used to detect the start and peak of the respiratory waveforms (see Fig. 3, A–F). Ten sequential breaths were quantified during room air breathing and at the start of each minute of the hypoxia or hypercapnia period. The pressure signal was corrected for the filtering characteristic of the chamber and used to calculate tidal volume based on the equation of Drorbaugh and Fenn (27). The interval between breaths was converted to respiratory rates. Minute ventilation values during room air breathing and every minute during exposure to test gases were obtained from the product of the tidal volume and respiratory rate. Values were expressed at means ± SE. Statistical comparison of vehicle-treated vs. riluzole-treated rats at the start and end of hypoxia, and at the middle and end of hypercapnia, was done using Student's t-test. A level of 0.05 was considered statistically significant.
Effect of riluzole on INaP expressed in petrosal chemoreceptor neurons.
Persistent currents were evoked by a slow depolarization ramp from an initial holding potential of −100 mV to +20 mV at a rate of 26 mV/s. In all cells tested (n = 25), the electrode current deviated from that expected from an ohmic load by demonstrating a negative resistance region near the expected resting potential of −60 mV (Fig. 2, A and C). The magnitude of the inward current was quantified as the holding current difference between −80 and −40 mV, and this averaged −186 ± 22 pA (n = 25). The inward current was likely due to Na+ influx through voltage-activated Na+ channels because it was fully antagonized by TTX (Fig. 2A). TTX also antagonized the transient Na+ current evoked by a depolarization step from a prepulse of −120 mV (500-ms duration) to −10 mV (Fig. 2B).
In initial experiments, riluzole was employed at a concentration of 5 μM, but it proved difficult to maintain constant recording conditions over the 15-min wash-in period. Cell input resistance often decreased over this period, and there was a slow drift toward negative potentials for activation of the fast Na+ current, which is likely due to washout of slowly diffusible anions. Thus riluzole was used at a higher concentration (10 μM) but a shorter duration of wash in (2 min). Riluzole at this concentration was, similarly, effective in reducing the INaP (Fig. 2C). The average decrease in magnitude of INaP before and after application of 10 μM riluzole applied for 2 min was −41 ± 8 pA (n = 9), representing a reduction of 78.0%. Unlike TTX, riluzole had no significant effect on the peak Na+ current evoked by a step depolarization from −120 mV (Fig. 2D).
Effect of riluzole and phenytoin on chemoreceptor function.
Chemoreceptor AP activity was recorded from the extracellular field developed around the soma in isolated rat chemoreceptors (n = 10 vehicle treatment; n = 14 riluzole treatment; n = 10 phenytoin treatment) (Fig. 1A). As expected, hypoxia (12% O2) increased the spontaneous AP rate from the recorded fiber (Figs. 1B and 3A). Above a threshold of 5 μM, riluzole caused a significant decrease in normoxia spiking activity compared with the vehicle-treated group (Fig. 3, B and D). Spiking frequencies were 0.2 ± 0.04 and 1.5 ± 0.5 Hz, respectively (P < 0.01), during administration of 5 μM riluzole or its equivalent vehicle concentration (Fig. 3D). A more pronounced inhibition of spontaneous spiking activity was observed at 10 and 20 μM riluzole concentrations (Fig. 3D), and, in some cases, spontaneous spiking activity ceased but returned to spontaneous activity during drug washout (not shown). Under hypoxia conditions (12% O2), vehicle-treated units discharged at 5.4 ± 1.0 Hz, whereas in units treated with 5 μM riluzole, peak discharge frequency during hypoxia was 0.8 ± 0.4 Hz (P < 0.01) (Fig. 3E). Similar to normoxia, the degree of depression in spiking activity in hypoxia was greater at higher drug concentrations (Fig. 3E). Phenytoin at 100 μM elicited a similar pattern of response (Fig. 3C). AP frequency (n = 10 phenytoin treatment; n = 10 vehicle treatment) during normoxia (phenytoin: 0.1 ± 0.03 Hz, vehicle: 0.9 ± 0.2 Hz; P < 0.01) and hypoxia (phenytoin: 2.4 ± 1.1 Hz, vehicle: 5.6 ± 1.4 Hz; P < 0.01) was significantly lower in the phenytoin-treated group compared with vehicle (Fig. 3, D and E).
Other AP characteristics were not altered with riluzole at 5 μM or less. At these doses, riluzole had no significant effect on the AP conduction time or AP amplitude as judged from the extracellular field (Fig. 3, F and G). At 10 μM riluzole, conduction time increased by 134.6 ± 8.6% of baseline (P < 0.01) (Fig. 3F). Riluzole below 10 μM had no significant effect on (inferred) spike amplitude, but at 10 μM it caused a decrease to 61.2 ± 15.1% of initial spike amplitude (P = 0.13; Fig. 3G). AP duration, which indirectly measures the effect of the drug on K+ channels (57), was not significantly changed by riluzole at any of the concentrations used (P = 0.14; Fig. 3H). Treatment with vehicle had no significant effect on conduction time (P = 0.59), spike amplitude (P = 0.31), or duration (P = 0.24) at all concentrations. AP conduction time, amplitude, and duration with phenytoin treatment were statistically similar to pretreatment values (Fig. 3, F–H). AP conduction, amplitude, and duration were 98.7 ± 7.2% (P = 0.44), 98.1 ± 7.7% (P = 0.41), and 104.0 ± 4.5% (P = 0.21) of baseline, respectively.
Effect of riluzole and phenytoin on catecholamine release.
Hypoxia-induced (0% O2 with glucose oxidase for 2 min) catecholamine secretion was used as a gauge of glomus cell function, and the magnitude of release after vehicle or drug treatment was normalized to the magnitude of release before treatment (Fig. 1D). Neither vehicle treatment (111.6 ± 19.4%, n = 7) nor riluzole treatment (5 μM, 111.8 ± 12.6%, n = 14) produced a significant change in the magnitude of hypoxia-induced catecholamine release (P = 0.50; Fig. 3I). Similarly, catecholamine secretion was not significantly altered by vehicle and phenytoin treatment (111.6 ± 19.4%, n = 7 vs. 107.4 ± 14.26%, n = 5; P = 0.57; Fig. 3I).
Effect of riluzole and phenytoin on hypoxic and hypercapnic ventilatory responses.
Minute ventilation was measured in 19 unanesthetized, unrestrained rats (n = 7 vehicle treatment; n = 7 riluzole treatment; n = 5 phenytoin treatment). Riluzole (2 mg/kg) and phenytoin (20 mg/kg) significantly decreased baseline ventilation with a concomitant decrease in metabolic rate and an obvious decrease in motor activity. Rate of CO2 production in vehicle treated rats was 6.1 ± 1.2 ml·min−1·100 g−1 and 3.7 ± 0.8 ml·min−1·100 g−1 after riluzole treatment (P = 0.06) and 3.6 ± 1.2 ml·min−1·100 g−1 after phenytoin treatment (Fig. 4, A, C, E, and G).
In the vehicle-treated group, ventilation promptly increased at the start of exposure to hypoxia, reaching a level of 86.1 ± 22.4 ml·min−1·100 g−1 at 1 min (Fig. 4, B and G). With continued hypoxia over 10 min, ventilation gradually fell to a level of 31.7 ± 3.5 ml·min−1·100 g−1 (Fig. 4G). In contrast to the vehicle-treated group, the riluzole-treated rats showed a blunted ventilatory response to acute hypoxia (Fig. 4, B and G). Although low inspired oxygen stimulated ventilation at 1 min into the hypoxia exposure, the increase was considerably less than that observed in vehicle-treated animals with an increase to 22.0 ± 5.2 ml·min−1·100 g−1 (P = 0.01; Fig. 4G). In the phenytoin-treated group, ventilation at 1 min of hypoxia (30.1 ± 4.7 ml·min−1·100 g−1) was similarly lower compared with vehicle treatment (P = 0.03; Fig. 4, F and G).
Unlike the blunted response to acute hypoxia, the response to acute hypercapnia was not significantly affected by riluzole treatment (Fig. 4H). In the vehicle-treated group, ventilation slowly increased over the first 5 min to 57.6 ± 11.8 ml·min−1·100 g−1 and was maintained at 10 min at a level of 55.1 ± 9.9 ml·min−1·100 g−1 (Fig. 4H). In the riluzole-treated group, ventilation rose to 42.3 ± 9.5 ml·min−1·100 g−1 at 5 min and was maintained at 41.2 ± 8.2 ml·min−1·100 g−1 at 10 min (Fig. 4H). Ventilation at 5 min (P = 0.17) and 10 min (P = 0.16) of hypercapnia was not significantly different from the vehicle-treated group. In contrast to riluzole, phenytoin blunted or delayed the hypercapnic ventilatory response at 5 min (25.8 ± 2.6 ml·min−1·100 g−1; P = 0.03 vs. control), but it was not significantly different at 10 min into the hypercapnic period (Fig. 4H).
The principal findings in this study are 1) petrosal neurons with projections to the carotid body express a significant INaP, which may be antagonized with TTX and riluzole; 2) riluzole and phenytoin inhibit spike generation in peripheral chemoreceptors without affecting glomus cell secretion; and 3) riluzole and phenytoin inhibit the acute respiratory stimulation by hypoxia, but riluzole has no significant effect on the hypercapnic ventilatory response. Taken together, the results suggest an important role for INaP in determining the functionality of the peripheral chemoreceptors, and its inhibition by therapeutic intervention may result in a reduced ability to sense systemic hypoxia.
INaP is generated from Na+ channel transitions from the inactive state to the open state (3), which occur with low probability and generate a noninactivating Na+ conductance with a magnitude of ∼1% of the peak Na+ conductance (16). This noninactivating current can be pharmacologically reduced by agents, such as riluzole and phenytoin, which stabilize the inactive state of the Na+ channel (41, 62). At low drug concentrations, riluzole causes a reduction in the INaP while having little effect on the transient Na+ current (INaT) evoked by a step depolarization (20, 60, 65). Based on this differential sensitivity for inhibition of INaP vs. INaT, riluzole has been used to ascertain the importance of INaP in pacemaker systems such as the pre-Botzinger complex (20, 58), hippocampus (32), suprachiasmatic nucleus (39), and hypothalamus (35); in stellate cells of the entorhinal cortex where it generates subthreshold oscillations that add to synaptic depolarization events, leading to AP generation (2); and in dorsal column nuclei where a riluzole-sensitive current leads to spontaneous AP generation (56).
The recordings obtained in this study appear to be the first whole cell voltage clamp recording from an intact ganglia with an intact peripheral field. This builds on two previous models that our laboratory developed in the rodent that were used to obtain voltage clamp recordings from intact, but denervated, ganglia (71) and from an intact chemoreceptor complex to obtain single-unit recordings from the soma of chemoreceptor neurons of rats and mice (26). Because the dense connective tissue of the ganglia precluded penetration of the patch pipette into the tissue, we utilized aspiration of cells located at the ganglia surface to obtain access to neurons with the required peripheral field. Although the soma could be reasonably voltage clamped, voltage ramps resulted in generation of multiple APs, presumably generated in the initial segment whose voltage was less well controlled.
Using this model, a slow ramp depolarization of the soma of neurons projecting to the carotid body resulted in a sustained inward current that activated near the resting potential of −60mV (17). This current was antagonized by low doses of both TTX and riluzole, and it is thus characterized as INaP (39, 56, 60, 65). However, riluzole, unlike TTX, did not affect the INaT developed by a rapid depolarization from a hyperpolarized potential. This differential block of the persistent vs. transient current by riluzole is consistent with that previously reported by Urbani and colleagues (65) in central neurons.
In addition to antagonism of INaP at the soma of chemoreceptor afferent neurons, riluzole and phenytoin caused a significant decrease in the ventilatory response to acute hypoxia and the response of isolated chemoreceptors to hypoxia. This is demonstrated by a reduction in AP generation frequency in isolated chemoreceptors by riluzole, a response also observed with phenytoin. In a number of excitable cells (3, 46, 60, 65), INaP is thought to raise the membrane potential to the brink of firing threshold, enhancing the ability of the cell to fire repetitively. Inhibition of the current attenuates membrane excitability, decreasing AP frequency. Consistent with an impairment of peripheral chemoreceptor function is the observation that animals treated with both drugs showed a reduced ventilatory response to acute hypoxia. However, the ventilatory response to acute hypercapnia, which is primarily mediated by central chemoreceptors (29), was not significantly altered by riluzole treatment and only slightly delayed by phenytoin treatment. The ventilatory pattern in the presence of riluzole and phenytoin is similar to that observed after surgical denervation of the peripheral chemoreceptors (47). In intact animals, hypoxia leads to an abrupt increase in ventilation due to chemoreceptor-evoked glutamate release within the brain stem (6, 45, 59). This is followed by a ventilatory fall off due to conversion of brain stem glutamate to γ-GABA and a depressive action of GABA (10, 59). Denervation would be expected to ablate the initial glutamate release, and thus the subsequent depressive effects of GABA, resulting in a sustained, albeit small, hyperventilation during prolonged hypoxia (47).
In addition to antagonism of Na+ channels, both riluzole and phenytoin have multiple pharmacological actions on other ion channels and neurotransmitter agents, which may have contributed to the observed results. For instance, riluzole inhibits glutamate release and blocks excitatory amino acid receptors such as NMDA and AMPA receptors (21). However, glutamate transmission appears to play no role in the peripheral chemoreceptors because physiological studies have commonly utilized high levels of glutamate as a metabolic substrate for chemoreceptors, in vitro, without any apparent biasing of the experimental results (64). Riluzole may also alter the characteristics of voltage-dependent K+ channels by 1) slowing of voltage-dependent K+ channel inactivation (70), 2) inhibition of Ca2+-activated K+ channels (4), and 3) inhibition of a rapidly inactivating K+ conductance (70). However, these are unlikely to significantly alter peripheral chemoreceptor function. Studies from several laboratories demonstrate little to no effect of specific (charybdotoxin) or broad-spectrum (TEA, 4-AP) K+ blocking agents on chemoreceptor function (7, 8, 22, 24, 42). In addition, riluzole had no significant effect on AP duration, which would be expected to occur if significant alteration in nerve repolarization were caused by riluzole (57). Phenytoin, like riluzole, has other pharmacological actions such as inhibition of delayed rectifier K+ currents (18), Ca2+-activated K+ currents (51), and certain isoforms of Ca2+ channels (63). However, like riluzole, it failed to alter AP duration, but it only decreased the spontaneous discharge frequency and ventilatory response to hypoxia. Nerve conduction AP parameters was also unchanged or only slightly changed by phenytoin or riluzole treatment. This includes AP amplitude and AP conduction velocity. Thus it is unlikely that the agents caused a decrease in nerve excitability by altering the conductance in the other channels affected by the study drugs.
The site of action of riluzole and phenytoin appears to be localized to the afferent nerve terminal. This conclusion is based on the observation that neither agent significantly altered the magnitude of hypoxia-induced catecholamine release within the carotid body. Catecholamine is released by glomus cells, which are presynaptic to the afferent nerve terminals and are generally believed to be essential for the hypoxia transduction process. Although the role of catecholamines and other transmitters is not well resolved, hypoxia-induced catecholamine release from these cells has been widely used as an index of transduction (53, 54).
Taken together, the results suggest that INaP plays an important role in spike generation in chemoreceptor afferent fibers, a result consistent with previous modeling studies and measurement of nerve terminal excitability. Spontaneous discharge activity of chemoreceptor fibers is highly sensitive to reductions in excitability produced by an isosmotic reduction in Na+ concentration, despite causing only slight changes in nerve terminal excitability as assessed using orthodromic electrical stimulation. This led to the conclusion that spike generation was a high-event-rate process suggestive of ion channel flicker compared with a low-event-rate process such as synaptic depolarizing potentials (25). INaP caused by episodic openings of hundreds to thousands of channels would have these characteristics of a high-event-rate process. These depolarizing events confined to the nerve terminals may explain the generation of spontaneous APs observed from sinus nerve neuromas in the absence of glomus cells (37, 50). In addition, recent modeling studies demonstrated that the noise developed by channel flicker in small fibers may give rise to APs whose pattern is similar to that observed from peripheral chemoreceptors (13).
Although INaP may underlie the process of AP generation, it does not obviate a role for neurotransmitter in the spike generation process. The glomus cells release, in response to hypoxia, multiple candidate transmitters. Recent studies developed compelling evidence that acetylcholine and/or ATP is important for initiating or maintaining chemoreceptor function (1, 11, 36, 48, 52, 61, 66, 67, 69, 72). Previously, substance P, dopamine, and adenosine have been implicated as also serving important modulatory functions (1, 15, 36, 38, 40). The present results shed no light on the role of any of these candidate transmitters, but any of these agents may modulate the spike generation function of INaP by changing membrane potential, changing the input resistance against which INaP changes resting potential, or perhaps through modulation of INaP characteristics. In other systems, G protein-coupled receptors change the activation and inactivation characteristics of Na+ channels, which may change the frequency and/or magnitude of INaP developed at any given resting potential (12).
The present results may have important implications for patients treated with riluzole or phenytoin therapeutically. Riluzole, for instance, is used to treat spasticity associated with amyotrophic lateral sclerosis, and the therapeutic dosages are within the levels used in the present study (21, 43). Our results would suggest that such patients may have an impaired ability to detect hypoxia and, thus may be more susceptible to experience a prolonged apnea. At present, we are not aware of any studies reporting an increased incidence of central apnea or sudden cardiac death, which may be precipitated by apnea in this population, but it is unclear whether this has been examined. However, Groeneveld et al. (33) report an increased incidence of somnolence, dizziness, and fainting among patients on riluzole. The association of these symptoms to the drug's effect on the peripheral chemoreceptors is unknown (33). Similarly, phenytoin is widely used for the suppression of seizures, albeit at concentrations considerably lower than that used in this study (28). It would also be of interest if this patient group evidences a decreased sensitivity to acute hypoxic events.
In conclusion, we report that a riluzole-sensitive, Na+ current is present in petrosal chemoreceptor neurons and that riluzole and phenytoin decrease the rate of AP generation of peripheral chemoreceptors as well as the ventilatory response to acute hypoxia. These results when taken together suggest an important role of INaP in mediating AP generation in peripheral chemoreceptors.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-073500.
We thank Dr. Clifford Bogue for helpful suggestions.
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