J Appl Physiol 101: 1076 1084, 2006.

First published June 15, 2006; doi:10.1152/japplphysiol.00090.2006.

An important functional role of persistent Na current in carotid body

hypoxia transduction
Edward Vincent S. Faustino and David F. Donnelly
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut
Submitted 24 January 2006; accepted in final form 6 June 2006

Faustino, Edward Vincent S., and David F. Donnelly. An

important functional role of persistent Na current in carotid body
hypoxia transduction. J Appl Physiol 101: 1076 1084, 2006. First
published June 15, 2006; doi:10.1152/japplphysiol.00090.2006.
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: 301311, 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 M, and by phenytoin at 100 M, 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.
riluzole; phenytoin; hypoxic ventilatory response; chemoreceptor;
petrosal neuron

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).

HYPOXIA INITIATES A PROMPT

Address for reprint requests and other correspondence: E. V. Faustino, Sect.

of Critical Care and Applied Physiology, Dept. of Pediatrics, Yale Univ.
School of Medicine, 333 Cedar St., PO Box 208064, New Haven, CT
06520-8064 (e-mail: vince.faustino@yale.edu).
1076

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.
METHODS

Experimental subjects. The experiments were performed on 2-wkold 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.
Patch-clamp recording. 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 37C 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,
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PERSISTENT NA CURRENT AND CAROTID BODY TRANSDUCTION

Fig. 1. A: photomicrograph of experimental preparation showing a stimulus

electrode placed in the carotid body and whole cell recording obtained from a
surface cell of the petrosal ganglia. Inset, a magnified view of a patch clamped
petrosal soma. A carbon fiber amperometer advanced into the carotid body
measures catecholamine secreted by glomus cells. B: spontaneous action
potentials (APs) (presumably) arising from nerve endings within the carotid
body. Note the increase in AP activity during hypoxia. C: orthodromic AP
elicited by an electrical stimulus delivered to the carotid body. D: catecholamine secretion increases with hypoxia.

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 currents 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.
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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 36C 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 37C 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-M impedance) was placed in the
center of the carotid body, and a cathodal electrical stimulus (100 A,
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 M),
phenytoin (100 M), or equivalent vehicle concentrations. The doses
used were based on previous studies (19, 60, 65).
Catecholamine secretion. 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-m 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

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PERSISTENT NA CURRENT AND CAROTID BODY TRANSDUCTION

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.
Ventilatory recording. 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 M. 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 M. 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 M, 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 M, 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.
Data analysis. 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).
J Appl Physiol VOL

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
Students 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 Students
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, AF). 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 Students t-test. A level of 0.05 was considered
statistically significant.
RESULTS

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 M, 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 M) 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 M riluzole
applied for 2 min was 41 8 pA (n 9), representing a
reduction of 78.0%. Unlike TTX, riluzole had no significant effect

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PERSISTENT NA CURRENT AND CAROTID BODY TRANSDUCTION

Fig. 2. Ramp-evoked inward current is antagonized by tetrodotoxin (TTX). A:

ramp depolarizations from 120 mV to 20 mV at 26 mV/s evoked a
voltage-dependent inward current starting around 50 mV. In the presence of
TTX (1 M), no inward current is evoked. B: TTX inhibited the fast, transient
Na current evoked by a depolarization to 10 mV from a prepulse potential
of 120 mV. The ramp-evoked inward current is also antagonized by riluzole.
C: ramp depolarizations from 120 mV to 20 mV at 26 mV/s evoked a
voltage-dependent inward current starting around 60 mV. Riluzole (10 M,
perfused for 2 min) reduced the magnitude of the evoked inward current. D:
unlike TTX, riluzole did not reduce the magnitude of the transient Na current
evoked by a depolarization to 10 mV from a prepulse potential of 120 mV.

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 M, 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 M riluzole or its
equivalent vehicle concentration (Fig. 3D). A more pronounced inhibition of spontaneous spiking activity was observed at 10 and 20 M 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 M
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 M
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:
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1079

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
M 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 M riluzole,
conduction time increased by 134.6 8.6% of baseline (P
0.01) (Fig. 3F). Riluzole below 10 M had no significant effect
on (inferred) spike amplitude, but at 10 M 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, FH). 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 M, 111.8 12.6%, n 14)
produced a significant change in the magnitude of hypoxiainduced 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 mlmin1 100
g1 and 3.7 0.8 mlmin1 100 g1 after riluzole treatment
(P 0.06) and 3.6 1.2 mlmin1 100 g1 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 mlmin1 100 g1 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 mlmin1 100 g1 (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 mlmin1 100 g1 (P 0.01; Fig. 4G). In the
phenytoin-treated group, ventilation at 1 min of hypoxia
(30.1 4.7 mlmin1 100 g1) 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

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Fig. 3. Sample traces from single unit recordings after superfusion with vehicle (A), riluzole at 5 M (B), and phenytoin at
100 M (C). Hypoxia elicited a significant increase in the
discharge frequency that is not altered with superfusion of
vehicle. In contrast, relative to the vehicle-treated group, superfusion with riluzole at 5 M and phenytoin at 100 M
depressed discharge frequency at normoxia (D) and hypoxia
(E). More significant decreases in the discharge rate were noted
at higher concentrations at both oxygen concentrations. In
contrast to AP frequency, when normalized to AP conduction
times (F), amplitude (G), and duration (H), riluzole at 5 M
and phenytoin did not significantly affect any of these AP
parameters. The effect of both drugs on AP frequency is
postsynaptic as neither drug resulted in any significant effect on
hypoxia-induced catecholamine secretion (I). Values are
means SE. *P 0.05. P 0.01.

slowly increased over the first 5 min to 57.6 11.8

mlmin1 100 g1 and was maintained at 10 min at a level of
55.1 9.9 mlmin1 100 g1 (Fig. 4H). In the riluzole-treated
group, ventilation rose to 42.3 9.5 mlmin1 100 g1 at 5
min and was maintained at 41.2 8.2 ml min1 100 g1 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 min1 100 g1; P 0.03 vs. control), but
it was not significantly different at 10 min into the hypercapnic
period (Fig. 4H).
DISCUSSION

The principal findings in this study are 1) petrosal neurons

with projections to the carotid body express a significant INaP,
J Appl Physiol VOL

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

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1081

Fig. 4. Sample recording of ventilation during normoxia with

vehicle (A), hypoxia (at 1 min of 12% O2) with vehicle (B),
normoxia with riluzole (C), hypoxia with riluzole (D), normoxia
with phenytoin (E), and hypoxia with phenytoin (F). In the
vehicle-treated rat, ventilation promptly increased with exposure
to low inspired oxygen concentration. Treatment with riluzole (2
mg/kg) and phenytoin (20 mg/kg) depressed the rats resting
ventilation and its response to acute hypoxia. Relative to treatment
E) at room air and at 1 min of
with vehicle, minute ventilation (V
hypoxia was significantly lower in the riluzole- and phenytointreated groups. Riluzole, unlike phenytoin, did not affect the
E values at 5 and 10 min were
hypercapnic ventilatory response. V
E at
similar in the vehicle- and riluzole-treated groups, whereas V
5 min was lower in the phenytoin-treated group. Values are
means SE. *P 0.05. P 0.01.

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
J Appl Physiol VOL

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

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PERSISTENT NA CURRENT AND CAROTID BODY TRANSDUCTION

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 -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), Ca2activated 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 transducJ Appl Physiol VOL

tion 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
drugs 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

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PERSISTENT NA CURRENT AND CAROTID BODY TRANSDUCTION

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.
ACKNOWLEDGMENTS
We thank Dr. Clifford Bogue for helpful suggestions.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-073500.
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