Ileitis modulates potassium and sodium currents in guinea pig dorsal root ganglia sensory neurons

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Abdominal pain is a major cause of morbidity for patients

who suffer from intestinal disorders, such as inflammatory

bowel disease (IBD) and irritable bowel syndrome.

Antecedent infection or inflammation has been implicated

in the visceral hyperalgesia associated with the irritable

bowel syndrome (IBS). The cell bodies of sensory neurons

which initiate these sensations originate in the dorsal root

ganglia (DRG) (Mayer & Gebhart, 1994). Sub-populations

of DRG neurons respond to innocuous and noxious

mechanical stimulation, as well as chemical and thermal

stimulation, and thus are thought to be polymodal sensory

neurons (Mayer & Gebhart, 1994). These polymodal

neurons play a central role in responding to conditions

that are potentially injurious to tissues. Inflammatory

mediators cause a reduction in the threshold and an

increase in the gain of the transduction process of these

neurons in a process referred to as ‘peripheral

sensitization’. Also, under certain pathological conditions,

there is evidence that an additional population of normally

mechanically insensitive nociceptive fibres can be

recruited (Gebhart, 2000). Following inflammatory

stimulation these ‘silent afferent’ fibres become active at

rest and begin respond to mechanical stimulation.

Together, these afferent pathways have been suggested to

contribute to disproportionate pain states in response to

injury and may persist even after the inflammatory state

has resolved (Al Chaer et al. 2000; Collins et al. 2001).

In studies in the somatic nervous system, peripheral

sensitization appears to involve both acute and chronic

mechanisms (Woolf & Costigan, 1999). Acute

mechanisms of increased nociceptive stimulation include

both direct depolarization of nerve terminals by

neuroactive agents and alterations in ionic currents in the

membrane terminals (Rang et al. 1991). Also, later and

longer lasting transcription-dependent changes occur and

appear to be evoked by either signalling molecules, such as

nerve growth factor and/or activity-dependent second

messenger cascades (McCleskey & Gold, 1999). These

transcription-dependent events can result in altered

expression of voltage-gated ion channels, such as increased

expression of TTX-resistant sodium channels, and

increased TTX-resistant currents (TTX-R INa) (Khasar et

Ileitis modulates potassium and sodium currents in guineapig dorsal root ganglia sensory neuronsTimothy Stewart*, Michael J. Beyak* and Stephen Vanner

Gastrointestinal Diseases Research Unit, Queen’s University, Kingston, Ontario, Canada

Intestinal inflammation induces hyperexcitability of dorsal root ganglia sensory neurons, which has

been implicated in increased pain sensation. This study examined whether alteration of sodium

(Na+) and/ or potassium (K+) currents underlies this hyperexcitability. Ileitis was induced in guinea

pig ileum with trinitrobenzene sulphonic acid (TBNS) and dorsal root ganglion neurons

innervating the site of inflammation were identified by Fast Blue or DiI fluorescence labelling.

Whole cell recordings were made from acutely dissociated small-sized neurons at 7–10 days.

Neurons exhibited transient A-type and sustained outward rectifier K+ currents. Compared to

control, both A-type and sustained K+ current densities were significantly reduced (42 and 34 %,

respectively; P < 0.05) in labelled neurons from the inflamed intestine but not in non-labelled

neurons. A-type current voltage dependence of inactivation was negatively shifted in labelled

inflamed intestine neurons. Neurons also exhibited tetrodotoxin-sensitive and resistant Na+

currents. Tetrodotoxin-resistant sodium currents were increased by 37 % in labelled neurons from

the inflamed intestine compared to control (P < 0.01), whereas unlabelled neurons were unaffected.

The activation and inactivation curves of these currents were unchanged by inflammation. These

data suggest ileitis increases excitability of intestinal sensory neurons by modulating multiple ionic

channels. The lack of effect in non-labelled neurons suggests signalling originated at the nerve

terminal rather than through circulating mediators and, given that Na+ currents are enhanced

whereas K+ currents are suppressed, one or more signalling pathways may be involved.

(Received 7 May 2003; accepted after revision 12 August 2003; first published online 15 August 2003)

Corresponding author S. Vanner: 166 Brock Street, Hotel Dieu Hospital, Kingston, Ontario, Canada K7L 5G2. Email: [email protected]

J Physiol (2003), 552.3, pp. 797–807 DOI: 10.1113/jphysiol.2003.046409

© The Physiological Society 2003 www.jphysiol.org

* Timothy Stewart and Michael J. Beyak contributed equally to this work.

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al. 1998; Tanaka et al. 1998) or decreased availability of

voltage-gated K+ channels (Yoshimura & de Groat, 1999).

Compared to the somatic nervous system, there is

considerably less known about the ionic mechanisms

underlying inflammation-induced changes in sensory

neurons innervating the gastrointestinal tract. Recent

studies in the rat stomach have shown that experimentally

induced gastric ulcers increase TTX-R INa in vagal and

DRG neurons innervating the stomach (Bielefeldt et al.2002a)and to a lesser degree in mild gastritis (Bielefeldt etal. 2002b). This latter study, however, found that outward

K+ currents were unchanged. In contrast, studies in viscera

outside the gastrointestinal (GI) tract suggest that

inflammation may also modulate K+ currents (Yoshimura

& de Groat, 1999) Taken together, it is likely that changes

in ionic mechanisms underlying inflammation-induced

plasticity depend on the organ involved and the

inflammatory repertoire which follows the initiation of

inflammation.

We have recently used trinitrobenzene sulphonic acid

(TNBS) ileitis in the guinea pig, as a model of

inflammatory bowel disease (Moore et al. 2002), to

examine the effects of intestinal inflammation on DRG

neurons. We found hyperexcitability of dissociated DRG

neurons innervating the intestine, manifested by

decreased threshold and repetitive firing, properties

consistent with hyperalgesia and allodynia. This study

employs whole cell voltage clamp techniques in this model

to determine if changes in Na+ and/ or K+ currents underlie

this hyperexcitability.

METHODS Animal model and neuron isolationGuinea pigs (140–225 g) of either sex were obtained from CharlesRiver Laboratories (Montreal, Quebec, Canada). Experimentswere performed according to the guidelines of the CanadianCouncil of Animal Care and approved by the Queen’s Universityanimal care committee.

Animals were anaesthetized using a combination of Hypnorm(0.315 mg ml_1 fentanyl citrate and 10 mg ml_1 fluanisone) andmidazolam (5 mg ml_1) (0.0025 ml g_1 each, I.P.), and surgery wasperformed under aseptic conditions to exteriorize the terminalileum as described previously (Moore et al. 2002). Ileal-projectingneurons were labelled by injecting the retrograde tracer Fast Blue(dissolved in distilled water) or DiI (dissolved in EtOH) into theileal wall using a Hamilton syringe fitted with a 30 Ga needle. Atotal injection volume of 15–20 ml was given via 10–15 injectionsites. DiI was used in later experiments since Fast Blue becameunavailable. Ileitis was induced by injection of 0.5 ml of TNBS(25 mg ml_1 in 25 % EtOH; TNBS kindly provided by Dr G.Morris, Department of Biology, Queen’s University, Kingston,Ontario, Canada K7L 5G2) into the ileal lumen using a 30 Ganeedle. The intestine was replaced in the abdominal cavity and thewound sutured with 4–0 silk. Buprenex (Buprenophine0.0225 mg g_1

I.P., Reckitt and Colman) was given to all animals tocontrol post-operative pain. Animals recovered from the

anaesthetic in a quiet environment, on an electric thermal blanketto maintain normothermia. The post-operative condition of theanimals was monitored at least twice daily by trained animal carestaff. Animals that were failing to thrive or showed behavioursuggestive of persistent pain were killed. After 7–10 days, animalswere anaesthetized by inhalation of isofluorane and killed bycervical dislocation and exsanguination.

The terminal ileum was removed from each animal to establishthe degree of TNBS induced ileitis. The ileum was opened alongthe mesenteric border and pinned flat with the mucosal surfaceuppermost. Inspection of the mucosa revealed mucosalhemorrhage, ulceration and thickening of the tissue, as describedin our previous studies (Moore et al. 2002). In the current study,inspection of the mucosa revealed similar damage followinginflammation.

DRG neurons were isolated from thoracic vertebra Th10_13 asdescribed previously (Moore et al. 2002). Briefly, isolated DRGwere dissected free of adherent connective tissue and thenincubated at 37 °C in HBSS with 0.2 mg ml_1 papain and0.4 mg ml_1 cysteine for 10 min. This was followed by washing inL-15 medium (GIBCO-BRL) with 10 % fetal bovine serum. TheDRGs were then incubated in HBSS containing 1 mg ml_1 Type 1collagenase (Worthington) and 4 mg ml_1 dispase II (BoeringerManneheim). The ganglia were then titurated with a fire-polishedPasteur pipette. Neurons were maintained in MEM culturemedium with Earle’s salts and HCO3 (GIBCO-BRL) containing1 % penicillin–streptomycin, 2 mM glutamine and 0.2 % (w/v)glucose. The cell suspension was plated onto rat tail collagen-coated glass coverslips and stored in a humidified incubator at37 °C under 95 % air and 5 % CO2 until they were retrieved for usein electrophysiological experiments 4–24 h later.

SolutionsFor current clamp experiments, the control solution used tosuperfuse the cells contained (mM): NaCl, 140; KCl, 5; MgSO4, 1;CaCl2, 1; Hepes, 5; pH adjusted to 7.4 using NaOH. Identicalcontrol solutions were used to superfuse the cells in K+ currentvoltage clamp experiments, except for the equimolar replacementof NaCl with N-methyl-D-glucamine. Potassium channelrecording solutions were adjusted to pH 7.4 using HCl. Thecontrol filling solution contained (mM): KCl, 140; Hepes, 5;MgSO4, 1; EGTA, 1; pH adjusted to 7.2 using KOH. For therecording of sodium currents, solutions of the followingcomposition were used: extracellular solution (mM): NaCl, 100;NMDG, 50; Hepes, 10; MgCl2, 10; D-Glucose, 10; pH adjusted to7.4 with HCl. Pipette solution (mM): Cs, 115; NaCl, 25; Hepes, 10;MgCl2, 3; EGTA, 11; pH adjusted to 7.2 with CsOH.

Electrophysiological proceduresCoverslips supporting adherent DRG neurons were placed in aRC-26 recording chamber (Warner Instrument Corporation) andmounted on an inverted microscope (IX-70, Olympus, Japan)fitted for both fluorescence and bright field microscopy. Labelledneurons were identified by their fluorescence under brief (< 15 s)exposure to ultraviolet light, using a U-MWIG2 filter for Fast Blueor a U-MWU2 (Olympus, Japan) for DiI, after which cells wereviewed under bright field illumination. Whole cell recordingswere made using variations of the patch clamp technique (Hamillet al. 1981) and an Axopatch-200B amplifier (Axon Instruments).Patch electrodes were fabricated using thin-walled borosilicateglass (Kimble Products) and a PP-830 electrode puller (NarishigeInstruments) or P-97 (Sutter Instruments). After fire polishing,pipettes used for whole cell voltage clamp experiments had DC

T. Stewart, M. J. Beyak and S. Vanner798 J Physiol 552.3

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resistances of 1.5–3.0 MV when filled with the control fillingsolution. Electronic compensation of total series resistance wasused in all experiments. Capacitive transients were correctedusing analog circuitry. Drugs were added to the bath solution, ordelivered by a fast-flow solution switching system (VC-6, WarnerInstrument Corporation). Membrane currents were filtered at2 kHz and sampled at 5 kHz and stored on a microcomputer forlater analysis using pCLAMP 8.2 software (Axon Instruments).Cells were included for analysis if in current clamp mode theresting membrane potential was more negative than _40 mV andthe cells displayed overshooting action potentials or in voltageclamp mode the series resistance error (after compensation) wasless than 5 mV and seal and access resistances remained stable.The mean number of cells studied per animal was 3 (range 1–10).

Drugs and chemicalsAll chemicals and drugs were from Sigma Chemical Co.4-Aminopyridine (4-AP) was dissolved in distilled water to a stockconcentration of 100 mM and the pH was adjusted to 7.4 using 2 N

HCl. Tetrodotoxin (TTX) was dissolved in water at a stockconcentration of 1 mM. Drugs were diluted to their finalconcentration in the bath solution. Drugs were administeredeither by direct addition to the bath solution, or directly to the cellunder study using a fast-flow solution switching system (WarnerInstruments).

Statistical analysisStudent’s t test or ANOVA with Student-Newman-Keuls post hoctest were used where appropriate. P values < 0.05 were consideredsignificant.

RESULTSGeneral propertiesSuccessful recordings were obtained from 149 DRG

neurons. Three groups of neurons were examined in this

study; Fast Blue- or DiI-labelled neurons from control

animals (Control) or TNBS-treated animals (TNBS) and

neurons from TNBS-treated animals which were not

labelled (non-labelled TNBS). Approximately 3–5 % of

the dissociated cells were labelled with Fast Blue or DiI.

The mean resting membrane potential in current clamp

recordings from labelled control neurons was

–58.1 ± 1.1 mV (range _48 to _64 mV; n = 13). Our

previous studies of TNBS ileitis using intracellular

recording techniques demonstrated that small cell size

correlated very closely with the following properties: TTX-

resistant action potentials with inflections on the

repolarizing phase and capsaicin sensitivity (Moore et al.2002). These properties have been shown to be present in

small-diameter somatic and visceral unmyelinated

afferents, a proportion of which are nociceptive afferents

(Sengupta & Gebhart, 1994; Blackshaw & Gebhart, 2002).

Using whole cell current clamp recordings, we found that

small neurons had similar properties, i.e. TTX-resistant

action potentials with a prominent inflection of the

repolarizing phase (5/5). Measurements of cell capacitance

were used to monitor size, and only cells with capacitance

< 40 pF were examined, as these cells consistently

expressed these properties. Larger cells (mean capacitance

81 ± 7.4 pF) had narrow, TTX-sensitive action potentials

(4/4). In our previous work using the same model we

demonstrated inflammation-induced changes in

excitability of this sub-population of small neurons. In

these studies, intracellular recordings were obtained from

neurons from control (n = 12) and animals with ileitis

(n = 17) and we found that TNBS ileitis caused significant

reductions in rheobase, and increased the number of

action potentials evoked at 2 w rheobase. Furthermore

TNBS ileitis resulted in increased action potential

upstroke velocity and increased input resistance. (Moore

et al. 2002). In the present study, using whole cell

recording techniques, we demonstrated a similar effect of

TNBS ileitis on rheobase (> 65 % decrease compared to

control and non-labelled cells) and number of action

potentials elicited at twice rheobase (> 2.7 times compared

to control and non-labelled cells) (Fig. 1).

Characterization of voltage-gated K+ currentsPrevious studies of unidentified DRG neurons have

identified two kinetically distinct voltage-dependent K+

currents, a transient ‘A-type’ current (IA) and ‘sustained

delayed rectifier type’ (IK) (McFarlane & Cooper, 1991;

Akins & McCleskey, 1993; Gold et al. 1996; Everill et al.1998). In the present study, using a voltage clamp protocol

with 5 mV steps (400 ms) from a holding potential of

–100 mV, both the IA and sustained IK were evident

(Fig. 2A). The reversal potential of these currents was

_75.5 ± 2.2 mV, near the predicted reversal potential for

potassium (_84 mV). Inactivating IA currents were

adequately fitted by a monoexponential decay with a mean

time constant of 171 ± 16 ms (range 133–210 ms).

IA and IK currents were isolated based on their contrasting

biophysical and pharmacological properties. In

preliminary experiments, we established that neither

current was significantly inactivated when the membrane

potential was held at –100 mV (Fig. 2). However, the IA was

selectively inactivated when the membrane potential was

held at –60 mV, whereas inactivation of IK at this holding

potential was minimal (Fig. 2). Thus, the sustained current

was isolated by holding the membrane potential at –60 mV,

with the sustained IK measured isochronally, 400 ms after

the onset of the pulse, at which time IA was largely

inactivated, minimizing contamination by this current

(Fig. 2). The IA was isolated by subtracting the sustained IK

from the total K+ current recorded from a holding potential

of –100 mV. Peak IA was measured as the peak of the

transient component of this subtracted current (Fig. 2).

IA was also isolated pharmacologically using the K+

channel blocker 4-aminopyridine (4-AP) (Fig. 2B).

Preliminary experiments demonstrated that 100 mM 4-AP

(n = 6), and 600 mM 4-AP (n = 3) caused partial

suppression of the IA currents whereas 2 mM (n = 3)

completely suppressed IA and caused significant

suppression of the IK currents. 4-AP most selectively

Ileitis modulates sensory nerve K+ and Na+ currentsJ Physiol 552.3 799

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blocked the IA at a concentration of 1 mM. Therefore the IA

was isolated by subtracting the current in the presence of

1 mM 4-AP from the total control current, yielding the

4-AP-sensitive IA (Fig. 2B). Experiments using both of

these approaches in the same control neuron revealed no

significant difference in the IA current density obtained by

either method (80.3 ± 19.7 vs. 99.1 ± 10.0 pA pF_1, n = 5)

IA and IK current density following TNBS-inducedinflammationThe density of IA and IK elicited by a depolarizing pulse to

+50 mV was compared in labelled neurons from control

animals and both labelled and unlabelled neurons from

TNBS animals (Fig. 2C and D). Current density was


Figure 1. Effects of TNBS ileitis on intestinal sensory neuron excitabilityA, representative current clamp traces of action potentials elicited at rheobase and two times rheobase incontrol labelled, TNBS labelled and TNBS non-labelled neurons. TNBS results in a reduction of therheobase, while increasing the number of spikes evoked by a 2 w rheobase current injection. Inflections onthe falling phase of the action potential are not obvious due to the time scale. B, mean rheobase in controllabelled (n = 13), TNBS labelled (n = 4) and TNBS non-labelled (n = 5) neurons (* P < 0.05). C, meannumber of spikes elicited by a 2 w rheobase current injection in these same groups of neurons (* P < 0.05).

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yIleitis modulates sensory nerve K+ and Na+ currentsJ Physiol 552.3 801

Figure 2. Voltage-gated potassium currents and the effect of TNBS ileitis on current densityRepresentative traces from one cell showing currents separated biophysically by manipulating the holdingpotential (A) and pharmacologically by 4-AP (B). Currents were elicited in response voltage steps from_90 mV to +50 mV in 5 mV increments. A, left: at holding potentials of _100 mV, two currents wereapparent, a transient, inactivating ‘A’ type current, and a non-inactivating sustained IK type current. A,middle: resultant current when membrane potential is held at _60 mV. Note disappearance of the transientcomponent. Only the sustained component remains, and was measured at 400 ms. A, right: subtraction ofthe sustained from the total current yields IA. IA amplitude was measured as the peak of the transientcomponent. B, left: in the pharmacological experiments, the holding potential was _100 mV. B, middle:when the voltage steps were repeated in the presence of 1 mM 4-AP the transient component was significantlyinhibited. B, right: results of subtracting current obtained in the presence of 4-AP from control, revealing the4-AP-sensitive IA current. C, effect of TNBS ileitis on mean peak IA density obtained using either biophysical(left) or pharmacological separation (right). TNBS ileitis results in a significant reduction in IA densitycompared to control, or non-labelled TNBS neurons (* P < 0.05). D, effect of TNBS ileitis on mean peak IK

density. TNBS ileitis results in a significant reduction in peak IK density, compared to control and non-labelled cells (* P < 0.05).

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determined by normalizing the current amplitude by the

cell’s capacitance. The IK amplitude was measured 400 ms

following the onset of a depolarizing pulse from a holding

potential of –60 mV. The IA amplitude was measured as

the peak current amplitude of the subtracted current as

described above (Fig. 2A and B). Figure 2C–E illustrates

the reduction of IA and IK in labelled neurons from TNBS

animals. When IA was isolated biophysically, the IA density

was 42 % less (P < 0.05) in labelled neurons from TNBS

animals (n = 22) than in labelled neurons from control

animals (n = 14) and 65 % less than in unlabelled neurons

from TNBS animals (n = 19) (Fig. 2C). IA was also isolated

pharmacologically using 4-AP (1 mM). The 4-AP-sensitive

IA in labelled neurons was reduced by 33 % in labelled

TNBS neurons (n = 6, P < 0.05) compared to the currents

in labelled neurons from control animals (n = 4) and was

43 % less than in non-labelled TNBS neurons (n = 7,

P < 0.01, Fig. 2D). The current density of IK from labelled

TNBS neurons (n = 20) was 34 % less (P < 0.05) than in

labelled neurons from control animals (n = 15) and 31 %

less (P < 0.05) than in unlabelled neurons from TNBS

animals (n = 20) (Fig. 2E).

We also attempted to isolate IK pharmacologically, using

TEA. However, we found, as have others (Everill et al.1998; Gold et al. 1996) that TEA in doses sufficient to block

IK, also cause significant blockade of the IA.

Voltage dependence of activation and inactivationVoltage dependencies of activation and inactivation were

compared in labelled control cells and labelled TNBS cells

to determine if changes in TNBS animals could contribute

to the reduced current density.

To determine IA K+ conductance and IK K+ conductance,

peak IA amplitudes and isochronally measured end-pulse

current amplitudes were measured, respectively.

Conductance was then determined using the relation

G = I/(Vm – EK), where G is the conductance, I is the

measured membrane current, Vm is the voltage step, and

EK is the equilibrium K+ potential, which was calculated to

be –84 mV in control solutions. Average K+ conductance

was plotted against membrane potential (Fig. 4) and the

continuous line is an average of individual fits to a

Boltzmann function of the form:

G/Gmax = 1/(1 + exp[VÎ _ Vm/k]),

where G is the conductance, Gmax is the fitted maximal

conductance, VÎ is the membrane potential for half-

activation, Vm is the command potential, and k is the slope

factor. No differences in the voltage dependencies of

activation for IA or IK were found between control and

TNBS neurons (Fig. 3A and B).

To examine the voltage dependencies of inactivation for IA

and IK, we employed two-pulse voltage protocols as

described by others (Philipson et al. 1991; Yoshimura & de

Groat, 1999). Residual IA currents were measured

following short conditioning pulses (1 s duration) which

allowed inactivation of only the rapidly inactivating IA

currents. Longer conditioning pulses (8 s duration)

allowed inactivation of both IA and IK currents. Therefore

residual currents were isochronally measured at the end of

a 1 s test pulse to minimize the contribution of IA currents

to the measured residual current. The residual current

amplitude was plotted against conditioning pulse


Figure 3. Activation and inactivationcurves of voltage-gated K+ currentsActivation curves were generated by voltage pulsesin 5 mV steps from -80 to +50 mV. Each curverepresents the mean of curves fitted to theBoltzmann equation (n = 8 cells each). A,activation curves for IA in control and TNBSneurons. TNBS did not affect the activationproperties of IA.B, inactivation curves for IA.

Inactivation curves were constructed using a two-pulse protocol, a 1 s prepulse varying between_120 and 0 mV, followed by a 400 ms test pulse of+50 mV. The peak of the transient component wasmeasured. TNBS treatment resulted in a slightleftward shift in the inactivation curve of IA.Activation (C) and inactivation (D) curves for IK.Inactivation curves for IK were generated using atwo-pulse protocol, with an 8 s prepulse varyingbetween _80 and 0 mV, followed by a 1 s test pulseof +50 mV. End-pulse IK amplitude was measured.TNBS had no effect on voltage dependence ofeither activation or inactivation of IK.

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potential and the continuous line is an average of fits to a

negative Boltzmann function:

I/Imax = 1/(1 + exp[VÎ _ Vm/k]),

where I is the current, Imax is the maximal current, VÎ is the

membrane potential for half-activation, Vm is the

command potential, and k is the slope factor (Philipson etal. 1991). Significant differences in IA were found between

control and TNBS neurons with VÎ (control

_65.4 ± 2.0 mV, TNBS 85.2 ± 2.4 mV, P < 0.05) and

slope factors (control 6.9 ± 0.9, TNBS 14.3 ± 1.1,

P < 0.05) (Fig. 3C). However, the voltage dependence and

voltage sensitivity of inactivation for IK were not different

between control and TNBS neurons (VÎ: control

_47.6 ± 2.8 mV, TNBS _50.2 ± 3.0 mV; slope: control

7.5 ± 1.5, TNBS 9.4 ± 1.2, P > 0.05; Fig. 3D).

We also employed a pharmacological approach using

4-AP (1 mM) to isolate IA and examine its voltage

dependence. Using the above two-pulse protocol, in the

absence and presence of 4-AP, the currents obtained in the

presence of 4-AP were subtracted from those obtained in

its absence (n = 10). These experiments indicated that

sensitivity to 4-AP varied significantly with the prepulse

voltage thus making analysis of the voltage dependence of

inactivation of the subtracted 4-AP sensitive current

impractical. This finding has been reported by others

(Thompson, 1982; Rasmusson et al. 1995).

Characterization of voltage-gated sodium currentsLarge inward currents were evoked in all neurons (n = 60)

by depolarizing +5 mV voltage steps from _80 to +40 mV

from a holding potential of _90 mV (Fig. 4A). These

currents were separated into a TTX-resistant (TTX-R INa)

and TTX-sensitive (TTX-S INa) components using TTX

(1 mM). TTX-S INa was obtained by subtracting the inward

currents in the presence of 1 mM TTX from the currents

recorded in the absence of TTX (Fig. 4A). All cells


Figure 4. Voltage-gated sodium currents and the effects of TNBS ileitisA, representative voltage-gated sodium currents from a small (25 pF) intestinal DRG neuron. Left: totalinward current generated by stepwise 30 ms voltage pulses between _80 and +40 mV from a holdingpotential of _90 mV. Middle: TTX-R INa currents obtained using the same protocol in the presence of 1 mM

TTX. Right: TTX-S INa currents obtained by subtracting TTX-R current from total. B, mean peak TTX-R INa

current density. TTX-R INa density was increased by TNBS ileitis, * P < 0.05 compared to control and non-labelled neurons (n = 7 cells for each group). C, TTX-S INa current density was not significantly affected byTNBS ileitis (P > 0.05, n = 7 cells for each group).

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examined exhibited both the TTX-R INa and TTX-S INa

currents.

Effects of TNBS ileitis on Na+ current densityTTX-R INa and TTX-S INa peak current densities were

obtained by normalizing the current amplitude by the

individual cell’s capacitance. TTX-R INa was increased

significantly in the labelled TNBS group (n = 10) compared

to labelled control neurons (n = 10) (37 %, P < 0.01) or

non-labelled control neurons (n = 10) (32 %, P < 0.05)

(Fig. 4B). TTX-S INa was not significantly different between

inflamed labelled neurons (282.0 ± 12.7 pA pF_1, n = 10),

labelled control neurons (312.2 ± 22.4 pA pF_1, n = 10,

P > 0.05) or TNBS non-labelled neurons (282.3 ±

17.0 pA pF_1, n = 10, P > 0.05) (Fig. 4C).

Activation and inactivation properties of TTX-R INa

We examined the voltage dependence of activation and

inactivation of TTX-R INa to determine if changes in these

properties could contribute to the increase in current

density induced by inflammation. Activation curves were

generated using depolarizing voltage steps in the presence

of TTX (1 mM). Normalized conductance (G/Gmax) was

plotted against test pulse voltage and the data were fitted

using a Boltzmann function of the form:

G/Gmax = 1/(1 + exp[VÎ _ Vm/k]).

Voltage of half-activation and slope were calculated from

the average of the fitted activation curves. Inflammation

did not alter the slope (control 4.0 ± 0.50, TNBS

5.2 ± 0.32, n = 7, P > 0.05) or the voltage of half-

activation of TTX-R INa (control _12.8 ± 2.0, TNBS

_16.9 ± 0.38, n = 7, P > 0.05) (Fig. 5A).

Steady-state inactivation curves were generated using a 1 s

prepulse from _120 to 0 mV, followed by a 30 ms test

pulse to 0 mV. The resultant normalized peak current

(I/Imax) was plotted against the prepulse voltage, and the

data were fitted to a negative Boltzmann function of the

form:

I/Imax = 1/(1 + exp[VÎ _ Vm/k]).

Mean voltage of half-inactivation and slope were

calculated from the average of the individual fitted curves

(Fig. 5B). Inflammation had no effect on the voltage of

half-inactivation of the TTX-R INa (control 26.0 ±1.6 mV,

TNBS 25.6 ± 2.3 mV, n = 7, P > 0.05), or the slope

(Control _2.03 ± 0.45 mV_1, TNBS _1.7 ± 0.42 mV_1).

DISCUSSIONVoltage-gated Na+ and K+ channels play a fundamental role

in controlling neuronal excitability (Hille, 1992) and

exhibit significant diversity in sensory neurons, depending

upon their innervation and functional properties. The

present study characterized K+ and Na+ currents in small-

diameter sensory neurons and tested the hypothesis that

alteration of one or more currents underlies the

hyperexcitability seen in these neurons during

inflammation. DRG neurons were functionally identified

by retrograde labelling from the intestine, and only small-

sized cells were selected for study, as we had previously

shown that small cell size correlates with properties such as

TTX-resistant action potentials, capsaicin sensitivity, and

inflections on the repolarizing phase of the action potential

(Moore et al. 2002). Sensory neurons innervating the

intestine exhibit transient IA and sustained outward IK K+

currents and both TTX-resistant and TTX-sensitive inward

Na+ currents. The major finding was that TNBS ileitis, a

model of inflammatory bowel disease caused a marked

reduction in the voltage-gated potassium currents, IA and

IK,whereas TTX-R INa currents were significantly increased.

These changes were not observed in unlabelled neurons

from animals with ileitis, suggesting that signalling

occurred at the nerve terminal rather than as result of

circulating inflammatory mediators. Moreover, the

opposing effects on K+ and Na+ currents would suggest that

multiple signalling pathways are involved.


Figure 5. Activation and inactivation curves for TTX-R INa

Activation curves in A were generated using voltage steps from _80to +20 mV. Normalized conductance (G/Gmax) was plotted againsttest pulse voltage and fitted to a Boltzmann function. Linesrepresent average of the individual curve fits. TNBS ileitis did notaffect the activation properties of the TTX-R INa. (P > 0.05, n = 7)B, inactivation curves for TTX-R INa. Inactivation curves weregenerated using a two-pulse protocol, with a 1 s prepulse between_120 and 0 mV, followed by a 30 ms test pulse to 0 mV.Normalized current (I/Imax) was plotted against prepulse voltageand fitted to a negative Boltzmann function. Line represents theaverage of individual curve fits. Inactivation properties were notsignificantly affected by TNBS ileitis (n = 7, P > 0.05).

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Voltage-gated K+ currents in neurons innervatingcontrol and inflamed ileumThe IA current plays an important role in setting high

thresholds for spike activation and suppression of

repetitive firing (Rudy, 1988; Hille, 1992; Yoshimura & de

Groat, 1999). Several types of IA current have been

described in DRG sensory neurons (Gold et al. 1996;

Yoshimura & de Groat, 1999) and can be separated into

those with relatively fast and slow inactivation rates based

on their biophysical and pharmacological properties

(Gold et al. 1996; Yoshimura & de Groat, 1999). Several

studies have shown that the fast inactivating IA currents are

confined to the large-sized neurons with TTX-sensitive

spikes (Yoshimura et al. 1996; Gold et al. 1996). The slowly

inactivating IA current has an inactivation time constant of

between 150 and 300 ms and the voltage of half-maximal

inactivation is displaced more positively than the fast

inactivating IA (Akins & McCleskey, 1993; Fjell et al. 1999).

In the present study the transient IA inactivation was fitted

by a single monoexponential function with a time constant

of 171 ms, typical of that described for the slowly

inactivating IA. These slowly inactivating IA currents, in

contrast to the fast inactivating currents, have been found

to be selectively expressed in the small-sized neurons

exhibiting TTX-resistant action potentials (Yoshimura etal. 1996). Taken together, the data suggest that slowly

inactivating IA current is the dominant form of IA in

sensory afferent DRG neurons innervating the small

intestine.

In addition to the transient IA current, studies of DRG

sensory neurons have identified a sustained delayed

rectifier current in all cells (Akins & McCleskey, 1993;

Gold et al. 1996). In the present study, these currents could

be separated from the IA currents by prepulse inactivation

or by 1 mM 4-AP which blocked the IA currents but had

little effect on the sustained current, as reported by others

(Gold et al. 1996). Some studies have suggested that this

sustained current may represent several different currents,

based largely on their steady-state inactivation properties

(Akins & McCleskey, 1993; Gold et al. 1996). These

sustained currents, however, often could not be separated

by voltage protocols or pharmacological agents (Gold et al.1996) making the study of changes in individual sustained

currents impractical.

TNBS ileitis caused a significant reduction in both IA and

sustained IK currents identified in this study and we have

previously shown that pharmacological suppression of K+

currents such as IA can significantly increase the

excitability of sensory neurons innervating the intestine

(Moore et al. 2002). It is unclear whether these changes in

K+ currents occur elsewhere in the GI tract because there

has been relatively little work done in this area and in

studies conducted in a gastritis model of inflammation

similar changes were not observed (Bielefeldt et al. 2002b).

It is possible that this difference can be explained by

variations in the degree of inflammation or experimental

protocols. Our finding that visceral inflammation

suppresses IA and correspondingly increases excitability of

sensory neurons is similar to previous studies examining

the inflamed urinary bladder (Yoshimura & de Groat,

1999). This reduction in peak current density was

associated with a hyperpolarizing shift in the inactivation

curve, as described in the current study, suggesting there

was an associated change in the biophysical properties of

the channels. The shift of the inactivation curve in a

hyperpolarizing direction would make fewer IA channels

available, at or near resting membrane potentials, and lead

to a further increase in excitability of the cell, leading to

repetitive firing. In contrast to the study in the rat urinary

bladder, we also observed a marked reduction in the

current density of IK. Whether these differences also reflect

degrees of inflammation in these organs or are unique to

the sensory neurons and the organs they innervate remains

to be established. Given that the role of these voltage-gated

potassium currents is to raise the action potential

threshold and limit firing (Hille, 1992) suppression of

these currents would be expected to increase excitability.

Voltage-gated Na+ currents in neurons innervatingcontrol and inflamed ileumThree types of sodium channels predominate in DRG

neurons; NaV1.7 channels, which are responsible for the

fast, rapidly inactivating TTX-S INa current, NaV1.8

channels, which result in a more slowly inactivating TTX-R

INa and are relatively selectively expressed in nociceptors,

and the recently described NaV1.9 which is responsible for a

TTX-R persistent current that is ultra-slowly inactivating

(Dib-Hajj et al. 2002).The activation and inactivation

properties of our TTX-R INa currents fit the known

properties of the NaV1.8 channel, although a detailed

molecular or immunocytochemical characterization of

guinea pig DRG sodium channels has yet to be performed.

We observed a significant increase in TTX-R INa in sensory

neurons innervating the inflamed ileum, as has been

implicated in other viscera (Yoshimura et al. 2001;

Bielefeldt et al. 2002a,b) and the somatic nervous system.

In the somatic nervous system (Khasar et al. 1998; Tanaka

et al. 1998) these changes were also accompanied by

neuronal hyperexcitability and molecular evidence of

increased expression of sodium channels (Gould et al.1998, 1999), specifically the TTX-R channel subtype,

NaV1.8 (Tanaka et al. 1998). Moreover, mice deficient in

Nav1.8 exhibit decreased neuronal excitability

(Renganathan et al. 2001) and decreased visceral pain

(Laird et al. 2002). Antisense NaV1.8 oligodeoxynucleotide

can also prevent inflammation-induced mechanical

hyperalgesia (Khasar et al. 1998) and cyclophosphamide

cystitis-induced bladder hyperreflexia (Yoshimura et al.2001). Taken together, these studies suggest that


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inflammation-induced increased expression of TTX-R INa

in nociceptive neurons is common to inflammation in

both the somatic and visceral organs, including the GI

tract, and is an important mechanism underlying

neuronal hyperexcitability and visceral hyperalgesia.

Potential mechanisms mediating inflammation-induced changes in ion channelsThe inflammatory milieu surrounding the nerve terminal

contains numerous inflammatory mediators, such as

PGE2, and 5-HT, which have been shown to augment

TTX-R Na+ currents in vitro, in both visceral (Gold et al.2002) and somatic afferents (Cardenas et al. 2001; Gold etal. 2002). PGE2 has also been shown to decrease IK

potassium currents through the same cyclic AMP–protein

kinase C-dependent mechanism which alters Na+ currents,

in unidentified DRG neurons (Nicol et al. 1997; Evans etal. 1999). These changes occur within minutes and are

dependent on the activation of intracellular second

messenger systems, and probably subsequent

phosphorylation of ion channels or other regulatory cell

signalling proteins. These actions cannot account for the

findings in the present study because recordings were

obtained from cell bodies of DRG neurons located in

ganglia outside the inflammatory milieu of the intestine

and recordings were made at least 6–8 h after removal of

the neurons from the animal at a time when unlabelled

neurons were unaffected. Consequently, changes in ionic

currents appear to result from longer term alterations such

as transcriptional events altering the number of channels,

their subunits or other biophysical properties of the

membrane channels themselves.

Both activity-dependent and growth factor signalling

pathways have been proposed to activate transcription

factors which can alter ion channel expression. In

particular there is considerable evidence that the

neurotrophin nerve growth factor (NGF) can increase

expression of TTX-R INa (Fjell et al. 1999; Bielefeldt et al.2003). In addition NGF and other neuotrophins have been

shown to modulate various potassium channels, via

activation of tyrosine kinases (Yang et al. 2001) or via

activation of ceramide (Zhang et al. 2002). Thus NGF

appears to be an important candidate for modulating ion

channel activity in visceral inflammation. However,

experiments specifically designed to test this hypothesis

are needed. It is also unknown whether the diverging

effects on these currents are due to signalling through a

single pathway or whether multiple pathways are involved.

ConclusionsThis study demonstrates that an animal model of

inflammatory bowel disease is associated with a

suppression of transient and sustained K+ currents and

augmentation of TTX-R INa currents in intestinal sensory

neurons. The implications are that these mechanisms are

important in the increased neuronal excitability seen in

these neurons as well as in the increased nociceptive

trafficking which occurs during intestinal inflammation,

such as in Crohn’s disease. Furthermore, there is

increasing evidence that visceral hyperalgesia underlies

many functional bowel disorders, such as post-infectious

irritable bowel syndrome. Whether the findings observed

in the current study are present following the resolution of

macroscopic inflammation, or whether low levels of

microscopic inflammation, which are suggested to persist

in these conditions (Collins et al. 2001; Chadwick et al.2002; Tornblom et al. 2002), are sufficient to sustain these

changes remains to be established.

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Acknowledgements This work was supported by a grant from The Crohn’s and ColitisFoundation of Canada. M. J. Beyak was supported by a CIHR/CAG/Astra-Zeneca Research Initiative Award. The authors wishto thank Margaret O’Reilly and Iva Kosatka for their experttechnical assistance in the performance of these experiments.


Ileitis modulates potassium and sodium currents in guinea pig dorsal root ganglia sensory neurons

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Transcript of Ileitis modulates potassium and sodium currents in guinea pig dorsal root ganglia sensory neurons