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Am J Physiol Gastrointest Liver Physiol 291: G424-G431, 2006. First published April 27, 2006; doi:10.1152/ajpgi.00560.2005
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MUCOSAL BIOLOGY

Enhanced excitability and suppression of A-type K+ current of pancreas-specific afferent neurons in a rat model of chronic pancreatitis

Guang-Yin Xu, John H. Winston, Mohan Shenoy, Huaizhi Yin, and Pankaj Jay Pasricha

Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas

Submitted 13 December 2005 ; accepted in final form 25 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic pancreatitis (CP) is a relatively common disorder, characterized by glandular insufficiency and chronic, often intractable, pain. The mechanism of pain in CP is poorly understood. We have previously developed a model of trinitrobenzene sulphonic acid (TNBS)-induced CP that results in nociceptive sensitization in rats. This study was designed to examine changes in the excitability and alteration of voltage-gated K+ currents of dorsal root ganglia (DRG) neurons innervating the pancreas. CP was induced in adult rats by an intraductal injection of TNBS. DRG neurons innervating the pancreas were identified by 1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate fluorescence labeling. Perforated patch-clamp recordings were made from acutely dissociated DRG neurons from control and TNBS-treated rats. Pancreas-specific DRG neurons displayed more depolarized resting potentials in TNBS-treated rats than those in controls (P < 0.02). Some neurons from the TNBS-treated group exhibited spontaneous firings. TNBS-induced CP also resulted in a dramatic reduction in rheobase (P < 0.05) and a significant increase in the number of action potentials evoked at twice rheobase (P < 0.05). Under voltage-clamp conditions, neurons from both groups exhibited transient A-type (IA) and sustained outward rectifier K+ currents (IK). Compared with controls, the average IA but not the average IK density was significantly reduced in the TNBS-treated group (P < 0.05). The steady-state inactivation curve for IA was displaced by ~20 mV to more hyperpolarized levels after the TNBS treatment. These data suggest that TNBS treatment increases the excitability of pancreas-specific DRG neurons by suppressing IA density, thus identifying for the first time a specific molecular mechanism underlying chronic visceral pain and sensitization in CP.

dorsal root ganglion; inflammation; visceral pain; trinitrobenzene sulfonic acid; membrane properties

PAIN is a cardinal feature of chronic pancreatitis (CP) and remains the most important clinical challenge in these patients (5, 34). Despite much speculation, little is known about the underlying mechanisms responsible for this symptom. By definition, CP is an inflammatory condition and is therefore expected to produce significant functional changes in the nociceptive system serving this organ, as has been observed in other forms of visceral inflammation such as cystitis, gastric ulcers, and ileitis (4, 21, 29, 40). However, until now, it has been difficult to demonstrate this in CP, in large part due to the lack of an appropriate animal model. We (35) have recently developed a novel and robust model of pain behavior and sensitization after the induction of CP with an intraductal injection of trinitrobenzene sulfonic acid (TNBS) in the rat. Three weeks after TNBS treatment, a robust chronic inflammation in the pancreas is seen, which is very similar to changes observed in human CP (9). Rats with TNBS-induced CP also exhibit hypersensitivity including pancreatic hyperalgesia and referred somatic allodynia and hyperalgesia, with the latter responding to conventional analgesics (35). These findings support the construct, face, and predictive validity of this model, rendering it suitable for studying the cellular and molecular responses of nociceptors in CP.

Voltage-gated Na+ channels and voltage-gated K+ (Kv) channels play a fundamental role in controlling neuronal excitability. Both the upregulation of Na+ currents and suppression of K+ currents appear to contribute to peripheral sensitization (2, 3, 29, 40). As part of an ongoing investigation, we first focused on Kv currents of dorsal root ganglion (DRG) neurons innervating the pancreas. A multitude of K+ channels are expressed by nociceptor neurons and have been characterized mainly by their biophysical properties with little molecular correlation. Kv currents are important modulators of spike frequency and activation thresholds (24, 29, 40, 41). Two important Kv currents are the transient A-type K+ current (IA) and the sustained delayed rectifier K+ current (IK) (1, 8, 10, 20). Recent studies in the guinea pig ileitis (29) and rat gastric ulcers (4) have shown that experimentally induced inflammation of the gastrointestinal tract enhanced the excitability of organ-specific sensory neurons, but the changes in voltage-gated ion channels were inconsistent. TNBS-induced ileitis greatly reduced both IK and IA density in DRG neurons innervating the ileal wall (29). The acetic acid-induced gastric ulcers reduced IA density in DRG neurons innervating the stomach, whereas the sustained IK density was not altered (4). Taken together, it is more likely that changes in ion channel function in DRG neurons are organ/disease specific.

In this study, we investigated changes in the membrane properties and excitability of pancreas-specific DRG neurons in response to chronic inflammation. Furthermore, we specifically examined changes in K+ channel conductance in these cells, a critical factor in determining the responsiveness of neurons to stimulation. Our findings indicate that CP results in increased spontaneous and evoked excitability of these neurons and that this is associated with a decrease in K+ channel currents of the IA type. Our study therefore provides the first mechanistic insight into painful CP from a neurobiological perspective and identifies a potential molecular target for therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell labeling and induction of chronic pancreatitis. Experiments were performed on adult male Sprague-Dawley rats (120–150 g). Care and handling of these animals were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and were in accordance with the guidelines of the International Association for the Study of Pain. Cell labeling and the induction of CP were performed as previously described (22, 35). The tracer was injected before a pancreatic duct infusion of TNBS or vehicle. In brief, animals were anesthetized with ketamine (80 mg/kg ip) plus xylazine (5–10 mg/kg ip). The abdomen was opened by a midline laparotomy, and the pancreas was exposed. The lipid soluble fluorescence dye 1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate (DiI; Molecular Probes; Eugene, OR), 25 mg in 0.5 ml methanol, was injected in 2-µl volumes at 8–10 sites on the exposed pancreas. To prevent leakage and possible contamination of adjacent organs with the dye, the needle was left in place for 1 min and each injection site was washed with normal saline after each injection. For the induction of CP, the common bile duct was closed temporarily near the liver with a small vascular clamp. A blunt 28-gauge needle with polyethylene-10 tubing attached was gently inserted into the duodenum and guided through the papilla into the biliopancreatic duct and secured with sutures. TNBS (0.5 ml of 2% solution of TNBS in 10% ethanol in PBS; pH 7.4) or vehicle was infused into the duct over a period of 2–5 min at a pressure of 50 mmHg. After 30 min, the needle and tubing were removed, the hole in the duodenum was sutured, and the vascular clamp was then removed, which restored the bile flow. The pancreas was gently swabbed before the abdomen was closed. Animals were returned to their housing and given free access to drinking water and standard food pellets.

Dissociation of DRG neurons and patch-clamp recording. Isolation of DRG neurons from adult control and TNBS-treated rats has been described previously (38, 39). Briefly, 21–26 days after TNBS or vehicle injection, rats were killed by cervical dislocation, followed by decapitation. DRGs (T9–13) were then bilaterally dissected out and transferred to an ice-cold, oxygenated fresh dissecting solution, which contained (in mM) 130 NaCl, 5 KCl, 2 KH2PO4, 1.5 CaCl2, 6 MgSO4, 10 glucose, and 10 HEPES, pH 7.2 (osmolarity: 305 mosM). After removal of the connective tissue, ganglia were transferred to 5 ml of dissecting solution containing collagenase D (1.8–2.0 mg/ml, Roche; Indianapolis, IN) and trypsin (1.2 mg/ml, Sigma; St. Louis, MO) and incubated for 1.5 h at 34.5°C. DRGs were then taken from the enzyme solution, washed, and transferred to 2 ml of the dissecting solution containing DNase (0.5 mg/ml, Sigma). A single-cell suspension was subsequently obtained by repeated trituration through flame-polished glass pipettes. Cells were plated onto acid-cleaned glass coverslips.

Coverslips containing adherent DRG cells were put in a small recording chamber (0.5-ml volume) and attached to the stage of an inverting microscope (Olympus) fitted for both fluorescence and bright-field microscopy. DiI-labeled neurons were identified by their fluorescence under the fluorescent microscope. For the patch-clamp recording experiments, cells were continuously superfused (1.5 ml/min) at room temperature with normal external solution containing (in mM) 130 NaCl, 5 KCl, 2 KH2PO4, 2.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH (osmolarity: 295–300 mosM). Recording pipettes were pulled from borosilicate glass tubing using a horizontal puller (P-97, Sutter Instruments) and typically had a resistance of 1.5–2.5 M{Omega} when filled with normal external solution. For perforated patch recording, 3 µl of a 50 mg/ml stock solution of amphotericin B (Calbiochem; La Jolla, CA) in DMSO (Sigma) were added to 0.5 ml of pipette solution to yield a final concentration of 200 µg/ml. The pipette tip was initially filled with amphotericin B-free solution, which contained (in mM) 100 KmeSO3, 40 KCl, and 10 HEPES, with pH 7.25 adjusted with KOH (290 mosM). The pipette was then back filled with the amphotericin B/pipette solution before being used immediately to obtain a gigaohm seal. Tip potentials were zeroed before membrane-pipette seals were formed. Perforation of the membrane patch, as revealed by the appearance of slow capacitance transients, occurred within 5–25 min, and recordings were only made when access resistance fell to <15 M{Omega}. The voltage was clamped at –60 mV by a Dagan 3911 patch-clamp amplifier (Dagan; Minneapolis, MN). Capacitive transients were corrected using capacitive cancellation circuitry on the amplifier that yielded the whole cell capacitance and access resistance. Up to 90% of the series resistance was compensated electronically. Considering the peak outward current amplitudes of <10 nA, the estimated voltage errors from the uncompensated series resistance would be <10 mV. The leak currents at –60 mV were always <20 pA and were not corrected. The currents were filtered at 2–5 kHz and sampled at 50 or 100 µs/point. Whole cell current and voltage were recorded with a Dagan 3911 patch-clamp amplifier; and data were acquired and stored on a Dell computer for later analysis using pCLAMP 9.2 (Axon Instruments; Sunnyvale, CA).

Isolation of Kv currents. To record Kv currents, Na+ in control external solution was replaced with equimolar choline and the Ca2+ concentration was reduced to 0.03 mM to suppress Ca2+ currents and to prevent Ca2+ channels becoming Na+ conducting (7). The reduced external Ca2+ would also be expected to suppress Ca2+-activated K+ current. The following two kinetically distinct Kv currents were isolated by the biophysical analysis and pharmacological approaches described in previous studies (1, 8, 10, 20, 29): IA and IK. IA and IK were separated biophysically by manipulating the holding potentials. The total outward currents (Itotal) were recorded in response to voltage steps from –100 to +30 mV in 5-mV increments with duration of 400 ms. IK was isolated when the membrane potential was held at –50 mV. Subtraction of IK from Itotal represented IA. To control for changes in cell size, the current density was measured by dividing the current amplitude by whole cell membrane capacitance (pA/pF), which was obtained by reading the value for whole cell input capacitance cancellation directly from the patch-clamp amplifier.

To study IA and IK channel conductance, activation curves were generated by voltage pulses in 5-mV steps from –80 to +30 mV. To determine the voltage dependence of the steady-state inactivation of IA, two-pulse voltage protocols as previously reported were employed in this experiment. Residual IA was measured after short conditioning pulses (1-s duration), which allowed inactivation of only rapidly inactivating IA. To ensure that IA was not significantly contaminated by slow inactivation of the delayed rectifier, we analyzed kinetic data on current decay. The decay phase of the currents was well fitted with a single exponential. The fitting correlation coefficient was much closer to 1 and the SD was much smaller compared with two or three exponentials. The time constant of inactivating IA was 145 ± 11 ms (n = 5) at a test pulse of 30 mV, which is close to previous data of inactivating IA of neurons innervating the rat urinary bladder (40) and guinea pig intestine (29). This excludes the possibility of contamination of our results by slow inactivation of the delayed rectifier. The peak IA was measured as the peak of the transient component of the subtracted current at every given voltage. All experiments were performed at room temperature (~22°C).

Data analysis. The membrane conductance (G) at each command potential (Vm) was determined by dividing the measured membrane current (I) by the driving force as follows: [G = I/(Vm – EK)], where EK is the equilibrium K+ potential and was calculated to be –76 mV (external [K+] = 7 mM and internal [K+] = 140 mM). Activation data (G-V curve) were fitted by the following modified Boltzmann equation: G/Gmax = 1/{1 + exp[–(V1/2 – Vm)/k]}, where Gmax is the fitted maximal conductance, V1/2 is the membrane potential for half-activation, and k is the slope factor. Steady-state inactivation of IA was fitted with the following negative Boltzmann equation: I/Imax = 1/{1 + exp[–(V1/2 – Vm)/k]}, where Imax is maximal current. Inactivation data were plotted as I/Imax versus the prepulse voltage used to generate the inactivation curves. No neuron with a resting membrane potential (RP) more depolarized than –40 mV was included in the data analysis. All data are expressed as means ± SE. Statistical significance was determined by Student's t-test or Fisher's exact test, as appropriate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Alteration in spontaneous activities and RPs. To determine the effect of TNBS treatment on the excitability of pancreas-specific DRG neurons, RPs of DRG neurons from control and TNBS-treated rats were first studied. DRG neurons were acutely dissociated; labeled pancreas-specific neurons were identified under fluorescent microscopy and studied using current-clamp techniques. In control rats, RPs of neurons recorded were very stable with <3-mV changes within 2 min of observation (Fig. 1A), and no spontaneous firing was seen (Table 1). In TNBS-treated rats, however, RPs of neurons (n = 7) recorded showed marked fluctuations, ranging from 5 to 15 mV within a 2-min observation period (Fig. 1B). In addition, four neurons displayed spontaneous firings (Fig. 1C). The spontaneous firing frequency varied between neurons and ranged from 0.5 to 6 Hz (average: 3.4 Hz, n = 4). The percentage of neurons with spontaneous oscillations (including spontaneous firings) was significantly higher than that of the control group (Table 1; P < 0.01). We also compared RPs of DRG neurons from both groups after excluding electrically unstable (fluctuation >5 mV) neurons. As a group, pancreas-specific DRG neurons from animals with CP were significantly more depolarized at rest than controls (Fig. 1D and Table 1; P < 0.005). These data suggest that a subset of pancreas-specific neurons is spontaneously active in the setting of CP.


Figure 1
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  		Fig. 1. Dorsal root ganglion (DRG) neurons from trinitrobenzene sulfonic acide (TNBS)-treated rats are spontaneously active and rest at more depolarized membrane potentials. A and B: example of resting membrane potential (RP) recorded from DRG neurons from control (Con; A) and TNBS-treated rats (B). The RP was very stable in the control group but exhibited a 5- to 15-mV fluctuation after TNBS treatment. C: spontaneous firings recorded from a neuron from a TNBS-treated rat. D: bar graph showing the average of RP of neurons from control and TNBS-treated rats. TNBS treatment significantly depolarized the RP. *P < 0.005.

 

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  		Table 1. Properties of spontaneous activities of pancreas-specific DRG neurons from control and TNBS-treated rats

 
Reduction of current threshold and increase in cell firing frequency. To further study the excitability of pancreas-specific DRG neurons in CP, we examined the current threshold (rheobase) and the pattern of firing in response to depolarizing current stimulation. The rheobase is the minimal current injection to induce one action potential (AP). In this study, the average rheobase of DRG neurons from TNBS-treated animals was markedly lower than that of controls (Fig. 2, A, B, top, and C, and Table 2; P < 0.01). In addition, the numbers of APs in response to a current stimulation (2 x rheobase) were also examined. The numbers of evoked APs were different in these two groups: 1.2 ± 0.3 APs/100 ms (range 1–2, n = 16; Fig. 2D) in the control group compared with 3.1 ± 0.8 APs/100 ms (range 2–5, n = 16; Fig. 2, E, top, and F, and Table 2; P < 0.02) in the CP group.


Figure 2
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  		Fig. 2. Reduction in rheobase and increase in firing frequency. Representative traces of action potentials were induced by 100-ms depolarizing current pulses injected through the patch pipette at rheobase (A and B) and two times rheobase (D and E) in labeled neurons from control and TNBS-treated rats under current-clamp conditions. TNBS treatment resulted in a marked reduction of the rheobase while significantly increasing the number of action potentials induced by a two times rheobase current injection. Note that in B and E, bottom traces, membrane potentials before current stimulation were corrected to the average level of neurons from the control group with current injection to better reveal the TNBS effect. C: bar graph showing the average of rheobase in pancreas-specific DRG neurons from control (n = 17) and TNBS (n = 20) rats (*P < 0.01). F: bar graph showing the average number of action potentials elicited by a two times rheobase current injection in these same groups of neurons (*P < 0.02).

 

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  		Table 2. Membrane characteristics of pancreas-specific DRG neurons in control and TNBS-induced chronic pancreatitic rats

 
To examine whether changes in rheobase and firing frequency of neurons from TNBS-treated rats could be explained simply by the observed differences in the RP, in further experiments, we adjusted the RPs to –58 mV (the average level of neurons from control rats) by injecting hyperpolarizing current into the cells recorded from TNBS-treated rats. After membrane potential adjustment, the rheobase for evoking an AP of neurons from the TNBS-treated group was not significantly altered (Fig. 2B, bottom) and was still markedly lower than that in the control group (TNBS correction: 0.20 ± 0.05 nA, n = 5; control: 0.61 ± 0.11 nA, n = 20, P < 0.05). The numbers of repetitive firings evoked by two times rheobase stimulation after membrane potential adjustment remained significantly higher than those in the control group (Fig. 2E, bottom; TNBS correction: 3.0 ± 0.8 APs/100 ms, n = 5; control: 1.2 ± 0.3 APs/100 ms, n = 16, P < 0.05).

Several additional electrical properties were also examined, with the results shown in Table 2. Notably, both AP duration (at 0 mV) and membrane input resistance in TNBS-treated rats were significantly greater than those in controls (P < 0.05). On the other hand, AP threshold and amplitude were not significantly changed after TNBS treatment (Table 2). TNBS treatment did not alter the cell size distribution of pancreas-specific neurons (Fig. 3 and Table 2). In both the control (n = 117) and TNBS-treated groups (n = 111), pancreas-specific DRG neurons were mainly medium-sized neurons (>20 and <35 µm) with small-size neurons (<20 µm) and large-size neurons (>35 µm) making a smaller contribution. To simplify our analysis, only small- and medium-sized cells were included in this study.


Figure 3
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  		Fig. 3. Cell size distribution of pancreas-specific DRG neurons. TNBS treatment did not significantly alter the size distribution among DRG neurons compared with controls (small: diameter ≤25 µm, medium: diameter >25 µm and <35 µm, and large: diameter ≥35 µm).

 
Suppression of IA density. Because changes in spike frequency and activation thresholds suggest an alteration in Kv channels (24, 29, 40, 41), we next performed perforated patch-clamp recordings to examine these currents under voltage-clamp conditions. Na+ in the control external solution was replaced with equimolar choline and the Ca2+ concentration was reduced to 0.03 mM. A depolarization step from –100 to +30 mV in 5-mV increments with a duration of 400 ms activated all Kv channels (Itotal; Fig. 4A). The peak current-voltage (I-V) curves are shown in Fig. 4D. TNBS treatment significantly reduced Itotal density in DiI-labeled neurons compared with the control (control: 114.0 ± 4.5 pA/pF, n = 6; TNBS: 88.1 ± 5.5 pA/pF, n = 9, P < 0.05; Fig. 4G). Because there were two main types of Kv currents (IA and IK) described in nociceptive DRG neurons, we then isolated these two kinetically different Kv currents by manipulating the holding membrane potential. A depolarization step from –50 to +30 mV in 5-mV increments with duration of 400 ms activated most of the sustained Kv channels but not A-type Kv channels (Fig. 4, B and E). Subtraction of IK from Itotal yields IA (Fig. 4, C and F). IA was further confirmed by the application of the A-type channel blocker 4-aminopyridine (4-AP; 5 mM, data not shown) and the reversal potential, which was determined for each individual current from its instantaneous tail current. Because the reversal potential of IA and IK was close (–71 mV) to the predicted reversal potential for K+, it is assumed that these currents are carried by potassium ions. The peak I-V curves are shown in Fig. 4, E and F. In this experiment, IA density was remarkably reduced in animals with CP (control: 72.8 ± 6.0 pA/pF, n = 6; TNBS: 48.0 ± 6.7 pA/pF, n = 9, P < 0.05; Fig. 4I), whereas IK density was not significantly changed (control: 51.7 ± 12.0 pA/pF, n = 6; TNBS: 50.4 ± 7.1 pA/pF, n = 9, P > 0.05; Fig. 4H).


Figure 4
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  		Fig. 4. TNBS-induced chronic pancreatitis (CP) significantly reduced the A-type K+ current (IA) density. Currents were measured at different holding potentials. A: for total voltage-gated K+ (Kv) current, the membrane potential was held at –100 mV and voltage steps were from –40 to +30 mV with 5-mV increments and 400-ms duration. B: for sustained Kv current, the membrane potential was held at –50 mV and the voltage steps were the same as above. C: currents generated by these two protocols were subtracted to produce IA. The peak currents of total current (ITotal; D), outward rectifier K+ current (IK; E) and IA (F) versus voltages (I-V) were plotted from the above representative cell. IA amplitude was measured as the peak of the transient component, and IK amplitude was measured at the ending point of 400-ms voltage steps. G–I: bar graphs showing the mean peak ITotal (G), IK (H), and IA (I) densities from control and TNBS-treated rats. The current density (in pA/pF) was calculated by dividing the current amplitude by cell membrane capacitance. TNBS-induced CP caused a significant reduction of total Kv current from 114.0 ± 4.5 pA/pF (n = 6) in the control group to 88.1 ± 5.5 pA/pF (n = 9) in the TNBS group (G; *P < 0.05). Current subtraction revealed that CP did not alter the IK density compared with controls (H; P > 0.05); however, the IA density was significantly reduced from 72.8 ± 6.0 pA/pF (n = 6) in the control group to 48.0 ± 6.7 pA/pF (n = 9) in the TNBS group (I; *P < 0.05).

 
Leftward shift in the inactivation curve of IA. Because a reduction in the peak current density of IA was evident, we then analyzed the voltage dependence of activation and inactivation of electrophysiologically isolated IA channels. Activation-voltage relationships were constructed from I-V curves of neurons from control and TNBS-treated rats. Currents at various test pulses were divided by the driving force for K+ (with –76 mV as the value for EK) and the resulting conductance was expressed as a percentage of that achieved at +30 mV for each condition (Fig. 5A). The G-V relationships were fitted with a Boltzmann equation to derive values for V1/2 and k. The G-V curve obtained from the control group had a V1/2 of –19.8 ± 3.1 mV and k of 12.7 ± 1.7 (n = 7; Fig. 5, C and D). TNBS treatment did not significantly change the V1/2 (–15.7 ± 1.8 mV) or k (14.6 ± 2.4) of the G-V curve (n = 8; Fig. 5, A, C, and D). To determine whether the reduction in IA involved a shift of the steady-state inactivation curve, a two-pulse voltage protocol was employed as described above. Inactivation curves were obtained by plotting IA during the test pulse against the membrane potential during the conditioning pulse. IA was normalized to Imax during the test pulse to 0 mV after a conditioning pulse to –160 mV (Fig. 5B). Data were fitted with the negative Boltzmann function. The I-V curve of steady-state inactivation obtained from the control group had a V1/2 of –73.6 ± 2.9 mV and k of 4.9 ± 0.4 (n = 3; Fig. 5, C and D). In contrast, the V1/2 and k of the I-V curve of inactivation from the TNBS-treated group were –94.5 ± 5.9 mV (n = 4; Fig. 5C) and 5.2 ± 0.6, respectively (n = 4; Fig. 5D). Thus TNBS treatment led to a ~20-mV negative shift in the V1/2 of the steady-state inactivation curve (P < 0.05); as such, at –60 mV, the IA in pancreas-specific DRG neurons from the TNBS-treated group was almost negligible. In contrast, ~20% of Imax could be available for activation in the control group. Therefore, this shift in the steady-state inactivation curve can account for the enhanced excitability of pancreas-specific DRG neurons in animals with CP.


Figure 5
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  		Fig. 5. Steady-state activation and inactivation curves for IA. A: for activation curves, IA was generated by two voltage pulses in 5-mV increment steps from –100 to 30 mV in one DRG neuron from a control rat and one from a TNBS-treated rat. The reversal membrane potential (Vrev) in this recording condition was –71 mV. Membrane conductances (G) at different test potentials were measured by dividing the peak IA by the current driving force (Vm – Vrev), and were normalized to that recorded at 30 mV (Gmax). Data were fitted with the following Boltzmann equation: G/Gmax = 1/{1 + exp[–(V – V1/2)/k]}, where V is membrane potential, V1/2 is the membrane voltage at which the current was half-maximally activated, and k is the slope factor. TNBS treatment did not change the activation curve compared with the control. V1/2 was –19.8 ± 3.1 mV (n = 7) for neurons from control rats and –15.7 ± 1.8 mV (n = 8) for neurons from TNBS-treated rats (C); k was 12.7 ± 1.7 (n = 7) and 14.6 ± 2.4 mV (n = 8) for neurons from control and TNBS-treated rats, respectively (D). B: for steady-state inactivation curves, a long conditional step of various voltages from –160 to 0 mV in 10-mV increment was followed by a testing pulse to +30 mV. These inactivation curves are representative curves of one neuron from a control rat and one neuron from a TNBS-treated rat, respectively. The peak current amplitude was normalized to that recorded at a –160-mV conditional step (Imax). Data were plotted as a function of conditional step potentials and fitted with the following Boltzmann equation: I/Imax = 1/{1 + exp[–(V1/2 – V)/k]}. TNBS treatment resulted in a significant leftward shift of the steady-state inactivation curve. V1/2 was –73.6 ± 2.9 mV for neurons (n = 3) from the control group and –94.5 ± 5.9 mV for neurons (n = 4) from the TNBS group (C; *P < 0.05); k was 4.9 ± 0.4 and 5.2 ± 0.6 mV for neurons from control (n = 3) and TNBS-treated rats (n = 4), respectively (D; P > 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Inflammation of an organ is characteristically accompanied by peripheral sensitization, a phenomenon in which sensory neurons respond in an abnormal and exaggerated manner to stimulation. The effect or processes by which sensitization is maintained include signal transduction, generation of APs, and neurotransmitter release. These functions are in turn subserved by several different classes of ion channels such as transient receptor potential and voltage-dependent Na+, K+, and Ca2+ channels. The purpose of our study was to examine the effects of CP on the electrophysiological properties of pancreatic sensory neurons and to provide insight into potential ionic mechanisms. To do this, we took advantage of our previously validated rat model for studying pain behavior in CP induced by intraductal TNBS. This model is accompanied by pancreatic allodynia and hyperalgesia as well as increases in neuropeptide expression and release by sensory neurons, providing strong evidence for peripheral sensitization (35). In this study, we provide the first direct evidence for hypersensitivity of pancreas-specific primary sensory neurons in the setting of CP. This conclusion is based on several findings shown in Figs. 1 and 2. First, pancreatic neurons from animals with CP displayed marked fluctuation of RP, spontaneous discharges, and more depolarized RPs than controls (Fig. 1 and Table 1), indicating that these neurons are spontaneously active. Second, these neurons exhibited lower current thresholds for initiating an AP compared with controls. Finally, these neurons had enhanced firing frequencies in response to a standardized stimulation compared with controls (Fig. 2 and Table 2).

Another interesting finding is that TNBS-treatment significantly suppressed IA density in pancreas-specific DRG neurons (Fig. 4). This is consistent with and an extension of previous reports of models of cystitis, ileitis, and gastric ulcers (4, 21, 26, 29, 40). Therefore, the reduction in IA density may well contribute to the enhanced excitability of pancreas-specific neurons in our model as well. Possible mechanisms for the reduction of IA density include somal hypertrophy, downregulation of IA channel expression, and changes in channel properties. Because the cell diameter was not altered after the TNBS treatment (Fig. 3 and Table 2), the reduction of IA density cannot be attributed simply to somal hypertrophy of pancreas-specific DRG neurons. Although single-channel properties and IA channel expression of pancreas-specific neurons after inflammation have yet to be studied, our analysis of the voltage dependence of steady-state inactivation showed that the I-V curve for IA channels shifted significantly in a hyperpolarized direction in the setting of CP (Fig. 5B). As such, this would make fewer IA channels available at or near RPs. According to our results, ~20% of IA channels were available for activation at –60 mV in the control group compared with only ~3% in the TNBS-treated group. Because there was no significant change of the activation-voltage relationship of IA (Fig. 5A), these data strongly suggest that the large shift of the steady-state inactivation toward a negative direction may be a major cause for the suppression of IA density after TNBS-induced inflammation (Fig. 4I) and could thus contribute to the enhanced excitability. The mechanism underlying this shift is yet unclear but may result from changes in either the function or expression of K+ channels. Nerve growth factor (NGF) could be a good candidate to regulate the function and expression of Kv channels because a large body of evidence shows that NGF plays an important role in producing sensitization in somatic pain models (37) and in acute and chronic CP (30, 35, 36) and affecting ion channels and membrane properties in afferent sensory neurons and pheochromocytoma-12 cells (18, 27, 42). To better understand the molecular basis of chronic changes in K+ currents in inflammatory states, further studies should be performed on the expression of Kv channels, especially the Kv1.4 channel, which may be the dominant IA channel protein in nociceptors (23) and is downregulated in DRGs in axotomy models (12, 15, 23).

In addition to the effect of IA, the contribution of other K+ channels to the enhanced excitability of pancreas-specific neurons has also to be considered. Sustained K+ currents have been reported to be altered after TNBS-induced ileitis (29). In this study, TNBS-induced CP had no effect on IK because the IK density was not significantly changed in the TNBS group compared with controls (Fig. 4H). Because IA was obtained by subtracting the current evoked from –50 mV from that evoked from –100 mV, it is possible that IA might be contaminated by IK subject to steady-state inactivation. However, this seems unlikely because IA was defined as that arising between the holding potential and potential minimal just after the capacity transient, 2–4 ms from the beginning of the depolarization steps. This point of measurement for IA was used to minimize contamination with IK. In addition, IA was completely blocked by 4-AP (2 mM, data not shown), whereas IK was resistant to 4-AP. Furthermore, the kinetic data on IA decay showed that the time constant of this current was 145 ms at a test potential of 30 mV, which is close to previous data of inactivating IA of neurons innervating the rat stomach (4), rat urinary bladder (40), and guinea pig intestine (29). Therefore, the contamination of IA with IK was minimal in this study. Pancreas-specific neurons from rats with CP also displayed an increased input membrane resistance compared with controls (Table 2). Changes in RP and membrane input resistance may also be mediated by modulation of hyperpolarization-activated cation current (IH; an inwardly rectifying current) or leakage current (IL; an outward resting current) (25). However, the depolarization of RP in our study is unlikely to be due to an increase in IH, as this would be expected to result in a decrease in input resistance. The depolarization of RPs in pancreatic DRG neurons in CP may have resulted from a decrease in IL, resulting in an increase in input membrane resistance. However, in our experiments, when the RPs of DRG neurons from TNBS-treated rats were corrected to the normal level (i.e., –58 mV), the rheobase and cell spike frequency were not significantly changed compared with those before the correction of RP (Fig. 2). Thus, even if there were changes in IL, they had a minimal effect on spike frequency and current threshold.

These changes in membrane properties and Kv currents thus provide a clear neurobiological explanation for the previously reported behavioral responses in this model (35) and set the stage for further mechanistic studies on pain in CP. Advances in knowledge in this area have been limited by the lack of a suitable experimental model. We (36) and others (6, 11, 16, 19, 28, 33) have previously reported on nociceptive changes in rat models of acute pancreatitis. Although important, these studies were limited by the fact that there are significant differences between the neurobiology of acute pancreatitis and CP. There has only been one previous report (32) measuring abdominal hypersensitivity in a "chronic" model using dibutyltin to induce inflammation. However, dibutyltin affects tetrodotoxin-resistant Na+ currents in sensory neurons in addition to its effect as a general neurotoxin (13, 14, 17, 31), thus seriously confounding the interpretation of the findings. In contrast, the TNBS-induced model of CP is morphologically similar to human CP (construct validity) and also displays face and predictive validity, thus representing a robust experimental approach for the establishment of working paradigms to explain the pathogenesis of pain in CP. Our findings of a lower current threshold and enhanced firing frequencies of pancreas-specific nociceptive DRG neurons (Fig. 2) are similar in some ways to those reported in experimental inflammation of other visceral organs such as the urinary bladder (26, 40), stomach (4), and intestine (21, 29). However, there are also notable differences. Thus no changes in RP were seen in either a mouse model of colitis (induced by TNBS) (2) or a rat model of cystitis (induced by cyclophosphamide) (40). Furthermore, we did not find any changes in neuronal size in our model, unlike the significant increases reported with interstitial cystitis in cats (26), chronic cystitis in rats (40), and TNBS ileitis in guinea pigs (21). Although some of these differences may be species related, our results also suggest that nociceptor changes may be organ and/or disease specific, further attesting to the importance of an appropriate model.

In conclusion, our data demonstrate that TNBS-induced CP is associated with an increased neuronal excitability and suppression of IA in pancreas-specific DRG neurons. Because changes in current threshold and spike frequency all suggest alteration of A-type K+ channels, the observed suppression of IA in our study assumes particular significance, potentially identifying for the first time a specific molecular mechanism underlying visceral pain and hypersensitivity in CP. This information and further studies hold the promise for providing new strategies for the treatment of pain in CP.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-62330-01 (to P. J. Pasricha).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. J. Pasricha, Div. of Gastroenterology and Hepatology, Dept. of Internal Medicine, Univ. of Texas Medical Branch, Galveston, Texas 77555 (e-mail: jpasrich{at}utmb.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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