Role of nerve growth factor in modulation of gastric afferent neurons in the rat

K. Bielefeldt1, N. Ozaki2, and G. F. Gebhart2

Departments of 1 Internal Medicine and 2 Pharmacology, University of Iowa, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies demonstrated that experimental ulcers are associated with changes in the properties of voltage-sensitive sodium currents in sensory neurons. We hypothesized that nerve growth factor (NGF) contributes to these changes. Gastric ulcers were induced by acetic acid injection into the wall of the rat stomach. NGF expression was determined by ELISA and immunohistochemically. Sensory neurons were labeled by injection of a retrograde tracer into the gastric wall. Sodium currents were recorded in gastric sensory neurons from nodose and dorsal root ganglia cultured for 24 h in the presence of NGF or a neutralizing NGF antibody, respectively. Gastric ulcer formation caused a rise in NGF concentration within the gastric wall and an increase in NGF immunoreactivity. Exposure to NGF caused a significant increase in the TTX-resistant sodium current, whereas the TTX-sensitive sodium current remained unchanged. This was associated with an acceleration of the recovery from inactivation in spinal sensory neurons. Production and release of NGF in the gastric wall may contribute to sensitization of primary afferent neurons during gastric inflammation.

visceral hyperalgesia; sodium current


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VISCERAL SENSATION PLAYS a central role in the physiology and pathophysiology of autonomic functions. Alterations in visceral sensation may be involved in the pathogenesis of a wide spectrum of diseases. They contribute to pain and discomfort experienced by patients with neoplastic or acute and chronic inflammatory diseases of the gastrointestinal tract and play an important role in functional disease, such as nonulcer dyspepsia or irritable bowel syndrome. We have recently demonstrated (30) that experimentally induced gastric ulcers trigger behavioral changes consistent with visceral hyperalgesia. This was associated with altered properties of voltage-sensitive sodium currents in primary sensory neurons within dorsal root (DRG) and nodose ganglion (4, 5). Similar to most models of hyperalgesia, the ulcer-induced gastric hyperalgesia relied on the induction of inflammation. Such inflammatory processes lead to the production and release of mediators and cytokines, which, in turn, may affect neuronal excitability and contribute to peripheral sensitization.

Neurotrophins are upregulated in the presence of tissue insult, including models of visceral inflammation associated with pain-related behaviors and in human disease (12, 37). It has long been appreciated that these growth factors play a central role in the development of the central and peripheral nervous systems. More recently, the importance of neurotrophins in maintaining and modulating the normal function of the nervous system in adults has been recognized (32). Neurotrophins, particularly nerve growth factor (NGF), normally act to maintain the sensitivity of nociceptors (33, 37). This is partly due to changes in the expression of ion channels in neurons (37). NGF produces prolonged pain when injected intradermally in humans (14). Intravesical instillation of NGF has been reported to sensitize pelvic nerve afferent fibers to bladder distension (13). We hypothesized that NGF contributes to the changes in sodium currents observed in gastric sensory neurons after induction of experimental ulcers.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals. Male Sprague-Dawley rats (ages 2-3 mo, Harlan Sprague Dawley, Indianapolis, IN) were used for the experiments. The animals were housed under a 12:12-h light-dark cycle with free access to water and food. Animal handling followed the guidelines of the American Physiological Society. The experimental protocol was approved by the Animal Care and Use Committee of The University of Iowa.

Food but not water was withheld for 12 h before surgery. To label gastric sensory neurons, animals were anesthetized with pentobarbital sodium (45-50 mg/kg ip). The stomach was exposed through a midline incision, and 16 µl of the dicarbocyanine dye 1.1'-dioctadecyl-3,3,3,'3-tetramethylindocarbocyanine methanesulfonate (DiI; 25 mg in 0.5 ml methanol) were injected (2 µl each in 8 sites) into the muscle of the stomach wall with a 30-gauge needle. DiI is incorporated into the lipid bilayer of nerve processes close to the site of injection and is transported to the cell body without transfer to adjacent cells. The epigastric incision was closed, and rats were allowed to recover for 7-10 days before harvesting the nodose or dorsal root ganglia.

Induction of experimental ulcers. Rats were anesthetized with pentobarbital sodium (45-50 mg/kg ip). The stomach was exposed through a midline incision and 5 µl of 20% acetic acid (vol/vol in sterile saline) were injected into 10 sites in the subserosal layer of the stomach in the glandular portion of the dorsal and ventral stomach wall. An equal number of same volume saline injections served as control.

Immunohistochemistry. The animals were deeply anesthetized, perfused with ice-cold 4% paraformaldehyde, and the stomach was removed. The tissues were postfixed overnight in fresh fixative and transferred to 30% sucrose. Inflamed areas of the glandular stomach and corresponding regions from control animals were sectioned on a cryostat at 10 µm. The sections were washed several times in PBS and treated with blocking solution containing 20% goat serum, 5% bovine serum albumin, and 0.1% Triton X-100. Endogenous peroxidase activity was eliminated by pretreatment with 3% H2O2. After repeated washings in PBS, the primary antibody (polyclonal anti-NGF; sc-549; dilution: 1:100; Santa Cruz Biotechnology, Santa Cruz, CA) was added for overnight incubation at 4°C. After repeated washings, the sections were incubated for 30 min with secondary anti-rabbit antibodies coupled to biotin. Commercially available reagents were used for development (Vector Laboratories, Burlingame, CA). Immunostaining was performed as outlined above for cryosections.

Quantitative determination of NGF in gastric tissue. Animals were anesthetized and decapitated at various points after intragastric injection of saline or acetic acid. The stomach was quickly removed and opened up along the greater curvature. Gastric contents were removed, and the stomach was quickly rinsed in ice-cold normal saline. The tissue was then frozen in liquid nitrogen and stored at -80°C for further analysis. The entire stomach was homogenized in ice-cold lysis buffer containing 137 mM NaCl, 20 mM Tris · HCl buffered to pH 8, 1% Nonidet P-40, 10% glycerol, 1 mM phenomethanesulfonyl fluoride, 10 µg/ml aprotinine, 1 µg/ml leupeptin, and 0.5 mM sodium vanadate. The protein concentration was determined with the Biuret method. The NGF concentration was measured using a commercially available assay following the manufacturer's instructions (NGF Emax Immunoassay, Promega; Madison, WI). All assays were done in triplicate.

Cell dissociation and culture. Rats were anesthetized and decapitated, and the nodose ganglia were quickly removed under a dissection microscope. For DRG neuron culture, rats were anesthetized with pentobarbital sodium (45-50 mg/kg ip) and the T9 and T10 dorsal root ganglia were removed, stripped of their connective tissue capsules, transferred into ice-cold culture media, and minced with microscissors. The tissue was minced with a surgical blade and incubated for 50 min in modified Leibowitz L-15 medium containing collagenase (type 1A, 1 mg/ml), trypsin (type III, 1 mg/ml), and DNase (type IV, 0.1 mg/ml) at 37°C. The enzymatic digestion was terminated by adding soybean trypsin inhibitor (2 mg/ml), 3 mM CaCl2, and bovine serum albumin (1 mg/ml). After gentle trituration, the tissue fragments were centrifuged at 800 rpm for 5 min and then resuspended in modified L-15 medium with 5% rat serum and 2% chick embryo extract (Life Technologies). The cells were plated on poly-L-lysine-coated glass coverslips and incubated for 24 h at 37°C before the electrophysiological studies. At the time of recording, the nerve cell bodies were round and did not have processes, reducing the potential for space-clamp problems during electrophysiological experiments. In the NGF group, 100 ng/ml NGF were added to the culture medium at the beginning of the 24-h period of incubation. To prevent effects of NGF produced by glia or neurons present in the medium, a neutralizing NGF antibody was added at a final concentration of 1:2,000. We have previously shown (4) that ~0.5% of the T9 and T10 DRG neurons are gastric neurons based on their incorporation of the retrograde label DiI. When we performed experiments with incubation times exceeding 24 h, the number of viable neurons decreased significantly and did not allow us to identify any surviving gastric sensory neurons in six independent experiments.

Electrophysiological recordings. The cells attached to the coverslips were transferred into a 0.5-ml recording chamber on the stage of an inverted microscope. DiI-labeled cells, identified by their red-orange color under Hoffman Contrast Optics (×400) in fluorescent light with a rhodamine filter (excitation wavelength ~546 nm and barrier filter at 580 nm), were studied. Sodium currents were recorded using the whole cell patch-clamp technique with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) interfaced with a personal computer. The patch pipettes were pulled from thin-walled borosilicate glass (TW150-4; World Precision Instruments, Sarasota, FL) with tip resistances of 1-3 MOmega after fire-polishing. Current recordings were digitized at 10 kHz using a Digidata 1200 interface (Axon Instruments). The series resistance and whole cell capacitance were compensated by >80%. The passive membrane properties were checked repeatedly during the course of the experiments. Cells were used for data analysis only if these properties remained stable. The leak current and residual capacitative transients were digitally subtracted using an online protocol for leak subtraction (p/n), where the test pulse (p) was followed by p/n hyperpolarizing pulses (with n = 4). The four current traces were added and digitally subtracted from the trace obtained with the test pulse. All experiments were performed at room temperature (21°C).

Solutions and chemicals. To measure sodium currents, the pipette solution contained (in mM) 100 Cs-Aspartate, 20 CsCl, 2.3 CaCl2, 4.8 MgCl2, 10 EGTA, 10 HEPES, 4 Mg-ATP, and 0.5 Na-GTP buffered to pH 7.2 with CsOH. The extracellular solution was composed of 20 mM NaCl, 120 nM choline chloride, 10 mM tetraethylammonium (TEA) chloride, 3 mM MgCl2, 10 mM HEPES, and 5.5 mM glucose buffered at pH 7.3 with TEA-OH. NGF and antibody against NGF were obtained from Sigma (St. Louis, MO). All other chemicals were analysis grade and were obtained from Sigma.

Data analysis. The software package pCLAMP6.0 (Axon Instruments) was used for data acquisition and analysis. TTX-sensitive currents were isolated by digital subtraction of current tracings before and after the administration of the sodium channel blocker. All data are expressed as means ± SE. Exponential fits were obtained using the curve-fitting routine of the Origin software package (Microcal, Northampton, MA). The two-tailed t-test for unpaired variables or analysis of variance was used to discern significant difference after intervention. Statistical significance was determined at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NGF and gastric inflammation. Consistent with previously published studies, acetic acid injection into the gastric wall induced inflammation and ulceration in all animals (n = 4), whereas saline treatment did not lead to macroscopic or microscopic changes (n = 4). Under control conditions, some immunostaining for NGF was found in the mucosa. Submucosa and muscularis did not show any signal (Fig. 1A). In contrast, we found strong NGF immunoreactivity in all layers of the stomach 7 days after the induction of gastric ulcerations with acetic acid (Fig. 1B). The immunoreactivity was typically found in round, mononuclear cells within the inflammatory infiltrate in the submucosa and muscularis propria (Fig. 1C).


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Fig. 1.   Ulcers increase nerve growth factor (NGF) expression within the gastric wall. Representative cryosections of the stomach (original magnification ×50). Saline treatment did not result in positive immunoreactivity for NGF (A), whereas there was significant wall thickening and increase with NGF immunoreactivity in all layers after acetic acid treatment (B). Counterstained sections (original magnification ×400) demonstrated NGF immunoreactivity in round mononuclear cells within the inflammatory infiltrate (C).

To quantify changes in NGF expression induced by the induction of gastric ulcer, we determined the concentration of NGF within the gastric wall. In untreated animals, 1.53 ± 1.1 pg NGF/mg protein was found (n = 3). Gastric surgery and saline injection caused a time-dependent increase that persisted for up to 2 mo. As shown in Table 1, induction of gastric ulcers with acetic acid further raised NGF levels within the gastric wall. Due to the severe inflammatory infiltrate, it was impossible to separate layers of gastric wall.

                              
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Table 1.   NGF concentrations in the gastric wall (pg/g protein) after induction of ulcers

NGF and sodium currents in nodose neurons. To assess whether NGF altered the properties of voltage-dependent sodium currents, we cultured nodose neurons in the presence of 100 ng/ml NGF for 24 h. Because a variety of growth factors are present in the culture medium and are produced by the neurons and glia cells, we exposed control cells to a neutralizing antibody to NGF for the same time period. The peak sodium current triggered by depolarization from -70 to 10 mV was -3,522 ± 395 pA under control conditions (n = 31) as opposed to -3,822 ± 492 pA for cells exposed to NGF (n = 29, not significant). Gastric sensory neurons can be differentiated based on their sensitivity to the neurotoxin TTX. In the presence of 1 µM TTX, the peak sodium current significantly differed between the two groups, with -741 ± 179 pA for controls compared with -1,969 ± 509 pA for NGF-treated cells (Fig. 2; P < 0.05). In contrast, the peak amplitude of the TTX-sensitive current was not significantly affected by the exposure to NGF (-3,370 ± 409 pA for control vs. -2,793 ± 398 pA for NGF-treated cells, not significant). The relative contribution of the TTX-resistant sodium current increased from 27 ± 6 to 44 ± 7% (P = 0.06) of the peak sodium current.


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Fig. 2.   Effect of NGF on TTX-sensitive (TTX-S) and -resistant (TTX-R) current in nodose neurons. The peak sodium current was measured in the absence and presence of 1 µM TTX. The peak amplitude of the TTX-sensitive component was calculated by subtracting the TTX-resistant from the total sodium current. The relative contribution of the TTX-S and -R sodium current was expressed as a fraction of the total sodium current. Compared with control cells (n = 31), culture in 100 ng/ml NGF (n = 29) caused a significant increase in the TTX-R current (*P < 0.05).

To determine the voltage dependence of activation, cells were stepped from -70 mV to various test potentials between -60 and 40 mV. The results were converted into normalized conductance [C = (I/Imax)/(V - Vreversal), where I is current and V is voltage] and fitted by the Boltzmann equation. As shown in Fig. 3, NGF did not alter the voltage dependence of activation for TTX-sensitive or TTX-resistant current.


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Fig. 3.   Effect of NGF on the voltage-dependence of activation of sodium currents in nodose neurons. The voltage dependence of activation was determined by stepwise depolarization from -70 mV to various test potentials as described in the text. The normalized results were fitted with the Boltzmann equation (broken lines). The voltage dependence of the TTX-resistant current (A) did not differ between control (closed symbols; n = 13) and NGF-treated cells (open symbols; n = 13) with half-activation voltages of -6.5 ± 0.8 (slope factor: 9.1 ± 0.7) and -7.5 ± 1.0 mV (slope factor: 8.1 ± 0.9), respectively. Similarly, the voltage-dependence of the TTX-sensitive current (B) did not differ between control (closed symbols; n = 21) and NGF-treated cells (open symbols; n = 19) with half-activation voltages of -17.5 ± 0.8 (slope factor: 7.4 ± 0.3) and -20.5 ± 0.5 mV (slope factor: 5.9 ± 0.5), respectively.

Finally, we tested whether NGF affected the recovery from inactivation. Cells were stepped from -70 to 10 mV for 15 ms and repolarized to -70 mV for 2-200 ms before a second test pulse to 10 mV. The peak inward current of the second pulse was normalized to the peak amplitude of the current triggered by the first depolarization. The experimental results were well described with a double exponential fit, with time constants of 5.4 ± 1 (weight: 58.5 ± 6.4%) and 101.7 ± 17.6 ms for control and 5.3 ± 0.9 (weight: 50.4 ± 6.6%) and 66.6 ± 12.4 ms for NGF (Fig. 4A). Similarly, the recovery from inactivation for the TTX-sensitive (time constants: 20.1 ± 4.4 vs. 13 ± 1.1 ms for control and NGF, respectively) and TTX-resistant current (1.5 ± 0.2 vs. 2.6 ± 0.3 ms for control and NGF, respectively) did not differ between neurons exposed to NGF compared with those cultured in the presence of anti-NGF (Fig. 4, B and C).


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Fig. 4.   Effect of NGF on the recovery from inactivation of sodium currents in nodose neurons. The recovery from inactivation was determined by stepping cells from -70 to 10 mV for 15 ms, followed by a second test pulse to 10 mV after variable periods of repolarization to -70 mV. The normalized peak current triggered by the second depolarization was plotted as a function of the time after repolarization and fitted with a double exponential (broken line). A: the results for the total sodium current. The time course of current recovery did not differ between control cells (closed symbols; n = 27; time constants: 6.2 and 54.7 ms) and cells cultured in the presence of NGF (open symbols; n = 25; time constants: 3.6 and 47.6 ms). The recovery from inactivation of the TTX-R (B) and TTX-S (C) current was fitted with a single exponential. The recovery of the TTX-R current did not differ between control cells (closed symbols; n = 10; time constant: 2.1 ms) and cells cultured in the presence of NGF (open symbols; n = 7; time constant: 2.7 ms). The recovery of the TTX-S current (C) did not differ between control cells (closed symbols; n = 9; time constant: 13 ms) and cells cultured in the presence of NGF (open symbols; n = 10; time constant: 13 ms).

NGF and sodium currents in DRG neurons. DRG neurons were cultured in the presence of NGF or neutralizing antibodies as described above. The peak sodium current triggered by depolarization from -70 to 10 mV was -1,859 ± 493 pA under control conditions (n = 17) as opposed to -1,397 ± 538 pA for cells exposed to NGF (n = 16, not significant). In the presence of 1 µM TTX, the peak sodium current significantly differed between the two groups with -291 ± 107 pA for controls compared with -734 ± 225 pA for NGF-treated cells (Fig. 5; P < 0.05). In contrast, the peak amplitude of the TTX-sensitive current was not significantly affected by the exposure to NGF (-1,707 ± 507 pA for control vs. -862 ± 469 pA for NGF-treated cells; P = 0.27). The relative contribution of the TTX-resistant sodium current increased from 34 ± 9 to 68 ± 11% (P = 0.05) of the peak sodium current.


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Fig. 5.   Effect of NGF on TTX-S and -R current in dorsal root ganglia (DRG) neurons. The peak sodium current was measured in the absence and presence of 1 µM TTX. The peak amplitude of the TTX-S component was calculated by subtracting the TTX-R from the total sodium current. The relative contribution of the TTX-S and -R sodium current was expressed as a fraction of the total sodium current. Compared with control cells (n = 17), culture in 100 ng/ml NGF (n = 16) caused a significant increase in the TTX-resistant current (*P < 0.05).

To determine the voltage dependence of activation, cells were stepped from -70 mV to various test potentials between -60 and 40 mV. The results were converted into normalized conductance (see above). As shown in Fig. 6, NGF did not alter the voltage dependence of activation for TTX-sensitive or TTX-resistant current.


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Fig. 6.   Effect of NGF on the voltage dependence of activation of sodium currents in DRG neurons. The voltage dependence of activation was determined by stepwise depolarization from -70 mV to various test potentials as described in the text. The normalized results were fitted with the Boltzmann equation (broken lines). The voltage dependence of the TTX-R current (A) did not differ between control (closed symbols; n = 5) and NGF-treated cells (open symbols; n = 7) with half-activation voltages of 3.9 ± 1.1 (slope factor: 5.5 ± 1) and 1.1 ± 0.7 mV (slope factor: 6.4 ± 1.0), respectively. Similarly, the voltage-dependence of the TTX-S current (B) did not differ between control (closed symbols; n = 12) and NGF-treated cells (open symbols; n = 5) with half-activation voltages of -9.8 ± 0.5 (slope factor: 7.0 ± 0.4) and -11.0 ± 0.4 mV (slope factor: 5.6 ± 0.4), respectively.

To examine the recovery from inactivation, cells were stepped from -70 to 10 mV for 15 ms and repolarized to -70 mV for 2-200 ms before a second test pulse to 10 mV. The peak inward current of the second pulse was normalized to the peak amplitude of the current triggered by the first depolarization. The experimental results were well described with a double exponential fit with time constants of 4.7 ± 0.8 and 86.9 ± 26.0 ms for 3.3 ± 0.3 and 64.6 ± 31.3 ms for control and NGF, respectively. Although the time constants did not differ significantly between the groups, the relative contribution of the faster time constant increased from 50.7 ± 9.7 to 82.0 ± 5.7% when cells were cultured in the presence of NGF (P < 0.05; Fig. 7A). This was due to the significant increase in the relative contribution of the TTX-resistant current as the recovery from inactivation for the TTX-sensitive (time constants: 28.8 ± 8.2 vs. 19.1 ± 5.6 ms for control and NGF, respectively) and TTX-resistant current (3.8 ± 0.8 vs. 2.9 ± 0.6 ms for control and NGF, respectively) did not differ between neurons exposed to NGF compared with those cultured in the presence of anti-NGF (Fig. 7, B and C).


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Fig. 7.   Effect of NGF on the recovery from inactivation of sodium currents in DRG neurons. The recovery from inactivation was assessed as described in Fig. 4. The time course of current recovery was significantly faster in cells cultured in the presence of NGF. Although the time constants did not differ between control cells (closed symbols; n = 11; time constants: 4.2 and 90.1 ms) and cells cultured in the presence of NGF (open symbols; n = 10; time constants: 2.6 and 73.6 ms), the relative contribution of the faster time constant increased from 50.7 ± 9.7 to 82.0 ± 5.7% in the NGF group (P < 0.05). The recovery of the TTX-R current (B) did not differ between control cells (closed symbols; n = 5; time constant: 2.9 ms) and cells cultured in the presence of NGF (open symbols; n = 7; time constant: 2.9 ms). The recovery of the TTX-S current (C) did not differ between control cells (closed symbols; n = 5; time constant: 16.6 ms) and cells cultured in the presence of NGF (open symbols; n = 4; time constant: 11.1 ms).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

With the use of an animal model of visceral hyperalgesia, we have recently shown that gastric injury alters the properties of sodium currents in primary sensory neurons. To identify mechanisms that contribute to these important functional changes, we investigated the potential role of NGF in peripheral sensitization. The key results of this study are 1) that NGF can be found in the gastric wall and is upregulated during inflammation and 2) that exposure to NGF enhances the expression of the TTX-resistant sodium current in cultured gastric sensory neurons, leading to a significant acceleration of the recovery from inactivation of sodium currents in spinal neurons. These changes mimic the results seen after induction of severe gastric inflammation (4).

Inflammation and injury cause production and release of mediators. Many studies have established the importance of rapidly acting substances, such as prostanoids or bradykinin, in acute pain (15, 24). They quickly and transiently alter neuron excitability by activating G protein-mediated signaling pathways within the cell, changing the phosphorylation status of ion channels and other target proteins. Especially in chronic pain, changes in gene transcription may play a more important role. A large number of cytokines and growth factors is found at sites of inflammation. On the basis of its important role in the development and maintenance of the nervous system (16), we investigated the role of NGF in peripheral sensitization. Others (27) and our laboratory (4) have shown that acetic acid injection into the gastric wall triggers a significant inflammation with ulcer formation. With the use of this model of severe gastric injury, we demonstrated a significant rise in NGF levels within the gastric wall, which persisted for more than 2 wk. This was associated with an increase in NGF immunoreactive cells within the submucosa and muscularis of the stomach. We attribute the lack of immunoreactivity for NGF in control animals to a lower sensitivity of the immunohistochemical technique compared with the ELISA, because we were able to detect messages for NGF in the stomach of naïve and control animals (unpublished observations). Our results are consistent with other reports describing an increase in NGF expression in animal models of visceral inflammation and in human disease (12, 17, 23, 33). Lymphocytes, mast cells, and histiocytes, cells that are found within the lamina propria and submucosa in inflammatory processes, produce NGF and express the high-affinity receptor for this neurotrophin (2, 26). Although we did not attempt to identify the NGF-immunoreactive cells, the size, shape, and distribution suggest that these immunocompetent cells are the source of NGF in this model of gastric injury.

Messenger RNAs for the high- and low-affinity NGF receptor have been found in nodose and DRG neurons of adult rats (3, 19). With the use of radioactively labeled ligands, Helke et al. (19) confirmed that visceral afferent neurons indeed express functional NGF receptors. Consistent with these results, the addition of NGF to cultured primary sensory neurons changes morphological or neurochemical characteristics consistent with the functional importance of neurotrophins in regulating the function of peripheral neurons in adult animals (23, 25).

On the basis of the importance of sodium channels in nerve function, we tested the effects of NGF on sodium currents in cultured nodose neurons. Voltage-dependent sodium channels are responsible for the rapid upstroke of the action potential in neurons, establishing them as important determinants of cellular excitability. This is supported by experimental models of chronic pain, which are associated with changes in sodium currents (12, 21, 35). Nine distinct voltage-gated sodium channels have been identified in mammals and functionally expressed in recombinant systems (18). Most of these voltage-gated sodium channels exhibit nanomolar sensitivity to TTX, but several TTX-resistant channels have been characterized. Two TTX-resistant sodium channels (Nav1.8 and Nav1.9), previously labeled SNS or PN3 and SNS2 or NaN, respectively, are present only in sensory neurons. Nodose neurons express message for Nav1.8, and the TTX-resistant sodium current significantly contributes to the whole cell sodium current in these neurons (22). Both Nav1.8 and Nav1.9 can be found in spinal sensory neurons, where they are primarily expressed in small-diameter neurons, many of which correspond to the C-type neurons that play an important role in nociception (1). Although Nav1.8 contributes to the action potential generation in these cells, the role of Nav1.9 is less clearly defined. Initial studies with knockout mice lacking Nav1.8 did not demonstrate a residual TTX-resistant sodium current in DRG neurons (1). Long hyperpolarizing prepulses to a potential more negative than -100 mV were required to demonstrate the presence of a second TTX-resistant sodium current (10). The biophysical properties of this current argue against a role in the inward current during action potential generation. Experiments with knockout animals as well as the use of antisense oligonucleotides to decrease the expression of Nav1.8 have demonstrated a blunted response in some models of somatosensory pain (1, 31). Conversely, carrageenan-induced inflammation of the hindpaw and gastric inflammation in rats induced an upregulation of TTX-resistant current (4, 21), further supporting the role of this ion channel in nociception.

To assess the effects of NGF on the TTX-resistant current, we cultured neurons for 24 h in the presence of NGF. To eliminate NGF present in the culture medium or produced as an auto- or paracrine factor by neurons or glia cells, control cells were kept in a medium supplemented with a neutralizing antibody against NGF. Compared with control conditions, culture in NGF-containing medium for 24 h resulted in an increase of the TTX-resistant sodium current, whereas the properties of both TTX-resistant and TTX-sensitive sodium currents were not changed. Although longer exposure to the neurotrophin may result in additional changes, we could not properly examine the effects of prolonged exposure to NGF, because gastric neurons from adult animals did not survive sufficiently long in culture. The increase in TTX-resistant sodium current is reminiscent of results seen in neurons harvested from animals with gastric inflammation (4). However, gastric inflammation also accelerated the recovery from inactivation of the TTX-sensitive sodium current, which was not seen after culture in NGF-containing media for 24 h, indicating that prolonged exposure to the neurotrophin and/or additional factors may contribute to the modulation of neuron excitability during inflammation and nerve injury.

Neurotrophins have been shown to regulate the expression of sodium channels in neurons (7, 29). Our findings are consistent with reports by Black et al. (7), who demonstrated an increase in Nav1.8 expression in somatosensory neurons after exposure to NGF. Similar effects of neurotrophins on voltage-sensitive channels have been reported in studies using cell lines (36). The TTX-resistant sodium current rapidly recovers from inactivation. Fast recovery rapidly restores the excitability after action potential firing, whereas slow recovery leads to a long refractory period. The shift in the relative contribution of the TTX-resistant current may therefore decrease the refractory period and allow repeated action potential generation during prolonged stimulation. This important role of the TTX-resistant sodium current in repetitive firing was recently confirmed experimentally (22). However, it is likely that NGF and other neurotrophins and cytokines affect the expression of more than one single ion channel, leading to more complex changes in neuronal excitability. Recently, Winston et al. (37) reported an upregualtion of VR-1 mRNA in dorsal root ganglion neurons cultured in the presence of NGF. Others have reported changes in the expression of both ligand- and voltage-gated ion channels (8, 34), which will also affect the functional properties of afferent neurons and alter their firing patterns.

Together, our data show that inflammation increases the expression of NGF within the gastric wall. NGF, in turn, can interact with primary afferent neurons, causing an upregulation of the TTX-resistant sodium current. The resulting changes in neuron excitability may contribute to peripheral sensitization. The use of specific NGF receptor antagonists or neutralizing antibodies may therefore offer therapeutic benefit in diseases associated with inflammatory pain of the viscera (9, 20, 28).


    ACKNOWLEDGEMENTS

This study was supported by grants from the National Institutes of Health (DK-01548 and NS-35790).


    FOOTNOTES

Address for reprint requests and other correspondence: K. Bielefeldt, Univ. of Iowa, Dept. of Internal Medicine, 4614 JCP, 200 Hawkins Dr., Iowa City, IA 52242 (E-mail: klaus-bielefeldt{at}uiowa.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.

10.1152/ajpgi.00356.2002

Received 22 August 2002; accepted in final form 1 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 284(3):G499-G507
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