1 Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, Vermont, 05405; and 2 Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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PGE2 is a proinflammatory mediator that can influence many cell types. This study was conducted to determine whether PGE2 alters the electrical activity of distal colonic myenteric neurons, because colitis is typically associated with altered motility and changes in neural signaling may be involved. The electrical properties of intact myenteric neurons were evaluated with intracellular microelectrodes. Acute application of PGE2 elicited a prolonged depolarization in both AH and S neurons with little effect on input resistance or electrical excitability. PGE2 effects were suppressed by tetrodotoxin (TTX) or neurokinin (NK) receptor antagonists, indicating that PGE2 acts directly and indirectly to depolarize colonic neurons. PGE2-evoked depolarization was concentration dependent (~3 µM EC50) and was attenuated by the E prostanoid (EP)1/2 receptor antagonist, AH-6809. When preparations were maintained for 48 h in the presence of the stable PGE2 analog PGE2-ethanolamide (10 µM), neurons exhibited a significant membrane depolarization and enhanced excitability. These results suggest that PGE2 can play a role in altered motility in colitis by evoking changes in the electrical properties of myenteric neurons.
motility; inflammation; colitis; innervation; enteric nervous system
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INTRODUCTION |
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THE NERVOUS SYSTEM OF THE bowel, which is known as the enteric nervous system (ENS) regulates visceromotor, secretomotor, and vasomotor activities in the wall of the intestines. Given that much of the morbidity associated with inflammatory bowel disease is caused by disordered gastrointestinal motor function and that colonic smooth muscle function is regulated by the ENS, it is quite possible that alterations in the excitability of the enteric neurons contribute to intestinal dysmotility. A variety of immune-mediated inflammatory disorders exist, including Crohn's disease and ulcerative colitis, and dysmotility, hypersecretion, and enhanced perception of pain are common features (17, 22, 23, 46). It is now clear that cutaneous and visceral inflammations lead to enhanced excitability of extrinsic primary afferent neurons and altered reflex activity (5, 8, 14, 48). Changes may also occur in the afferent components of intrinsic enteric neural circuitry, and such changes could contribute to altered motor reflex activity in the inflamed bowel.
In guinea pig bowel, it is possible to identify the functional role of a given neuron on the basis of consensus knowledge regarding the electrical, morphological, chemical coding, and axonal projection patterns of these cells. For example, a class of cells known as AH neurons, on the basis of electrical properties, functions as the afferent limb of the peristaltic reflex circuit (19) and is considered to have roles as intrinsic primary afferent neurons and interneurons (for review, see Ref. 30). These cells have a Dogiel type II morphology (multiple processes) and extend projections to other ganglia and to the lamina propria (7, 34, 40, 42), where they could respond to stimuli originating at the mucosal surface. Therefore, changes in the electrical properties of AH cells would likely alter motility and secretory patterns in the gut. Other neurons of the myenteric plexus, consisting of interneurons and motor neurons, have a Dogiel type I morphology (single process) and share a common electrophysiological phenotype (7, 34). These cells are known as S cells and project to other enteric neurons and to intestinal smooth muscle (35, 41). Because intestinal inflammation is typically centered in the lamina propria of the mucosal layer, components of the reflex circuitry that are most likely to be affected by inflammatory mediators are those that pass into this region. In the case of motility reflex circuits, this includes the processes of AH cells, which, as described above, project to the mucosa.
During the inflammatory process, a plethora of compounds are released that have actions on many cell types, including neurons. A common feature of intestinal inflammation is the upregulation of the cyclooxygenase-2 (COX-2) enzyme, leading to increased synthesis of several eicosanoids (16, 46, 47, 50), including prostaglandin E2 (PGE2). Within guinea pig submucosal plexus neurons in the colon, PGE2 application has been shown to lead to membrane depolarization and increased action potential frequency, as well as increased chloride ion secretion (18). PGE2 has also been shown to activate myenteric plexus neurons in the guinea pig ileum (14) and augment acetylcholine release from myenteric neurons of the small intestine (9, 13) and colon (27, 39). Moreover, in ileal myenteric AH cells, activation of adenylyl cyclase increases excitability by causing a membrane depolarization and suppression of the prolonged afterhyperpolarization (45), consistent with the known effects of Trichinella-induced inflammation in the small intestine (44).
In the present study, we conducted studies designed to elucidate the effects of PGE2 on colonic myenteric neurons in an effort to resolve whether PGE2-induced changes in neuronal activity could contribute to changes in motility that are associated with colitis. Intracellular voltage recordings were obtained from randomly impaled colonic myenteric neurons in acutely dissected and organ-cultured preparations. Responses to PGE2 were evaluated after pressure microejection or addition to the circulating Krebs solution. To assess the effects of prolonged exposure of myenteric neurons to PGE2, longitudinal muscle-myenteric plexus preparations were maintained in organ culture conditions in the presence or absence of the stable PGE2 analog, PGE2 ethanolamide.
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METHODS |
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Tissue Preparation
Adult guinea pigs of either sex, weighing 250-300 g, were anesthetized with isofluorane and exsanguinated. This method has been reviewed and approved by the Institutional Animal Care and Use Committee of the University of Vermont. The distal colon was removed and immediately placed in an iced Krebs solution [chemical components (in mM): 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 8 glucose, aerated with a 95% O2-5% CO2]. Nifedipine (5 µM) and atropine (200 nM) were added to the Krebs solution to eliminate smooth muscle contraction. The colon preparation was dissected by pinning both free ends, then carrying a longitudinal incision along the mesenteric border. Tissue was then pinned flat in a Sylgard polymer-lined dish while being continuously perfused by circulating iced Krebs. The mucosal and submucosal layers of the colon were removed under a dissecting microscope, exposing the underlying muscularis externa. Circular muscle bundles of the muscularis externa were subsequently removed, exposing the myenteric ganglionated plexus. This dissected tissue was then placed into a tissue chamber for electrophysiological recording.Electrophysiological Recordings
Methods for electrophysiological recording were similar to those previously described (36). The colon preparation was pinned flat in a low-volume (6-ml) tissue chamber and continuously perfused with warm Krebs solution (37°C, aerated by 95% O2-5% CO2). Ganglia were visualized using Hoffman modulation contrast microscopy.Intracellular recordings were done using an Axoclamp 2B electrometer
(Axon Instruments, Foster City, CA) with 2 M KCl-filled microelectrodes. Tips of the microelectrodes were backfilled with 1%
(wt/vol) neurobiotin (Vector Laboratories, Burlingame, CA) to allow for
subsequent reacquisition and immunohistochemical and morphological
characterization. These microelectrodes had resistances between 60 and
80 M for voltage recording. Capacitance neutralization was adjusted
as needed for optimum current injection and voltage recording. Results
were monitored on a separate oscilloscope, whereas data acquisition and
analysis were performed using MacLab Chart software to emulate
oscilloscope readings (MacLab hardware and software; ADInstruments,
Castle Hills, NSW, Australia). Electrodes used to stimulate fiber
tracts were extracellular monopolar Teflon-coated platinum wires
(25-µm diameter) placed against interganglionic fiber bundles and
used to stimulate synaptic transmission to impaled cells. Activation of
fast excitatory postsynaptic potentials (EPSPs) involved a
single 0.5-ms current pulse applied to interganglionic fiber bundles.
Baseline neuronal excitability was assessed by determining the threshold current necessary to activate at least one action potential in a given cell (threshold strength in nA) during a 500-ms current pulse. Potential alterations in neuronal excitability were assessed by determining the number of action potentials generated during a depolarizing current pulse delivered at two times the threshold strength.
Compounds to be tested on colonic myenteric neurons were applied by pressure microejection or addition to the circulating Krebs solution. Pressure ejections of compounds involving glass micropipettes (15- to 20-µm tip diameter) placed 50-100 µm from the target ganglia were induced by pulses of nitrogen gas (300 kg/cm2; 900 ms). The distance of pressure ejection micropipette was maintained at 50-100 µm from the tissue, if selection of a new target ganglion was necessary. Washout periods (>15 min) were done between subsequent administrations of test compounds to allow for the recovery of responses in the event of receptor desensitization.
Classification of Impaled Neurons
Originally, neurons in the myenteric plexus of the guinea pig distal colon were called type 1 or 4 if they exhibited multiple or single action potentials, respectively, with no after-spike hyperpolarization, and they were called type 2 neurons if they exhibited a single action potential on depolarization and did display a prolonged postspike hyperpolarization (53). Subsequent investigations combined evaluations of electrophysiological and morphological properties and concluded that single axonal Dogiel type I neurons exhibited properties similar to S cells of the small intestine, whereas multiaxonal Dogiel type II neurons exhibited AH cell properties (35, 49).In the present study, we used the same classification scheme used to
describe neurons in the guinea pig small intestine, because, as in the
small intestine, there are two primary types, S or AH, of spiking
neurons in the myenteric plexus of the colon (35, 37, 49).
The most important criteria for identifying a given cell as an AH
neuron were the presence or absence of a shoulder on the repolarizing
phase of the action potential, its morphology, and the duration of the
prolonged afterhyperpolarization (usually lasting seconds). Particular
attention was paid to the repolarizing phase of the action potential,
because the shoulder caused by an inward Ca2+ conductance
is retained in activated AH neurons, although their other electrical
properties become indistinguishable from S neurons when they are
activated synaptically or pharmacologically (6, 31, 44). S
neurons exhibited action potentials after the onset of a depolarizing
current pulse, often exhibited anodal break spikes on the offset of a
hyperpolarizing current pulse, and, in some cases, generated
spontaneous action potentials. Prostaglandin actions were evaluated in
AH and S cells. All cells exhibiting a mean input resistance of <50
M were excluded from data analysis.
Immunohistochemical Labeling
After intracellular recording, tissues were fixed in a 2% paraformaldehyde, 0.2% picric acid solution at 4°C overnight. Tissues were then blocked with a 4% goat serum in 100 mM PBS/Triton-X (0.5%) for 30 min at room temperature. Primary antisera to the calcium-binding protein, calbindin (Swant, Bellinzona, Switzerland), were used at 1:1,000 diluted in 4% goat serum/PBS-Triton-X and incubated at 4°C overnight. Tissues were then washed 3 × 15 min each and placed in secondary antibodies. Neurobiotin-filled cells were visualized by adding streptavidin-conjugated FITC (1:400; Jackson Laboratories) to the secondary antibody solution. Immunohistochemical labeling was then visualized using an Olympus fluorescent microscope, and images were captured using an Olympus digital camera utilizing Magnafire digital imaging software (Optronics, Goleta, CA).In Vitro Organ Culture
To determine the effects of prolonged exposure to PGE2, colon explants were placed in organ culture for ~48 h before electrophysiological recording. Organ culture techniques were similar to those previously reported (28). In these experiments, colon was removed from experimentally naive animals, as above. This tissue was then dissected under sterile conditions using Krebs solution containing 5 µM nifedipine filtered through a 0.22-µm-diameter Fisherbrand filter disc (Fisher Scientific, Springfield, NJ). Tissue was pinned flat in a Sylgard-lined petri dish sterilized before dissection by soaking in 100% ethanol, and mucosal and submucosal layers were removed. After dissection, the tissue was bathed with a DMEM/F-12 culture media containing 10% horse serum, antibiotic-antimycotic (1 ml/100 ml), gentamicin (10 mg/100 ml), amphotericin B (12.5 µg/100 ml), and nifedipine (1 µM) (all compounds from Sigma, St. Louis, MO). Experimental tissues were bathed in culture media containing 10 µM PGE2-ethanolamide (Cayman, Ann Arbor, MI), a synthetic analog of endogenous PGE2 that exhibits higher stability and slower degradation in aqueous environments than the endogenous form. Culture medium was replaced at ~12-h intervals in all experiments. After organ culture for 48 h, preparations were washed with normal Krebs solution, the circular layer of the muscularis externa was removed, and the underlying myenteric plexus was exposed. Preparations were then repinned in an electrophysiology culture dish. Myenteric neurons were then tested for passive and active electrical properties and the presence and/or absence of synaptic inputs. Measures of input resistance, number of action potentials per depolarizing current pulse, threshold to action potential, presence of anodal break action potentials, and presence or absence of fast or slow excitatory postsynaptic potentials were recorded. Impaled cells were iontophoretically labeled with neurobiotin, and preparations were immunohistochemically stained with rabbit calbindin antibody to determine cell morphology and calbindin immunoreactivity. Control tissues were dissected and maintained in organ culture as above, but no PGE2-ethanolamide was added to the culture media.Compounds
PGE2, AH-6809, and PGE2 ethanolamide were all obtained from Cayman. PGE2, AH-6809, and PGE2 ethanolamide were dissolved in 100% ethanol to 60 mM, 60 mM, and 100 mM stock solutions, respectively, and serially diluted in normal Krebs to final working concentrations. Ethanol concentrations were never >0.1% in final solutions. Stock solutions (1 mM) of SR-140333 and SR-142801 (courtesy of Sanofi Recherche, Montpellier, France) were prepared in DMSO, and these compounds were serially diluted in normal Krebs to a final working concentration of 100 nM. Tetrodotoxin (TTX) was obtained from Sigma and diluted in 100% ethanol to a stock concentration of 0.5 mM. Final working concentrations were made by diluting the stock in normal Krebs.Statistical Analysis
Averaged values are presented as means ± SE. Statistical analysis was performed using paired or unpaired Student's t-test when appropriate to determine the difference between control and experimental values (Prism v. 3.0a for Macintosh; Graphpad Software, La Jolla, CA). Differences were considered to be significant if P < 0.05. ![]() |
RESULTS |
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In the present study, intracellular recordings were obtained from
a total of 120 neurons from 52 preparations. Out of a total of 120 neurons, 77 neurons were S cells (49.4%) and 43 were AH cells
(27.6%). Of the 77 S neurons, 73% were successfully characterized morphologically, and all of these were classified as having Dogiel type
I morphology, with the remainder of cells not classified due to
insufficient iontophoretic filling of the cell. None of the S/Dogiel
type I cells were calbindin immunoreactive. Of the 43 AH cells,
all of the cells successfully filled with neurobiotin (88%) were
classified as Dogiel type II cells. Seventy-four percent of the cells
identified electrophysiologically and morphologically as AH/Dogiel type
II cells were calbindin immunoreactive (Fig. 1). No cells exhibiting S cell
electrophysiological properties were Dogiel type II in morphology, and
no AH cells exhibited a Dogiel type I morphology.
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In recordings from S cells, resting membrane potential was 57.9 ± 1.7 mV and resting membrane input resistance was 211 ± 40 M
(n = 77). In AH cells, resting membrane potential was
58.9 ± 2.1 mV and resting membrane input resistance was
105 ± 23 M
(n = 43).
PGE2 Depolarizes AH and S Cells
AH cells.
When PGE2 was applied to AH cells by pressure microejection
(10 µM in pipette, 900 ms), 20 of 21 AH cells responded with a maximal depolarization that had an amplitude of 8.9 ± 1.1 mV and a duration to half-maximal repolarization (dur50) of
49.4 ± 13.6 s (n = 20) (Fig.
2). Membrane input resistances were
unaffected during this depolarization (control = 96 ± 22 M, PGE2 = 97 ± 21 M
; P is not
significant, n = 8). The threshold strength to elicit
action potentials by intracellular depolarizing current in AH cells was
not altered after acute PGE2 application and application of
direct current to return the membrane potential to resting levels
(control, 0.3 ± 0.07 nA; PGE2, 0.3 ± 0.05 nA).
Similarly, there was no difference in the number of action potentials
generated at two times threshold strength (control, 2 ± 1 action
potential; PGE2, 2 ± 1 action potential). Of
the AH cells responding to PGE2, only one generated
spontaneous action potentials during the sustained depolarization. This
cell also fired anodal break action potentials after PGE2
application, and generated more action potentials in response to a
depolarizing current pulse of the same amplitude.
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S cells.
In S cells, PGE2 evoked a prolonged depolarization with an
amplitude of 6.9 ± 0.8 mV and a dur50 of 27.6 ± 6.0 s (Fig. 2). Membrane input resistances were unaffected during
PGE2-mediated depolarization (control, 261 ± 76 M;
PGE2, 231 ± 39 M
; P is not significant,
n = 12). No difference was detected in threshold strength (control, 0.3 ± 0.04; PGE2 0.3 ± 0.06)
or number of action potentials generated by doubling threshold strength
(control = 2 ± 1, PGE2 = 2 ± 1) after
acute application of PGE2. Of the 38 S cells that responded
to PGE2 application, three exhibited an increase in
excitability as demonstrated by the generation of spontaneous action potentials.
Effects of TTX on PGE2-Mediated Depolarization
To determine whether the PGE2-induced depolarization was due to a direct action of the prostanoid on myenteric neurons, responses to PGE2 were measured in the presence of the sodium channel blocker TTX (0.5 mM) added to the perfusing solution. In these experiments, 22 neurons from 14 preparations were studied. In 12 AH cells tested, TTX inhibited both the amplitude and the dur50 of the PGE2-evoked depolarization to ~40-50% of control values (P < 0.05 for both; Fig. 3). In 10 S cells, both the amplitude and the dur50 of the PGE2-evoked depolarization were significantly inhibited in the presence of TTX, resulting in an approximate inhibition of 65-75% control values (P < 0.05 for both; Fig. 3).
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To eliminate the possibility that receptor desensitization contributed to the reduction in responsiveness to PGE2, two sequential applications of PGE2 were performed on impaled neurons in normal Krebs solution. Washout periods of ~10-15 min were done between prostaglandin applications in a given cell. In the five AH cells from two preparations tested, initial application of 10 µM PGE2 resulted in a maximal depolarization amplitude of 10.7 ± 4.6 mV and dur50 of 32.7 ± 5.1 s determined by comparing resting membrane potential with the maximum depolarization amplitude after PGE2 application. A second application of PGE2 after the washout period resulted in a depolarization of statistically similar mean amplitude, as determined by paired Student's t-test (6.9 ± 2 mV, n = 5) and dur50 (25.6 ± 6 s, n = 5). In 8 S cells from three preparations, initial and second applications of PGE2 were also statistically similar (amplitude: initial = 6.4 ± 2 mV, second = 6.5 ± 2 mV, n = 8; dur50: initial = 41.6 ± 26 s, second = 28.7 ± 12 s, n = 6).
PGE2-Induced Depolarization was Concentration-Dependent
Concentration-effect curves were generated in AH cells by adding PGE2 to the bathing solution at concentrations ranging from 100 nM to 30 µM with washout of
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Prostanoid Receptor Antagonist AH-6809 Attenuates PGE2-Induced Depolarizations
PGE2 acts as an agonist at E prostanoid (EP) receptors. The specific EP receptor(s) responsible for the effects of PGE2 in the myenteric plexus is currently unknown, but evidence suggests the presence of the EP1 receptor mRNA within rat myenteric plexus (15, 38). These same studies failed to detect a message for EP2 in the gut. To confirm that the PGE2-induced depolarization of colonic myenteric neurons was EP receptor mediated, PGE2 was applied to seven AH and eight S cells in the presence of the dual EP1/EP2-receptor antagonist, AH-6809 (receptor subtype antagonists specifically selective for the EP1 or EP2 receptor are currently not available). In the seven AH cells that were tested, AH-6809 (5 µM) significantly attenuated both the amplitude and the dur50 of the PGE2-evoked depolarization (P < 0.05 for both; Fig. 5). No significant differences in membrane input resistances were detected in the presence of AH-6809.
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S cells responded to initial pressure microejection application of PGE2 with a depolarization amplitude of 7.5 ± 1.3 mV and a mean dur50 of 19.0 ± 5.0 s (n = 8). The depolarization amplitude was significantly reduced in the presence of AH-6809 (amplitude = 3.6 ± 0.9 mV, n = 8), but the dur50 was not significantly altered (dur50 = 10.8 ± 3.2 s, n = 8). No changes in membrane input resistances were detected in the presence of AH-6809.
NK Receptor Antagonists Significantly Inhibit PGE2-Mediated Depolarizations in AH Cells
Previous investigations have determined that NK1 and NK3 receptors mediate prolonged depolarizations in the myenteric plexus of the intestine and that tachykinins likely contribute to slow EPSPs in myenteric neurons (1, 25, 26). Because the result of the experiments in the presence of TTX indicated that a component of the PGE2-mediated depolarization may be indirect and due to release of neuroactive compounds from nearby nerve terminals, additional experiments were conducted to investigate a possible role of tachykinins in the generation of the indirect or presynaptic effects of PGE2 observed in myenteric AH cells. In these experiments, antagonists of neurokinin receptors NK1 and NK3 (SR-140333 and SR-142801, respectively, 100 nM for each) were applied to impaled AH cells after a control application of PGE2 (10 µM in pipette, 900 ms). Application of the NK receptor antagonists significantly inhibited PGE2-mediated depolarization amplitude (control = 5.4 ± 0.4 mV, NK antagonists = 2.9 ± 0.5 mV, range = 36-54% decrease; n = 8, P < 0.05) and dur50 (control = 40 ± 10 s, NK antagonists = 26 ± 7 s, range = 30-48% decrease; n = 8, P < 0.05).Effects of Prolonged Exposure to PGE2-Ethanolamide
Inflammation is typically a long-lasting condition associated with a prolonged elevation of PGE2 levels. Therefore, we conducted additional experiments to determine the effects of prolonged exposure of myenteric neurons to PGE2 by maintaining colonic longitudinal muscle-myenteric plexus preparations in organ culture for 48 h in the presence or absence of the relatively stable PGE2 analog PGE2-ethanolamide (10 µM). Proportions of impaled S and AH cells cultured in the absence of PGE2-ethanolamide did not differ from those evaluated in acutely dissected preparations (S: naïve = 64%, cultured = 63%; AH: naïve = 36%, cultured = 20%).Significantly depolarized resting membrane potentials were detected in
PGE2-ethanolamide-cultured AH cells compared with cultured controls (control = 60.5 ± 1.7 mV, n = 11;
PGE2-ethanolamide =
50.8 ± 3.0 mV,
n = 9). No difference was observed in the threshold to
initiate an action potential (threshold strength in nA) in AH cells of
either treatment group (control, n = 11;
PGE2-ethanolamide, n = 9), although there
was a significantly higher number of action potentials elicited at two
times threshold strength in PGE2-ethanolamide-cultured AH
cells (Fig. 6). Spontaneous action
potentials were observed in 67% of
PGE2-ethanolamide-cultured AH cells, whereas spontaneous action potentials were not detected in any of the AH cells studied in
the control preparations. Peak amplitude of the prolonged
afterhyperpolarization in AH cells was significantly diminished in the
PGE2-ethanolamide-cultured cells (control,
n = 11; PGE2-ethanolamide,
n = 9) (Fig. 7). Time to
the peak of the prolonged afterhyperpolarization was also significantly attenuated in PGE2-ethanolamide-cultured AH cells (control,
n = 11; PGE2-ethanolamide,
n = 9) (Fig. 7). The dur50 of the prolonged afterhyperpolarization was also significantly inhibited (control, n = 11; PGE2-ethanolamide,
n = 8). The total duration of the prolonged afterhyperpolarization was also significantly diminished (control, n = 11; PGE2-ethanolamide,
n = 9). No difference was detected between control and
PGE2-ethanolamide-cultured AH cells in input resistance
(control = 108 ± 19 M
, n = 11;
PGE2 = 115 ± 19 M
, n = 7) or
action potential amplitude (control = 72 ± 4 mV,
n = 7; PGE2 = 64 ± 9 mV,
n = 7).
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PGE2-ethanolamide-cultured S cells had a significantly
depolarized resting membrane potential compared with control cultured cells (control = 55 ± 2 mV, n = 34;
PGE2 =
42 ± 3 mV, n = 8). Mean
input resistances were significantly decreased in
PGE2-ethanolamide-cultured S cells (control = 175 ± 22 M
, n = 34; PGE2 = 67 ± 17 M
, n = 8). Action potential amplitude was
also significantly decreased (control = 66 ± 3 mV,
n = 34; PGE2 = 41 ± 3 mV,
n = 8). No observable differences were seen between
control and PGE2-ethanolamide-cultured S cells in threshold
strength, maximum number of action potentials per depolarizing current
pulse generated at two times threshold strength, or presence of
spontaneous action potentials.
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DISCUSSION |
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The present study was conducted to test whether PGE2 can alter the electrical properties of colonic myenteric neurons as a means of contributing to changes in motility typically associated with colitis. Acute application of PGE2 elicited a depolarization of myenteric neurons but did not alter the excitability of either S or AH cells. Acute PGE2 application typically did not lead to the spontaneous generation of action potentials in myenteric neurons, did not increase the number of action potentials in response to a depolarizing current pulse, and did not alter the prolonged afterhyperpolarization in AH cells. However, chronic application of PGE2 to longitudinal muscle-myenteric plexus explants resulted in increased excitability of AH cells, with a membrane depolarization, an increase in the detection of spontaneous action potentials, more action potentials generated per depolarizing current pulse, and diminished prolonged afterhyperpolarization. These results suggest that myenteric neurons express receptors for PGE2 and that continuous activation of these receptors leads to enhanced excitability in AH cells.
Increase in AH cell excitability caused by prolonged exposure to PGE2 may contribute to the dysmotility associated with inflammatory bowel disease. Because AH cells are thought to act as intrinsic primary afferent neurons and interneurons in the motor reflex circuitry of the intestines, alteration of active and passive membrane properties of AH cells would affect the afferent portion of the peristaltic reflex arc (35, 40). AH cells have processes that extend into the lamina propria, as well as projections to other myenteric neurons (2-4, 40). Thus, because the inflammatory response is typically centered in the lamina propria of the mucosal layer in colitis, the processes of AH cells would be directly exposed to proinflammatory mediators such as PGE2. Consistent with this model, we have recently found that the excitability of AH cells is increased in trinitrobenzenesulfonic acid (TNBS)-induced colitis (32).
Although the PGE2-mediated depolarization in AH cells was significantly reduced in the presence of TTX, it was not completely inhibited. This suggests the possibility that the PGE2-mediated depolarization of colonic myenteric neurons involves actions of PGE2 both directly on AH cells and indirectly through the release of neuroactive compounds from nearby nerve terminals onto the impaled neurons. The family of bioactive peptides known as the tachykinins has been implicated in the generation of slow EPSPs in colonic myenteric ganglia (1, 25, 26). Present results suggest that tachykinin receptor blockade can alter PGE2-mediated depolarizations in myenteric AH cells, suggesting that the indirect effects of PGE2 may be due to the release of endogenous tachykinins, such as substance P.
Acute and chronic application of PGE2 depolarized S cells with negligible alteration of neuronal excitability. S cell depolarization in response to acute PGE2 application was also inhibited by TTX, suggesting that PGE2 also depolarizes S cells through direct and indirect mechanisms. S cells exhibit Dogiel type I morphology with single axonal processes that typically contact the circular or longitudinal muscle bundles, but S/Dogiel type I cells do not exhibit processes that extend into the lamina propria (10, 35, 40, 54). It is therefore unlikely that S cells are directly altered by the inflammatory response in colitis, but their activity may change due to upstream changes in the neuronal reflex circuitry. In support of this model, we have recently found that, whereas AH cells are hyperexcitable in the inflamed colon, the electrical properties of S cells in the inflamed colon are not different from those in the normal colon (32). The only change detected in S cells in colitis was an increase in the proportion that received slow excitatory synaptic input, which is consistent with a downstream effect of inflammation on primary afferent neurons.
In the myenteric ganglia of the distal colon, acute application of PGE2 evoked a depolarization that was not associated with a change in input resistance or neuronal excitability. In other regions of the bowel, brief application of PGE2 elicits a depolarization that is often accompanied by the generation of action potentials. This type of response has been observed in the myenteric plexus of the ileum (14) as well as the submucosal plexus of the distal colon (18). By contrast, application of PGE2 to gallbladder neurons results in a complex response typified by rapid membrane hyperpolarization, followed by depolarization and another, prolonged, hyperpolarization (24). Taken together, these findings indicate that the actions of PGE2 are tissue specific, and acute exposure of distal colonic myenteric neurons to PGE2 has a modulatory effect, but it does not itself activate the generation of action potentials.
The EP1-4 receptors are most sensitive to PGE1 and PGE2. In the present study, we found that the dual EP1/EP2 receptor antagonist AH-6809 attenuated the PGE2-mediated depolarization. EP1 and EP2 receptor mRNA is present in multiple physiological systems (for review see Ref. 12), but of these, only the EP1 receptor message is present within the digestive tract in any appreciable level (38). The EP1 receptor is linked to the heterotrimeric G protein Gq/11, which activates phospholipase C after prostaglandin binding and generates inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG). In addition, EP3 receptor mRNA has been detected in the muscularis of the rat colon (15), indicating that it is possible that myenteric neurons express this prostanoid receptor as well. This receptor is alternatively coupled either to Gq/11, leading to increased cytosolic Ins(1,4,5)P3/DAG, or Gi, which leads to inhibition of adenylyl cyclase and a decrease in cAMP. However, to date, no subtype-selective EP3 receptor antagonist has been developed. Therefore, barring the development of higher-specificity receptor agonists and antagonists for EP receptors, uncoupling of the receptor-intracellular signaling cascade may be one way of defining the receptor responsible for the actions of PGE2 in the myenteric plexus of the colon.
Evidence from the present study indicates that PGE2 likely acts on more than one type of receptor to depolarize AH cells in this system. A slight PGE2-induced depolarization persisted in the presence of the dual EP1/EP2 receptor antagonist AH-6809. Dissociation studies involving the displacement of [3H]PGE2 from EP receptors by AH-6809 have demonstrated Ki of ~350 nM (11, 29), suggesting that the concentration used in the present study (5 µM) would be sufficient to block the majority of EP1 receptors. The present experiments also failed to detect any change in membrane input resistance in response to acute PGE2 application. This result could be explained by activation of receptors coupled to opposing intracellular signaling cascades.
Upregulation of COX-2 activity and PGE2 synthesis are common features of intestinal inflammation (47), and PG levels typically remain elevated for an extended period during chronic inflammation (21, 47, 51, 52). We have recently found that inhibition of COX-2 activity reverses the increases in myenteric neuronal excitability seen in TNBS-induced colitis (33), suggesting that increases in PG synthesis that accompany inflammation may lead to altered myenteric neuronal activity. Results of the present study suggest that prolonged exposure of AH neurons to an analog of PGE2 has a dramatic effect on AH neuronal excitability. This result is important because it suggests that desensitization of myenteric neurons does not occur rapidly in the presence of PGE2, allowing PGE2 to maintain its effects throughout chronic inflammation. Furthermore, these findings indicate that changes observed in response to prolonged exposure to the agonist may involve changes in gene expression and protein synthesis, because acute application of PGE2 did not alter neuronal excitability. AH neurons normally have a steady-state Ca2+-activated K+ conductance that holds the membrane at a more negative potential, and this conductance also contributes to the prolonged afterhyperpolarization characteristic of these cells (20, 43). It is possible that prolonged exposure to the analog of PGE2 leads to a decrease in the Ca2+-activated K+ conductance in these cells. Alternatively, PGE2 could have similar effects by disrupting Ca2+ entry and/or mobilization of intracellular Ca2+ stores in AH cells. Further studies will be required to test whether changes in protein synthesis occur in response to prolonged exposure to PGE2, and to determine what changes in these neurons are responsible for the changes in excitability that were observed.
Results of this study support the theory that PGE2 can contribute to dysmotility in colitis by modulating the activity of myenteric neurons in the distal colon. Although acute application of PGE2 simply caused a depolarization of myenteric neurons, chronic application, which more closely reflects the inflamed condition, depolarized the neurons and significantly increased the excitability of AH cells. Inhibition of the prolonged afterhyperpolarizing potential and decrease in action potential accommodation of myenteric AH cells, which act at the afferent limb of the reflex circuitry, would allow these neurons to respond more vigorously to luminal stimuli, and in turn, alter motor patterns, resulting in dysmotility.
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ACKNOWLEDGEMENTS |
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We thank Dr. David Linden for valuable discussion.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-62267, National Institute of Neurological Disorders and Stroke Grant NS-26995, and the Crohn's and Colitis Foundation of Canada (to K. A. Sharkey and G. M. Mawe). K. A. Sharkey is an Alberta Heritage Foundation for Medical Research Medical Scientist.
Address for reprint requests and other correspondence: Gary M. Mawe, Given D 403A, Dept. of Anatomy and Neurobiology, Univ. of Vermont, Burlington, VT 05405 (E-mail: gary.mawe{at}uvm.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.
August 21, 2002;10.1152/ajpgi.00141.2002
Received 11 April 2002; accepted in final form 25 July 2002.
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