Background: Increasing evidence suggests that chronic stress plays an important role in the pathophysiology of several functional gastrointestinal disorders. We investigated whether cannabinoid receptor 1 (CB1) and vanilloid receptor 1 (TRPV1; transient receptor potential vanilloid 1) are involved in stress-induced visceral hyperalgesia.
Methods: Male rats were exposed to 1 h water avoidance (WA) stress daily for 10 consecutive days. The visceromotor response (VMR) to colorectal distension (CRD) was measured. Immunofluorescence and western blot analysis were used to assess the expression of CB1 and TRPV1 receptors in dorsal root ganglion (DRG) neurons.
Results: WA stressed rats demonstrated a significant increase in the serum corticosterone levels and faecal pellet output compared to controls supporting stimulation of the hypothalamic–pituitary–adrenal (HPA) axis. The VMR increased significantly at pressures of 40 and 60 mm Hg in WA stress rats compared with controls, respectively, and was associated with hyperalgesia. The endogenous CB1 agonist anandamide was increased significantly in DRGs from stressed rats. Immunofluorescence and western blot analysis showed a significant decrease in CB1 and a reciprocal increase in TRPV1 expression and phosphorylation in DRG neurons from stressed rats. These reciprocal changes in CB1 and TRPV1 were reproduced by treatment of control DRGs with anandamide in vitro. In contrast, treatment of control DRGs in vitro with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats in situ with WIN 55,212-2 or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the upregulation of TRPV1.
Conclusions: These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia.
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Stress is an adaptive response that affects endocrine, autonomic and visceral functions in order to maintain homeostasis of the organism in the face of internal or external threats (stressors). Stress is associated with symptom onset, exacerbation and perpetuation in patients with functional gastrointestinal disorders.1 A variety of stressors appear to have an important role in the pathophysiology of irritable bowel syndrome (IBS), which is characterised by altered bowel evacuation, bloating and visceral pain, in the absence of anatomical or biochemical abnormalities.2 Thus, based on both human and animal studies, stress-induced visceral hyperalgesia has been suggested as an important biological feature of IBS.1 3
Accumulating evidence suggests that the endogenous cannabinoid system is involved in the regulation of the hypothalamic–pituitary–adrenal (HPA) axis.4 The endogenous cannabinoid system consists of two receptors: cannabinoid receptor 1 (CB1), which is expressed primarily in the central and peripheral nervous system; and the CB2 receptor, which is localised primarily in immune cells. The CB1 receptor is known to play a role in modulating a number of important body functions including pain perception4 and is expressed at high levels in the brain. CB1 agonists suppress the secretion of adrenocorticotropic hormone and plasma corticosterone levels.5 Mice deficient in CB1 display increased levels of adrenocorticotrophin and corticosterone and an enhanced susceptibility to chronic variable stress.6 Furthermore, CB1 inhibits gastrointestinal motility in the enteric nervous systems, mainly by inhibition of contractile neurotransmitter release.7 Therefore, there is a rationale for the use of cannabinoid drugs to treat IBS in humans.
Apart from activation of cannabinoid receptors, the endocannabinoid anandamide directly activates the vanilloid receptor 1 (TRPV1; transient receptor potential vanilloid 1) in nociceptive neurons.8 TRPV1 is a ligand-gated ion channel that plays a key role in modulation of the sensation of pain and thermal hyperalgesia.9 Activation of TRPV1 leads to influx of the cations and consequently results in depolarisation, neuronal hyper-excitability and pain sensation. In the gastrointestinal tract, TRPV1 is located on the visceral primary afferent neurons.10 Electrophysiological recordings in retrograde labelled neurons innervating the colon reveal that a significant number are capsaicin- and TRPV1-sensitive,11 supporting a role for the TRPV1 channel in visceral sensation and nociception. Taken together, the available data support the involvement of CB1 and TRPV1 receptors in the modulation of visceral nociception. However, the potential relationship between visceral hypersensitivity and CB1 and TRPV1 receptors in chronic psychological stress has not been examined.
In the present study, we investigated whether CB1 and TRPV1 receptor expression and function play roles in the regulation of sustained visceral hyperactivity in a validated rat model of psychological stress that demonstrates many characteristics of IBS in the human.12
MATERIALS AND METHODS
Male Sprague–Dawley rats (200–220 g) were obtained from Charles River Laboratories (Wilmington, Massachussets, USA). Animals were housed in the animal facility, which was maintained at 22°C with an automatic 12 h light/dark cycle. The animals received a standard laboratory diet and tap water ad libitum. The experimenter was blinded to animal treatment during behavioural experiments.
Water avoidance stress
Repeated water avoidance (WA) stress to adult rats was conducted as described previously.12 The rats were placed on the glass platform in the middle of a test Plexiglas tank that was filled with water (25°C) to 1 cm below the height of the platform. The animals were maintained on the block for 1 h daily for 10 consecutive days. This repeated WA procedure represents a potent psychological stressor.13 The sham control rats were placed similarly for 1 h daily for 10 days in the container without water. In a separate study, a group of eight rats were subcutaneously injected with WIN 55,212-2 (2 mg/kg) consecutively for 10 days after rats were subjected to WA stress. A group of nine WA stress rats were injected intraperitoneally with capsazepine (5 mg/kg) 1 h before the asssessment of visceromotor response (VMR). WIN 55,212-2 and capsazepine were dissolved in 10% dimethylsulfoxide (DMSO)/5% Tween 80/85% saline and the doses were selected based on published reports.14 15
Measurement of serum corticosterone
Blood was collected from the rat after 1 h WA stress on day 10 of the stress procedure. Total corticosterone in serum samples was analysed by the Correlate–EIA corticosterone enzyme immunoassay kit (Assay Designs, Ann Arbor, Michigan, USA) according to the manufacturer’s instructions. All samples were run in triplicate with standard corticosterone controls and kit calibrators in each analysis. Absorbance at 405 nm was measured with a reference wavelength of 680 nm.
Measurement of faecal pellet output
Faecal pellet output was used to estimate the regulation of distal colonic motility as a validated procedure as described previously.12 Faecal pellets found in the tank were counted at the end of 1 h WA stress. Control rats were left in the home cage for 60 min to count the faecal pellets.
Visceromotor response to colorectal distension
Rats were deeply anaesthetised with a subcutaneous injection of a mixture of ketamine (60 mg/kg) and xylazine (5 mg/kg), and Teflon-coated, 32-gauge stainless steel wires were inserted into the external oblique pelvic muscles superior to the inguinal ligament 5–7 days before the beginning of the experimental procedures. Measurement of VMR to colorectal distension (CRD) was conducted in rats on the following day after the WA stress procedure was completed as described.12 16 A flexible latex balloon (4–5 cm) was inserted intra-anally with its end 1 cm proximal to the anus. Animals with a balloon inserted were placed in the animal cage for 30 min before CRD was initiated. The VMR was quantified by measuring electromyographic activity in the external oblique musculature. A series of CRDs was conducted to constant pressures of 20, 40, 60 mm Hg by a custom-made distension control device. Each distension consisted of three segments: a 20 s predistension baseline period, a 20 s distension period, and a 20 s period after termination of CRD with a 4 min inter-stimulus interval. The responses were considered stable if there was less than 20% variability between two consecutive trials of CRD at 60 mm Hg. Spike bursts higher than 0.3 mV were regarded as significant and therefore used to estimate the pain response. Rats showing a VMR signal/ratio <0.05 were excluded from the study. The increase in the area under the curve (AUC), which is the sum of all recorded data points multiplied by the sample interval (in seconds) after baseline subtraction, was presented as the overall response during the course of the CRD test.
Dorsal root ganglion (DRG) samples used for mass spectrometry analysis (liquid chromatography–atmospheric pressure chemical ionisation/mass spectrometry (LC–APCI/MS–MS)) were prepared according to the method described.17 Briefly, L6–S2 DRGs from control and WA stressed rats (n = 5 each group) from rats that had not undergone surgery or CRD were dissected on day 11 of the WA stress procedure, combined and homogenised in 2.0 ml acetonitrile containing 114 pmol of deuterated arachidonic acid. The supernatants were concentrated under a constant stream of nitrogen and subjected to LC-APCI/MS analysis. Analysis of anandamide was performed using a ThermoFinnigan Surveyor HPLC system in conjunction with a triple quadrupole mass spectrometer (ThermoFinnigan TSQ Quantum Ultra AM; Thermo Scientific, Waltham, Massachusetts, USA). For quantitative analysis, the peak area of anandamide ions from the test samples was compared and normalised. The standard curve was obtained by injecting known amounts of anandamide ranging between 1 pmol/l and 100 μmol/l and plotting peak area versus molar concentration.
Administration of cannabinoids in vitro
L6–S2 DRGs from control rats were isolated, chopped and incubated in minimal essential medium (Gibco, Gaithersburg, Maryland, USA) supplemented with 3% fetal bovine serum and 50 ng/ml nerve growth factor in 95% air + 5% CO2 at 37°C. A series of doses of cannabinoids containing 0–2 μg/ml anandamide or 0–20 μg/ml of the CB1 agonist WIN 55,212-2 were added to the incubation chambers and incubated for 16 h. The DRGs were then collected for western blot analysis.
Western blot analysis
L6–S2 DRGs from rats that had not undergone surgery or CRD were taken on day 11 of WA stress procedure and homogenised in ice-cold lysis buffer containing 50 mmol/l Tris, pH 8.0, 150 mmol/l NaCl, 1 mmol/l ethylene glycol-bis(β-aminoethylether)N,N,N′,N′-tetraacetic acid (EGTA), 50 mmol/l NaF, 1.5 mmol/l MgCl2, 10% v/v glycerol, 1% v/v Triton X-100, 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l Na3VO4, and “Complete” protease inhibitor mixture (Roche Diagnostics, Indianapolis, Indiana, USA). Proteins were separated and transblotted to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, New Jersey, USA). The membranes were blocked and incubated with CB1 antibody raised against amino acids 1–77 of the rat CB1 receptor (1:2000; Sigma–Aldrich, St Louis, Missouri, USA) or TRPV1 antibody raised against the N-terminus of TRPV1 of rat origin (1:3000; Santa Cruz Biotechnology, Santa Cruz, California, USA), at 4°C, overnight, and subsequent with secondary antibodies (1:5000; Santa Cruz Biotechnology) for 1 h and developed using x ray films by the West Dura Supersignal chemiluminescence kit (Pierce, Rockford, Illinois, USA). In some experiments, equal amounts of total proteins were mixed with anti-TRPV1 antibody (1:50) for immunoprecipitation and probed with anti-phosphorylation antibodies (1:4000; Chemicon, Temecula, California, USA) as described previously.18
L6–S2 DRGs from rats that had not undergone surgery or CRD were removed on day 11 of WA stress procedure and fixed for 2–3 h in 4% paraformaldehyde in 0.1 mol/l phosphate buffer. Transverse sections through the DRG (10 μm) were cut on a cryostat for immunohistochemistry. Sections were permeabilised with 0.3% Triton X-100 for 2 h, and then blocked with 10% normal goat serum in phosphate-buffered saline with 0.3% Triton X-100 for 4 h at room temperature. Primary antibodies used for overnight incubation were anti-TRPV1 raised against the C-terminus of TRPV1 of rat origin (1:400; Chemicon) and anti-CB1 (1:400; Sigma) antibodies. Secondary antibodies Alexa Fluor 488, 594 (1:500) from Molecular Probes (Eugene, Oregon, USA) were used with a 2 h incubation. Sections were mounted with an anti-fade fluorescence mounting medium for microscope viewing.
Immunostained DRG sections were viewed under a Zeiss Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany), and digitised images were acquired covering the entire DRG in one field under low magnification. Images from four to five different DRG sections from each rat were pooled for counting in each group. In each rat, 1000–1500 total DRG neurons were counted. Neurons were judged to be positive if they had mean brightness values greater than the corresponding control value stained with the secondary antibody alone. For quantitative analysis of immunofluorescence intensity in DRG neurons, stained DRG sections were randomly selected and the intensity of immunofluorescence staining from the digitised images were converted to grey values ranging from 0 to 255. An appropriate threshold was set such that only specific immunoreactivity was accurately represented and light non-specific background labelling was not detected. The threshold was the same for all images. The density limited to threshold in the stained cell area was measured for each section and normalised to cell area (100 μm2) by NIH Image-J software V1.41.
To examine the VMR in response to pressure, the electromyographic amplitudes, represented by calculating the AUC, were normalised as a percentage of baseline response for the highest pressure (60 mm Hg) for each rat and then averaged for each group of rats. This type of normalisation has been validated in a similar model to account for individual variations of the electromyographic signals.19 The effects of stress and/or pharmacological treatment on the VMR to CRD was analysed by comparing the post-stress or post-treatment measurements with the baseline or pretreatment values at each distension pressure using a repeated-measures two-way ANOVA followed by Bonferroni post-test comparisons. The VMR measurements at day 11 for rats treated with compound or vehicle were expressed as the mean change from baseline for different pressures of distension as described.20 The unpaired Student t test was used to examine the data for the expression of TRPV1 and CB1 obtained from western blot and immunohistochemistry, for corticosterone and faecal pellet output measurements. Results were expressed as means with the SEM. A value of p<0.05 was considered statistically significant.
Chronic water avoidance stress resulted in increased visceral nociception
The serum corticosterone level was significantly higher in chronic WA rats compared with the controls (p<0.05). In control rats, the serum corticosterone level was 97.8 (SEM 23.2) ng/ml, while it was 310.8 (SEM 30.9) ng/ml in stressed rats (fig 1A). The VMR in response to the intensities of CRD was recorded on day 0 before the start of WA stress and sham stress as the baseline level and recorded again on day 11 after chronic stress. In response to CRD, the VMR mean change, expressed as AUC percentage, was significantly higher in the WA stress rats at day 11 compared with the baseline level for the pressures of 20 and 40 mm Hg (fig 1B; p<0.05). The AUC in the WA stress rats increased 109.7 (SEM 37.2)% compared with the controls for the pressure 60 mm Hg (p<0.01). The VMR in rats following sham stress did not change significantly at day 11 compared with the baseline level. All eight rats after repeated WA stress showed increased VMR to CRD, which is consistent with visceral hyperalgesia observed in the rats following repeated WA stress.12 Moreover, faecal pellet outputs were significantly increased in rats on days 1, 5 and 10 of WA stress compared with the controls (fig 1C, p<0.05), indicating the altered colonic motor function following repeated WA stress.
Chronic water avoidance stress increased the level of the endocannabinoid anandamide in DRGs
Representative chromatographs depicting the content of anandamide in DRG tissue extracts are shown in fig 2A. The value from control L6–S2 DRGs was 111.3 ng/g tissue weight for anandamide, whereas it was 160.3 ng/g tissue weight in chronic WA stress rats, corresponding to 44% higher anandamide content than that of the controls (fig 2B). This percentage increase in anandamide content was similar to the significant increase in anandamide observed in lumbar DRG in a model of neuropathic pain.21 We also measured the content of 2-arachidonylglycerol. However, the levels of 2-arachidonylglycerol in DRG extracts from both control and WA stressed rats were too low to be detected, possibly due to the natural significant lower content of 2-arachidonylglycerol as compared with anandamide in DRGs.21
Chronic water avoidance stress decreased CB1 expression in DRG neurons
To determine the expression of CB1 receptors, the colonic DRGs (L6–S2) corresponding to the distension area for VMR measurement were identified by retrograde labelling (data not shown). As shown in fig 3A,B, the CB1 immunoreactivity (IR) signal was greatly reduced in L6–S2 DRGs from chronic WA stress rats compared with controls. In chronic WA stress rats, the CB1 labelling intensity was 43.2 (SEM 6.7), whereas it was significantly decreased to 12.3 (SEM 2.4) in DRGs from control animals. The number of CB1 IR-positive neurons was also significantly decreased in stress rats (57.5 (SEM 5.8)%) compared with the control (84.3 (SEM 4.2)%) (fig 3C; p<0.05). The decreased CB1 expression was further confirmed by western blotting analysis. The mean immunoblot band density for CB1 in stressed rats was significantly decreased to 39.8 (SEM 11.5)% of the control (fig 4A,B; p<0.05). However, the expression level of CB2 was not altered in the rats following repeated WA stress compared with the controls (fig 4C,D).
Chronic water avoidance stress increased TRPV1 expression in DRG neurons
As shown in fig 5A,B, the TRPV1 immunoreactivity signal was increased in L6–S2 DRGs from chronic WA stress rats compared with controls. The number of TRPV1 IR-positive neurons was 43.2 (SEM 1.4)% in controls rats and increased significantly to 57.3 (SEM 4.1)% in chronic WA stress rats, while the labelling intensity was increased to 125.3 (SEM 7.9) in stress rats compared with the control (78.3 (SEM 4.6)) (fig 5C; p<0.05). The changes in TRPV1 expression in chronic WA stress rats was confirmed using western immunoblot analysis. The protein level of TRPV1 and the level of phosphorylated TRPV1 increased 81% and 45% in L6–S2 DRGs in chronic WA stress rats, respectively, compared with controls as shown by quantitative densitometry of the immunoblots (fig 5D,E; p<0.05).
Treatment of control DRGs in vitro with anandamide resulted in decreased CB1 and increased TRPV1 receptor levels
It has been reported that the function of the TRPV1 receptor is modulated by the cannabinoid system. We examined whether CB1 and TRPV1 receptor levels in control DRGs were affected by the preferential CB1 agonist anandamide. Isolated DRGs from control rats were incubated with anandamide (0–2 μg/ml) in the culture media for 16 h. As shown in fig 6A,B, anandamide treatment (2 μg/ml) induced significant upregulation of TRPV1 receptor in control DRGs. Moreover, the levels of CB1 were significantly decreased in DRGs after treatment of anandamide at the concentration of 0.5 μg/ml and 2 μg/ml (p<0.05). Moreover, the phosphorylated TRPV1 significantly increased 85% in DRGs treated with 2 μg/ml anandamide compared with the non-treated control (data not shown). These results support the interpretation that the endogenous preferential CB1 agonist anandamide can induce reciprocal alterations in the levels of CB1 and TRPV1 receptors in isolated healthy DRG neurons similar to the pattern observed in the WA stressed rats in vivo.
Effect of the exogenous CB1 agonist WIN 55,212-2 on CB1 and TRPV1 receptor protein levels in situ and in vitro
It has been shown that cannabinoid agonists have antinociception effects in several pain models.4 In view of our results demonstrating upregulation of TRPV1 receptors in the stressed rat in situ and that anandamide upregulates the level of TRPV1 in control DRGs in vitro, we next examined whether the stable CB1 receptor agonist WIN 55,212-2 affects the expression and function of TRPV1 in situ in stressed rats. After rats were injected with WIN 55,212-2 (2 mg/kg, subcutaneous injection) daily for 10 days during the WA stress-induction period, CB1 and TRPV1 receptor levels were examined in L6–S2 DRGs. As shown in fig 7B, injection of WIN 55,212-2 prevented the upregulation of TRPV1 but had little effect on CB1 expression in stress rats. Quantitative densitometry of the immunoblot bands showed that the level of TRPV1 protein after WIN 55,212-2 treatment in chronic WA stress rats was 92.9 (SEM 9.1)% of the vehicle-treated controls (p = 0.48) and that phosphorylated TRPV1 was 112.6 (SEM 8.7)% of the vehicle-treated controls (p = 0.19; fig 7A). The effects of WIN 55,212-2 treatment on TRPV1 expression in situ was confirmed by in vitro studies. As shown in fig 7C,D, WIN 55,212-2 treatment in isolated control DRGs in vitro significantly decreased the levels of TRPV1 and phosphorylated TRPV1 compared with the control (p<0.05). Thus, the effects of anandamide and WIN 55,212-2 on the expression TRPV1 receptors in L6–S2 DRGs were quite different, anandamide causing upregulation and WIN 55,212-2 downregulation in TRPV1 receptor levels and phosphorylation.
Administration of the CB1 agonist WIN 55,212-2 or TRPV1 antagonist capsazepine prevented hypersensitivity in chronic water avoidance stress rats
To further assess the potential therapeutic effect of the CB1 agonist WIN 55,212-2 on visceral hyperalgesia in WA stress rats, we measured VMR to CRD in rats that received WIN 55,212-2 (2 mg/kg) daily for 10 days during the WA stress-induction period. Another group of rats received an intraperitoneal injection of capsazepine (5 mg/kg) 1 h before the measurement of VMR. Consistent with previous results, repeated WA stress resulted in an increase in the VMR to CRD (fig 8A). As shown in fig 8A,B, repeated treatment with WIN 55,212-2 significantly decreased the stress-induced increase in the VMR at pressures of 40 and 60 mm Hg. The VMR amplitude was significantly decreased to 29.5 (SEM 2.9)% and 32.3 (SEM 17.8)% at CRD pressures of 40 and 60 mm Hg compared with vehicle-treated stress rats (77.8 (SEM 11.8)% over baseline level at 40 mm Hg and 102.7 (SEM 23.3)% over baseline level at 60 mm Hg), respectively (p<0.05). Similarly, the TRPV1 antagonist capsazepine significantly decreased the stress-induced increase in the VMR to CRD (35.4 (SEM 15.4)% at 40 mm Hg; 41.0 (SEM 14.0)% at 60 mm Hg, respectively) in stress rats. No significant effects of WIN 55,212-2 or capsazepine were observed on the VMR to CRD at all the pressures examined in sham control rats (fig 8C).
The present study supports a potentially important novel role for the TRPV1 receptor in mediating visceral hyperalgesia in a chronic stress rat model that is modulated by the endocannabinoid CB1 receptor pathway. Water avoidance-induced stress was associated with (1) increased serum corticosterone levels; (2) increased stool output; (3) visceral hyperalgesia in response to colorectal distension; (4) increased levels of the endocannabinoid anandamide in DRGs from stressed rats; (5) decreased levels of CB1 receptor and increased levels of TRPV1 receptor and TRPV1 receptor phosphorylation in L6–S2 DRGs from stressed rats in vivo and in anandamide-treated DRGs isolated from control rats in vitro; (6) decreased levels of TRPV1 receptor and TRPV1 phosphorylation in control L6–S2 DRGs treated with the selective CB1 receptor agonist WIN 55,212-2 in vitro; and (7) prevention of visceral hyperalgesia in WA stressed rats treated in vivo with WIN 55,212-2 or the TRPV1 receptor antagonist capsazepine.
Evidence supports the concept that visceral hypersensitivity measured in response to colorectal distension is an important factor in IBS.1 We observed that the expression of TRPV1 and TRPV1 phosphorylation were upregulated in L6–S2 DRG neurons in the WA stressed rats, as well as an increased number of TRPV1 IR-positive neurons. It is likely that the increased number of TRPV1 IR-positive neurons includes DRG neurons innervating the colon the in chronic WA stress rats since >50% of the rodent colon DRG neurons respond to capsaicin and this response is blocked by capsazepine.11 Upregulation of TRPV1 has been reported in a variety of gastrointestinal diseases,22 23 including IBS in human patients.24 Capsaicin-induced pain and hyperalgesia has also been reported in humans.25 It has been suggested that intrinsic modulators, such as substance P26 and 5-hydroxytryptamine (5-HT),27 can sensitise TRPV1 via phosphorylation and thereby enhance the probability of channel gating by heat or other stimuli.28 The substance P/neurokinin 1 receptor (NK1R) has recently been suggested to play an important role in the maintenance of visceral hyperalgesia in chronic WA stress rats.19 Thus, the increased expression of TRPV1 in L6–S2 DRG neurons may act to facilitate pain signals and maintain peripheral or central sensitization in chronic stress.
The antinociceptive effects of cannabinoids have an important physiological role in modulating pain sensitivity.4 Recent electrophysiological and neurochemical studies provide new evidence to support a role of endocannabinoids in the modulation of visceral pain. We hypothesised that in chronic stress-induced visceral hyperalgesia endocannabinoids such as anandamide act to downregulate the CB1 receptor in visceral nociceptive pathways which, in turn, results in upregulation of TRPV1 receptor expression and function. Consistent with this hypothesis, chronic WA stress was associated with increased endogenous anandamide , reduced levels of CB1 receptor and increased levels of TRPV1 receptor in L6–S2 DRGs. The majority of CB1 IR-positive DRG neurons also express the TRPV1 receptor.29 This co-localization of CB1 and TRPV1 in DRG neurons provides the anatomical basis for the interaction of these receptors. TRPV1 is a non-selective cation channel and the activity is modulated by its phosphorylation status through phosphokinase A (PKA)/PKC dependent pathways. Activation of CB1 activates inhibitory Gi/o proteins and leads to inhibition of adenylyl cyclase, reduces production of the second messenger cAMP and inhibits calcium channels,30 which enables the functional basis for interaction with the TRPV1 receptor. Recent reports suggest that application of a CB1 agonist inhibits TRPV1 activation when cAMP-mediated signalling is activated31 and reverses capsaicin-induced thermal hyperalgesia.32 We observed that in chronic WA stress rats, the CB1 receptor level decreased whereas the TRPV1 and its phosphorylation levels increased in colonic DRG neurons. This response was reproduced in control DRGs by treatment with anandamide in vitro, possibly through phosphorylation by G-protein-coupled receptor kinases followed by desensitisation, internalisation and degradation.33 We hypothesise that the downregulation of CB1 receptor levels is linked to enhanced expression and function of TRPV1 receptors in DRG neurons in WA stress rats. Of interest, the upregulation of TRPV1 receptor levels in WA stress rats was attenuated by serial treatment with the selective CB1 receptor agonist WIN 55,212-2 in vivo, supporting a potential therapeutic role of WIN 55,212-2 or another peripherally acting CB1 receptor agonist in stress-induced visceral hyperalgesia. Consistent with this, WIN 55,212-2 reduces visceromotor responses to distension and a CB1, but not CB2, antagonist enhances colitis-induced hyperalgesia.34 Thus, the endocannabinoid anandamide and CB1 receptor agonist WIN 55,212-2 had different effects on TRPV1 receptor expression and function and visceral nociception. Endocannabinoids such as anandamide are synthesised “on demand” at the cell surface membrane and act locally as paracrine/autocrine factors. Under conditions of chronic stress, our data suggests that endocannabinoids modulate CB1 (downregulate) and TRPV1 (upregulate) receptors in L6–S2 DRGs. Therefore, the endogenous expression of synthetic/degradation enzymes and receptor targets at the cellular level are likely to play a particularly important role in the differential, tissue-specific actions of endocannabinoids. On the other hand, exogenous delivery of cannabinoids such as WIN 55,212-2 appears to produce antihyperalgesic and antinociceptive effects via different mechanisms. For example, it has been reported that WIN 55,212-2 dephosphorylates and desensitises TRPV1 via the TRPA1 receptor and contributes to the peripheral antihyperalgesic effects of cannabinoids.31 32 Therefore, our data demonstrating the prevention of visceral hyperalgesia by WIN 55,212-2 in a chronic stress model is consistent with the reported analgesic effect of cannabinoids.4
Although numerous studies have demonstrated that activation of cannabinoid receptors can reduce nociceptive transmission in a variety of animal models, the clinical significance of the analgesic effect remains controversial, along with elucidation of the relevant site(s) of action. The increase in the content of anandamide as well as the reciprocal changes in CB1 and TRPV1 in L6–S2 DRGs in chronic WA stress rats supports a peripheral action for endocannabinoids. Consistent with this interpretation, specific loss of CB1 receptors in nociceptive neurons in the peripheral nervous system significantly reduces analgesic effects produced by endocannabinoids, as well as systemically administered cannabinoids. This suggests that peripheral CB1 receptors are the primary target for cannabinoid-mediated analgesia.35 Our study was not designed to dissect the relative contribution of central versus peripheral mechanisms to the visceral hyperalgesia observed in the water avoidance-stressed rat. We administered the CB1 agonist WIN 55,212-2 and TRPV1 antagonist capsazepine systemically; therefore, WIN 55,121-2 and capsazepine could act on receptors in the brain and spinal cord since CB1 and TRPV1 are broadly expressed in the brain and spinal cord. Previous studies support the suggestion that endocannabinoids can act at CB1 receptors in the central nervous system to modulate pain processing.36 It will be interesting to investigate whether both central and peripheral mechanisms involving CB1 and TRPV1 receptors contribute to chronic stress-induced visceral hyperalgesia in future studies.
Recent studies support the hypothesis that functional interactions occur between glucocorticoids and the endocannabinoid system in situ. For example, mice deficient in the CB1 receptor have high levels of corticotrophin releasing factor, enhanced circadian drive to the HPA axis as well as elevated plasma corticosterone levels,6 suggesting endocannabinoids provide a tonic feedback to control the HPA axis. Exposure of animals to chronic unpredictable stress causes a significant reduction in the expression of CB1 in hippocampus.37 These data support the hypothesis that glucocorticoids exert negative regulation of the expression of CB1 receptors. In accordance with this, we observed elevated serum corticosterone level and decreased expression of CB1 receptor in colonic DRG neurons in chronic WA stress rats. It is possible that the decreased expression of CB1 receptors might be linked to the increased corticosterone level in stressed rats. Supporting this, repeated administration of glucocorticoids or corticosterone increases the contents of anandamide and 2-arachidonylglycerol, both in vitro and in situ,38 and significantly reduces the CB1 protein level. Future studies will explore the detailed mechanism(s) underlying the neuroplastic alterations of CB1 and TRPV1 receptors that are associated with chronic stress-induced visceral hyperalgesia.
In summary, visceral hyperalgesia in response to psychological stress is associated with reciprocal changes in the expression and function of CB1 (decreased) and TRPV1 (increased) receptors in L6–S2 DRGs. This novel observation suggests that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play an important role in stress-induced visceral hyperalgesia.
Funding: This work was supported by grants R01DK052387 and R01DK056997 to JWW from the National Institutes of Health; by an ANMS/SmartPill grant to SH from the American Neurogastroenterology and Motility Society; and by a Pilot/Feasibility grant to SH from the Michigan Gastrointestinal Peptide Research Center (NIH grant 5 P30 DK34933).
Competing interests: None.
Ethics approval: All animal procedures were approved by the University of Michigan Committee on Use and Care of Animals according to National Institutes of Health guidelines, on 2 March 2005.
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