Reciprocal changes in vanilloid (TRPV1) and endocannabinoid (CB1) receptors contribute to visceral hyperalgesia in the water avoidance stressed rat

Abstract

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.

Figure 1. Effect of chronic water avoidance (WA) stress on serum corticosterone level, visceromotor response (VMR) to colorectal distension (CRD), and colonic motor function

(A) The level of serum corticosterone level was significantly increased in rats following chronic WA stress (n = 8). (B) Electromyographic amplitude expressed as area under curve (AUC) of the raw electromyographic response was significantly increased following chronic WA stress at day 11 compared with the baseline level and the controls (n = 8). (C) Effect of WA stress on distal colonic motor function. Fecal pellets were counted at the end of 1 hour WA stress session from day 1 to day 10. Data are expressed as mean ± SE, n = 12 in each group. *, P < 0.05; **, P < 0.01.

Figure 2. The level of the endocannabinoid anandamide was increased in L6-S2 DRGs from WA rats

(A) Representative chromatograph from DRG extracts from control and WA stress rats depicting the relative abundance of endocannabinoid anandamide. (B) The bar graph illustrated the increase in anandamide content in L6-S2 DRGs in rats following chronic WA stress compared to controls. L6-S2 DRGs from 5 rats in each group were combined for sample extraction to reach the adequate weight for LC-APCI/MS-MS analysis.

Figure 3. Immunofluorescence staining of endocannabinoid CB1 receptor was decreased in L6-S2 DRGs in WA stress rats

(A–B) Representative immunofluorescence images of CB1 immunoreactivity (IR)-positive neurons in L6-S2 DRGs from control rats (A) and rats after WA stress (B). Scale bar: 80 μm. (C) CB1 labeling intensity and the number of CB1 IR-positive neurons were significantly decreased in L6-S2 DRGs in rats after WA stress (WA) compared with controls (CT). Data are expressed as mean ± SE, n = 4 in each group. *, P < 0.05.

Figure 4. The protein level of CB1 receptor in L6-S2 DRGs was decreased in WA stress rats

(A) Levels of CB1 protein measured by Western immunoblot analysis. A significant decrease in the level of CB1 protein was observed in samples from WA stress rats compared with controls. (B) Representative Western blot for CB1 receptor in extracts from L6-S2 DRGs showing a prominent band at ~60 kDa. (C) Level of CB2 protein did not change significantly in the WA stress rats compared with the control (P = 3.87). (D) Representative Western blot for CB2 receptor in extracts from L6-S2 DRGs. Data are expressed as normalized density to β-actin, mean ± SE, n = 5 in each group. *, P < 0.05.

Figure 5. TRPV1 receptor protein level was increased in L6-S2 DRGs in WA stress rats

(AB) Representative immunofluorescence images of TRPV1 immunoreactivity (IR)-positive neurons in L6-S2 DRGs from control rats (A) and rats after chronic WA stress (B). Scale bar: 100 μm. (C) Quantification of TRPV1 IR-positive neurons and TRPV1 labeling intensity in L6-S2 DRGs from control and chronic WA stress rats (n = 5). (D) Significant increases in the levels of TRPV1 and phosphorylated TRPV1 (p-TRPV1) were observed in DRGs from chronic WA stress rats compared with controls. Data are expressed as normalized density to β-actin and expressed as % of controls, (E) Representative Western blot for TRPV1 in DRG extracts. *, P < 0.05.

Figure 6. Effects of the endogenous anandamide (AEA) on the expression of CB1 and TRPV1 receptor protein levels in control DRG neurons in vitro

(A) Treatment with AEA in isolated control DRGs significantly decreased the expression level of CB1 and increased the level of TRPV1 receptor protein levels. (B) Representative Western blotting for TRPV1 and CB1 in extracts from isolated DRGs after AEA treatment for 16 h. Data are expressed as normalized density to β-actin, mean ± SE, n = 4 in each group. *, P < 0.05.

Figure 7. Effects of the exogenous CB1 agonist WIN in vivo and in vitro on the expression of TRPV1 and CB1 receptor levels in DRGs

(A) Repeated WIN treatment in vivo prevents the changes of TRPV1 and phosphorylated TRPV1 (p-TRPV1), but not the CB1 protein, in L6-S2 DRGs in rats following chronic WA stress. (B) Representative Western blotting for TRPV1 and CB1 after repeated WIN treatment. (C) Treatment with WIN in isolated control DRGs in vitro significantly decreased the level of TRPV1 and phosphorylated TRPV1 (p-TRPV1). (D) Representative Western blotting for TRPV1 and CB1 in extracts from isolated DRGs after WIN treatment for 16 h. Data are expressed as normalized density to β-actin, mean ± SE, n = 4 in each group. *, P < 0.05.

Figure 8. Treatment of WA rats with the CB1 agonist WIN or TRPV1 antagonist capsazepine reversed visceral hyperalgesia

(A) Representative electromyography (EMG) recordings depicting the VMR to CRD at 40 mmHg in control rats, WA stress rats, WIN-treated stress rats, and capsazepine-treated stress rats. (B) EMG amplitude expressed as mean change from baseline after treatment with WIN for 10 days or one-time treatment with capsazepine in rats following chronic WA stress. Treatment with WIN or capsazepine abolished the WA stress-induced increase in the VMR to CRD at pressures of 40 and 60 mmHg. (C) EMG amplitude expressed as mean change from baseline after treatment with WIN or capsazepine in sham control rats. Data are expressed as mean ± SE, n = 8 in each group. *, P < 0.05