Local activation of cannabinoid CB₁ receptors in the urinary bladder reduces the inflammation-induced sensitization of bladder afferents

Abstract

Background: Systemic administration of cannabinoid agonists is known to reduce pain induced by bladder inflammation and to modulate cystometric parameters in vivo. We have previously reported that intravesical administration of a cannabinoid agonist reduces the electrical activity of bladder afferents under normal conditions. However, the effects of local activation of bladder cannabinoid receptors on afferent activity during inflammation are unknown. This study was aimed to assess the effects of intravesical administration of a cannabinoid agonist on the discharges of afferent fibers in inflamed bladders ex vivo. We also characterized the expression of CB1 receptors in the bladder and their localization and co-expression with TRPV1, a marker of nociceptive afferents.

Results: Compared to untreated animals, afferent fiber activity in inflamed bladders was increased for intravesical pressures between 10 and 40 mmHg. Local treatment with a non selective cannabinoid agonist (AZ12646915) significantly reduced the afferent activity at intravesical pressures above 20 mmHg. This effect was blocked by AM251 but not by AM630 (selective for CB1 and CB2 respectively). Finally, CB1 was co-expressed with TRPV1 in control and inflamed bladders.

Conclusion: These results demonstrate that sensitization of bladder afferents induced by inflammation is partly suppressed by intravesical activation of cannabinoid receptors, an effect that appears to be mediated by CB1 receptors. Also, TRPV1 positive fibers were found to co-express CB1, supporting the hypothesis of a direct action of the cannabinoid agonist on nociceptive afferents. Taken together, these results indicate a peripheral modulation by the cannabinoid system of bladder hypersensitivity during inflammation.

Figure 1

Behavior observed after injection of cyclophosphamide. A) Abdominal contractions observed during 5 min periods every 20 min post cyclophosphamide (CYP) or saline injection. B) Number of micturitions measured during 20 min periods every 20 min periods post CYP or saline injection. Results are mean ± SEM (Saline n = 8, CYP n = 11, **P < 0.01, ***P < 0.001, t-test).

Figure 2

Effects of cyclophosphamide and AZ12646915 on the bladder compliance and firing threshold of the afferent fibers. A and B) Compliance of the bladder calculated as the volume infused to reach 40 mm Hg in normal (29 mice) and cyclophosphamide (CYP) (19 mice) pooled groups (A) and pre- versus post-administration of AZ12646915 in CYP mice (7 mice) (B). C and D) Firing threshold (mm Hg) of afferent fibers responding to bladder filling in normal (146 fibers, 52 mice) and CYP (63 fibers, 19 mice) pooled groups (C) and pre and post AZ12646915 in CYP mice (29 fibers, 7 mice). Results are mean ± SEM. Normal versus CYP analyzed with t-test (*** P < 0.001) (A and C). Pre- versus post-drug analyzed with paired t-test (B and D).

Figure 3

Effects of cyclophosphamide and AZ12646915 on afferent activity in response to mechanical distension. A) Representative traces of one fiber activity in a normal mouse (left panel) and of another fiber from an inflamed mouse (CYP) before and after the intravesical administration of the cannabinoid agonist AZ12646915 (100 μM) (middle and right panels). The superimposed waveforms of each action potential are displayed in the boxes. B) Afferent activity (spikes/s) in response to an increase of intravesical pressure for normal and inflamed (CYP) before and after administration of AZ12646915. Results are mean ± SEM, * P < 0.05, ***P < 0.001 for normal versus CYP (t-test) and + P < 0.05, ++ P < 0.01, +++ P < 0.001 versus CYP pre AZ12646915 (paired t-test). Normal: n = 32 fibers, 10 mice. CYP: n = 29 fibers, 7 mice.

Figure 4

Activity of inflamed (CYP) bladder afferent fibers, normalized to the maximal activity of pre-drug stimulation. A) normalized activity before and after AZ12646915 alone (n = 29 fibers, 7 mice). B) normalized activity before and after AZ12646915 + AM251 (n = 19 fibers, 6 mice), C) normalized activity before and after AZ12646915 + AM630 (n = 19 fibers, 6 mice) and D) represents the percentage of the area under the curves from A, B and C. Data are mean ± SEM. Paired t-test was used for A, B and C and one sample t-test for D. * P < 0.05, **P < 0.01 and ***P < 0.001 versus pre-drug groups.

Figure 5

Control for the selectivity of CB₁ and TRPV1 receptors antibodies. Upper panels (A-D, green) show the immunoreactivity obtained in normal bladder sections incubated with the CB₁ antibodies alone (A, B) or with the specific blocking peptide (C, D). The lower panels (E-H, red) show the reactivity obtained with TRPV1 antibodies alone (E, F) or with the specific blocking peptide (G, H). U = urothelium, L = lumen, SU = suburothelial and M = muscular layers, scale bar = 20 μm.

Figure 6

Bladder immunoreactivity for CB₁ receptor (A, B) and TRPV1 (C, D). Mice were injected with saline (control, A, C and E) or cyclophosphamide (CYP, B, D and F) 300 mg/kg two hours before tissue harvest. Arrowheads = nerve fibres which co-express CB₁ receptors and TRPV1, U = urothelium, L = lumen, dotted line = separation between the suburothelial (SU) and the muscular (M) layers. The boxes focus on the co-expression of CB₁ with TRPV1 in fibers of the suburothelium layer, scale bar = 20 μm.