nerves are co-localised with the CB receptors, ChAT, a cholinergic nerve marker was used. Co-localisation of CB1 receptor with ChAT was detected in detrusor muscle but not in the mucosa (Figure 3). CB2 receptor was also co-localised with ChAT in both mucosa and detrusor (Figure 3). Negative controls were in the absence of primary antibodies and incubation with non-immunised IgG2A (data not shown). Saturation Binding assays Saturation binding experiments in human cystectomy samples, rat bladder and cerebellum showed that the binding of [3H]-CP55,940 (a synthetic cannabinoid) was concentration dependent and saturable (Figure 4). The Bmax for human bladder was 421.4 fmol[3H]-CP55,940/mg protein and the Kd 1.26 nM. For rat bladder, Bmax was 429.7 fmol[3H]-CP55,940/mg protein with the Kd being 0.39 nM. Binding of [3H]-CP55,940 to rat cerebellum (positive control) demonstrated a higher Bmax of 1974 fmol[3H]-CP55,940/mg protein and a similar Kd (0.45 nM), which is in agreement with a previous report [22]. Discussion Studies investigating differences in the expression and distribution of cannabinoid receptors between normal human bladders and those from patients with DO remain scarce. A recent study using bladder biopsies from MS patients with neurogenic DO and normal bladders, found a lower RNA expression of CB1 receptor and a higher expression of CB2 receptor in bladders with neurogenic DO [23]. Similarly, we showed, using qRT-PCR, that CB1 receptor was significantly upregulated 2.8 fold in the urothelium of DO samples and down regulated 3.2 fold in the detrusor of DO
nerves are co-localised with the CB receptors, ChAT, a cholinergic nerve marker was used. Co-localisation of CB1 receptor with ChAT was detected in detrusor muscle but not in the mucosa (Figure 3). CB2 receptor was also co-localised with ChAT in both mucosa and detrusor (Figure 3). Negative controls were in the absence of primary antibodies and incubation with non-immunised IgG2A (data not shown). Saturation Binding assays Saturation binding experiments in human cystectomy samples, rat bladder and cerebellum showed that the binding of [3H]-CP55,940 (a synthetic cannabinoid) was concentration dependent and saturable (Figure 4). The Bmax for human bladder was 421.4 fmol[3H]-CP55,940/mg protein and the Kd 1.26 nM. For rat bladder, Bmax was 429.7 fmol[3H]-CP55,940/mg protein with the Kd being 0.39 nM. Binding of [3H]-CP55,940 to rat cerebellum (positive control) demonstrated a higher Bmax of 1974 fmol[3H]-CP55,940/mg protein and a similar Kd (0.45 nM), which is in agreement with a previous report [22]. Discussion Studies investigating differences in the expression and distribution of cannabinoid receptors between normal human bladders and those from patients with DO remain scarce. A recent study using bladder biopsies from MS patients with neurogenic DO and normal bladders, found a lower RNA expression of CB1 receptor and a higher expression of CB2 receptor in bladders with neurogenic DO [23]. Similarly, we showed, using qRT-PCR, that CB1 receptor was significantly upregulated 2.8 fold in the urothelium of DO samples and down regulated 3.2 fold in the detrusor of DO