Shelby N. Morrisroe, Brian J. Philips, Christian H. Coyle, Erin P. Gibbons, Immaculada Ballesteros, Fernando de Miguel, Pradeep Tyagi, William C. de Groat, Naoki Yoshimura, Michael B. Chancellor, University of Pittsburgh Medical Center, Pittsburgh, PA

Introduction and Objective: Cannabinoids (CBs), the active components of Cannabis sativa (marijuana) and their derivatives, have shown clinical promise as effective pain medications, though studies have been few in number. We reasoned that the CB system may represent an innovative target for genitourinary tract pain associated with interstitial cystitis (IC)/painful bladder syndrome (PBS). Consequently, as a proof-of-principle, we evaluated CB1 and CB2 mRNA and protein expression in female rat bladder and in cultured human bladder urothelial cells.

Methods: Bladder tissues were grossly dissected from adult female Sprague-Dawley rats. Immunofluorescence staining for CB1 and CB2 receptors was performed in rat bladder and in normal human (UROtsa) bladder urothelial cells. Expression profiles of CB1/CB2 mRNA and protein in (normal) UROtsa/(tumorigenic) T24 human bladder urothelial cells were analyzed by Real-Time quantitative PCR and Western blot analyses, respectively.

Results: Immunofluorescence staining for CB1 and CB2 proteins was localized predominantly to the urothelium of rat bladder. Cultured UROtsa bladder cells also demonstrated positive staining for both receptors. Analysis of CB1 mRNA expression revealed an approximate 13-fold increase in T24 bladder tumor cells in comparison to normal UROtsa bladder cells. In agreement, Western blot analysis indicated elevated CB1 protein expression in T24 cells compared to UROtsa cells, though only a 3-fold difference was observed. In contrast, relative to levels of CB2 mRNA expression in UROtsa cells, we noted a 3.5-fold reduction in CB2 mRNA levels in T24 cells, further confirmed by Western blot (protein) analysis.

Conclusions: Our results demonstrate that mRNA and protein for CB1/CB2 cannabinoid receptors are expressed in rat bladder and in normal/tumorigenic human bladder urothelial cell lines. The differential expression of CB1 and CB2 in normal/malignant bladder cells suggests a biological role for these receptors in bladder disease/dysfunction. We hypothesize that CB receptors play a critical role in the irritative voiding symptoms of IC/PBS and that administration of non-addictive synthetic and semi-synthetic cannabinoids can help alleviate these painful symptoms. Current studies are ongoing that will determine whether or not: 1) CB1 and CB2 receptors are differentially expressed in the urothelium of normal bladder versus bladder of IC/PBS patients and 2) cellular signaling mechanisms differ between normal and irritated (IC/PBS) human bladder tissues following CB receptor binding/activation.

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