Yan Sun1, Toby C Chai. 1. Department of Surgery, University of Maryland School of Medicine, Baltimore, 21201, USA.
Abstract
PURPOSE: Extracellular adenosine triphosphate (ATP) can act through the purinergic receptor subtype P2X3 to transmit a pain signal to the central nervous system. Bladder urothelial cells (BUCs) from patients with interstitial cystitis (IC) patients have augmented release of ATP during in vitro stretch. We queried whether P2X3 receptor subtype exists on cultured BUCs. Furthermore, we asked whether P2X3 expression can be altered by in vitro stretch and whether there exists a difference in expression between IC and control BUCs. MATERIALS AND METHODS: BUCs from 4 IC and 5 control subjects were cultured according to established cell culture techniques. After cells were grown to confluence they were stretched on an in vitro stretch machine at 20% elongation for up to 96 hours. Cells were harvested at various times, and immunofluorescence and Western blots were performed for P2X3 expression. Band densitometry normalized to the actin band was performed using software. To confirm P2X3 expression in a whole cell preparation flow cytometry and fluorescence activated cell sorter analysis were used. RESULTS: P2X3 was expressed in cultured BUCs. With stretch P2X3 expression increased in IC BUCs more than in control BUCs. Using relative densitometry units normalized for actin expression in the Western blots stretch of IC BUCs resulted in 66% more expression of P2X3 than any other experimental condition (p = 0.003). By fluorescence activated cell sorter analysis stretched IC BUCs had the highest P2X3 fluorescence activity (30%) compared with unstretched IC (2.4%, p = 0.003), stretched control (9.7%, p = 0.009) and unstretched control (2.3%, p = 0.003) BUCs. CONCLUSIONS: These data show that P2X3 subunits expressed by cultured IC BUCs are up-regulated during in vitro stretch. Augmented ATP signaling in the bladder may explain IC symptoms. Furthermore, this study further supports the hypothesis that urothelial cells can phenotypically mimic sensory neurons.
PURPOSE: Extracellular adenosine triphosphate (ATP) can act through the purinergic receptor subtype P2X3 to transmit a pain signal to the central nervous system. Bladder urothelial cells (BUCs) from patients with interstitial cystitis (IC) patients have augmented release of ATP during in vitro stretch. We queried whether P2X3 receptor subtype exists on cultured BUCs. Furthermore, we asked whether P2X3 expression can be altered by in vitro stretch and whether there exists a difference in expression between IC and control BUCs. MATERIALS AND METHODS: BUCs from 4 IC and 5 control subjects were cultured according to established cell culture techniques. After cells were grown to confluence they were stretched on an in vitro stretch machine at 20% elongation for up to 96 hours. Cells were harvested at various times, and immunofluorescence and Western blots were performed for P2X3 expression. Band densitometry normalized to the actin band was performed using software. To confirm P2X3 expression in a whole cell preparation flow cytometry and fluorescence activated cell sorter analysis were used. RESULTS:P2X3 was expressed in cultured BUCs. With stretch P2X3 expression increased in IC BUCs more than in control BUCs. Using relative densitometry units normalized for actin expression in the Western blots stretch of IC BUCs resulted in 66% more expression of P2X3 than any other experimental condition (p = 0.003). By fluorescence activated cell sorter analysis stretched IC BUCs had the highest P2X3 fluorescence activity (30%) compared with unstretched IC (2.4%, p = 0.003), stretched control (9.7%, p = 0.009) and unstretched control (2.3%, p = 0.003) BUCs. CONCLUSIONS: These data show that P2X3 subunits expressed by cultured IC BUCs are up-regulated during in vitro stretch. Augmented ATP signaling in the bladder may explain IC symptoms. Furthermore, this study further supports the hypothesis that urothelial cells can phenotypically mimic sensory neurons.
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