Electroporation-mediated muscarinic M receptor gene transfer into rat urinary bladder.

摘要

Abstract Background: Muscarinic M(3) (M(3)) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M(3) receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M(3) receptor gene into rat bladders using electroporation (EP) and evaluated the functional expression of the transferred gene. Methods: Plasmids expressing luciferase, a green fluorescence protein and M(3) receptor were injected into the rat bladder and square-wave electric pulses were immediately applied. Two days after gene transfer, we analyzed gene expression. Immunohistochemical staining was performed and the contractile responses from isolated bladder strips, which were induced KCl, carbachol and electrical field stimulation (EFS), were evaluated. Results: The optimal conditions of electroporation were 8 pulses, 45 voltages, 50 milliseconds/pulses and 1 Hz. Under these conditions, luciferase gene expression was enhanced approximately 300-fold, compared to an injection of DNA only. Regarding immunohistochemistry with an anti-M(3) receptor, an increase in immunoactivity was observed in the M(3) receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the transferred M(3) receptor gene, carbachol- and EFS-induced maximum contractile responses of bladder smooth muscle strips significantly increased. Conclusions: These findings suggest that an in vivo EP procedure is an useful method for gene transfer into the bladder and that an overexpression of M(3) receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity.