Electroporation-mediated muscarinic M3 receptor gene transfer into rat urinary bladder.
BACKGROUND
Muscarinic M3 (M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M3 receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M3 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 M3 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-M3 receptor, an increase in immunoactivity was observed in the M3 receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the transferred M3 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 M3 receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity.
Otani
Masayuki
M
Department of Urology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan.
Yoshida
Masaki
M
Iwashita
Hitoshi
H
Kawano
Yoshiaki
Y
Miyamae
Kouichi
K
Inadome
Akito
A
Nishi
Toru
T
Ueda
Shoichi
S
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D004558:Electric Stimulation; D018274:Electroporation; D005240:Feasibility Studies; D015870:Gene Expression; D018014:Gene Transfer Techniques; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D043587:Receptor, Muscarinic M3 / Q000235:genetics* / Q000378:metabolism*; D001743:Urinary Bladder / Q000378:metabolism* / Q000503:physiopathology
D018679:Cholinergic Agonists; D043587:Receptor, Muscarinic M3; D002217:Carbachol; D008156:Luciferases