Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP).
OBJECTIVE
To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra.


METHODS
Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of β-actin. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity.


RESULTS
MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P = 0.09]. MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P = 0.001]. Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to β-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra.


CONCLUSIONS
In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone.
Knuppe Molecular Urology Laboratory, Department of Urology, University of California School of Medicine, San Francisco, CA 94143-0738, USA.
Guiting
G
Lin
Thomas M
TM
Fandel
Alan W
AW
Shindel
Guifang
G
Wang
Lia
L
Banie
Hongxiu
H
Ning
Tom F
TF
Lue
Ching-Shwun
CS
Lin
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D005260:Female; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D009129:Muscle Tonus / Q000502:physiology; D009130:Muscle, Smooth / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014521:Urethra / Q000201:enzymology*; D001743:Urinary Bladder / Q000201:enzymology*
D000199:Actins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009219:Myosin-Light-Chain Kinase; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1