Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP)

Abstract

Objective: To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra.

Materials and 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.

Conclusion: 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.

FIG. 1

Rat bladder and urethra (n=6 each) were analyzed by real-time PCR for MLCK and MYPT1 mRNA expression. Expression level is relative to GAPDH expression and based on 3 independent experiments.

FIG. 2

Rat bladder and urethra (n=6 each) were analyzed by western blot for MLCK and MYPT1 expression. A. Representative gel showing the MLCK, MYPT1, and β-Actin bands of 3 bladder and 3 urethra samples. B. Graph representation of the compiled densitometry data from A. Expression level is relative to β-Actin expression and based on 3 independent experiments.

FIG. 3

Rat bladder and urethra (n=6 each) were analyzed for MLCK activity at 1, 3, and 5-min time points. MLCK activity was calculated as pMole of ³²P bound to MLC20 per g of cell extract protein. * indicates p<0.01.

FIG. 4

Rat bladder and urethra (n=6 each) were analyzed for MLCP activity at 1, 3, and 5-min time points. MLCP activity was calculated as pMole of ³²P released from ³²P-labled MLC20 per g of cell extract protein. * indicates p<0.05.