Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells.
OBJECTIVE
Lines of evidence suggest that Rho-associated protein kinase (ROCK)-mediated myosin phosphatase-targeting subunit 1 (MYPT1) phosphorylation plays a central role in smooth muscle contraction. However, the physiological significance of MYPT1 phosphorylation at Thr696 catalyzed by ROCK in bladder smooth muscle remains controversial. We attempt to directly observe the quantitative protein expression of Rho A/ROCK and phosphorylation of MYPT1 at Thr696 after carbachol administration in rat bladder smooth muscle cells (RBMSCs).


METHODS
Primary cultured smooth muscle cells were obtained from rat bladders. The effects of both concentration and time-course induced by the muscarinic agonist carbachol were investigated by assessing the expression of Rho A/ROCK and MYPT1 phosphorylation at Thr696 using Western blot.


RESULTS
In the dose-course studies, carbachol showed significant increase in phosphorylation of MYPT1 at Thr696 (p-MYPT1) from concentrations of 15-100 μM based on Western blot results (p < 0.05, ANOVA test). In the time-course studies, treatment of cells with 15 μM of carbachol significantly enhanced the expression of p-MYPT1 from 3 to 15 h (p < 0.05, ANOVA test) and induced the expression of Rho A from 10 to 120 min (p < 0.05, ANOVA test).


CONCLUSIONS
Carbachol can induce the expression of ROCK pathway, leading to MYPT1 phosphorylation at Thr696 and thereby sustained RBSMCs contraction.
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA.
Benchun
B
Liu
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA.|Department of Urology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
Yung-Chin
YC
Lee
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA.
Amjad
A
Alwaal
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA.
Guifang
G
Wang
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA.
Lia
L
Banie
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA.
Ching-Shwun
CS
Lin
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA.
Guiting
G
Lin
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA. tlue@urology.ucsf.edu.
Tom F
TF
Lue
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects*; D043263:Myosin-Light-Chain Phosphatase / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects; D001743:Urinary Bladder / Q000166:cytology; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*
D002217:Carbachol; D054460:rho-Associated Kinases; D043263:Myosin-Light-Chain Phosphatase
Bladder smooth muscle cell; Carbachol; Phosphorylation; Rat; Rho-associated protein kinase (ROCK)