Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo.
Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.
Dept. Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040, USA.
Yusuke
Y
Mizuno
Eiji
E
Isotani
Jian
J
Huang
Hailei
H
Ding
James T
JT
Stull
Kristine E
KE
Kamm
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007537:Isometric Contraction / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009124:Muscle Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D001743:Urinary Bladder / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
D000577:Amides; D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D009281:Naphthalenes; D010750:Phosphoproteins; C491520:Ppp1r14a protein, mouse; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D011189:Potassium Chloride; D002217:Carbachol; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; C510645:Ppp1r12a protein, mouse; C058819:calphostin C; D002118:Calcium