Acetylcholine-induced phosphorylation of CPI-17 in rat bronchial smooth muscle: the roles of Rho-kinase and protein kinase C

2005 ◽  
Vol 83 (4) ◽  
pp. 375-381 ◽  
Author(s):  
Hiroyasu Sakai ◽  
Tomona Hirano ◽  
Hisao Takeyama ◽  
Yoshihiko Chiba ◽  
Miwa Misawa
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Dependent Manner ◽  
Rock Inhibitor ◽  
Bronchial Smooth Muscle ◽  
Calphostin C ◽  
Kinase C

It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca2+ sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca2+ sensitization were both attenuated by 10–6 mol Y-27632 /L, a ROCK inhibitor, 10–6 mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca2+ sensitization in α-toxin-permeabilized bronchial smooth muscle. In this case, the Ca2+ sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca2+ sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca2+ sensitization of bronchial smooth muscle contraction.Key words: CPI-17, bronchial smooth muscle, acetylcholine, ROCK, protein kinase C.

Inhibition of myosin light chain phosphatase during Ca(2+)-independent vasocontraction

1993 ◽  
Vol 265 (5) ◽  
pp. C1319-C1324 ◽  
Author(s):  
H. Itoh ◽  
A. Shimomura ◽  
S. Okubo ◽  
K. Ichikawa ◽  
M. Ito ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Water Soluble ◽  
Two Dimensional ◽  
Kinase C ◽  
Rat Thoracic Aorta ◽  
Phosphopeptide Mapping

Phorbol 12,13-dibutyrate (PDB) induced a sustained contraction of rat thoracic aorta strip in Ca(2+)-free buffer without significant change in intracellular free Ca2+ concentration. NKH477, a water-soluble forskolin derivative, markedly relaxed the PDB-induced contraction. The PDB-induced contraction was associated with the phosphorylation of 20-kDa myosin light chain (MLC). Two-dimensional phosphopeptide mapping of 20-kDa MLC revealed that approximately 90% of the phosphopeptides was derived from an MLC kinase-catalyzed reaction and approximately 10% was due to phosphorylation by protein kinase C. NKH477 inhibited the PDB-induced phosphorylation of 20-kDa MLC. MLC phosphatase activity of intact aorta strips was inhibited by the treatment with PDB, and the inhibition was recovered by the application of NKH477. These results suggest that the regulation of MLC phosphatase in vascular smooth muscle may play important roles in the PDB-induced contraction and the NKH477-induced relaxation in Ca(2+)-free buffer.

Purified rabbit brain protein kinase C relaxes skinned vascular smooth muscle and phosphorylates myosin light chain

1987 ◽  
Vol 254 (1) ◽  
pp. 136-141 ◽  
Author(s):  
Masaki Inagaki ◽  
Hisayuki Yokokura ◽  
Takeo Itoh ◽  
Yuichi Kanmura ◽  
Hirosi Kuriyama ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Rabbit Brain ◽  
Brain Protein ◽  
Kinase C

scholarly journals The involvement of protein kinase C in myosin phosphorylation and force development in rat tail arterial smooth muscle

2000 ◽  
Vol 352 (2) ◽  
pp. 573-582 ◽  
Author(s):  
Lynn P. WEBER ◽  
Minoru SETO ◽  
Yasuharu SASAKI ◽  
Karl SWÄRD ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Light Chain Phosphatase ◽  
Arterial Smooth Muscle ◽  
Kinase C ◽  
Detergent Treatment ◽  
Rat Tail

Myosin light-chain phosphorylation is the primary mechanism for activating smooth-muscle contraction and occurs principally at Ser-19 of the 20kDa light chains of myosin (LC20). In some circumstances, Thr-18 phosphorylation may also occur. Protein kinase C (PKC) can regulate LC20 phosphorylation indirectly via signalling pathways leading to inhibition of myosin light-chain phosphatase. The goal of this study was to determine the relative importance of myosin light-chain kinase (MLCK) and PKC in basal and stimulated LC20 phosphorylation in rat tail arterial smooth-muscle strips (RTA). Two MLCK inhibitors (ML-9 and wortmannin) and two PKC inhibitors (chelerythrine and calphostin C) that have different mechanisms of action were used. Results showed the following: (i) basal LC20 phosphorylation in intact RTA is mediated by MLCK; (ii) α1-adrenoceptor stimulation increases LC20 phosphorylation via MLCK and PKC; (iii) Ca2+-induced LC20 phosphorylation in Triton X-100-demembranated RTA is catalysed exclusively by MLCK, consistent with the quantitative loss of PKCs α and β following detergent treatment; (iv) very little LC20 diphosphorylation (i.e. Thr-18 phosphorylation) occurs in intact or demembranated RTA at rest or in response to contractile stimuli; and (v) the level of LC20 phosphorylation correlates with contraction in intact and demembranated RTA, although the steady-state tension–LC20 phosphorylation relationship is markedly different between the two preparations such that the basal level of LC20 phosphorylation in intact muscles is sufficient to generate maximal force in demembranated preparations. This may be due, in part, to differences in the phosphatase/kinase activity ratio, resulting from disruption of a signalling pathway leading to myosin light-chain phosphatase inhibition following detergent treatment.

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