Ca(2+)-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment

1995 ◽  
Vol 268 (1) ◽  
pp. C21-C29 ◽  
Author(s):  
S. Morimoto ◽  
Y. Ogawa

After an acidic treatment in the presence of ADP, Triton X-100-skinned rabbit aortic smooth muscle strips were found to develop a large sustained, Ca(2+)-insensitive tension when returned to a relaxing solution with neutral pH. The presence of ADP during treatment was essential for the manifestation of the Ca(2+)-insensitive contraction. This contraction was reversibly eliminated by withdrawal of MgATP or addition of vanadate and was found to be accompanied by an extraordinarily high level of 20-kDa myosin light-chain (MLC20) phosphorylation. The rate constant for dephosphorylation of MLC20 in treated strips was about one-twenty-fifth that in untreated control, when determined after removal of Ca2+, Mg2+, and ATP. Two-dimensional phosphopeptide mapping of tryptic digests of MLC20 showed that most incorporated phosphate was in the peptides which would be phosphorylated by myosin light-chain kinase. These results provide strong evidence that ADP inactivates myosin light-chain phosphatase under acidic conditions.

1994 ◽  
Vol 72 (11) ◽  
pp. 1386-1391 ◽  
Author(s):  
Yawen Zhang ◽  
Suzanne Moreland ◽  
Robert S. Moreland

Ca2+-dependent myosin light chain (MLC) phosphorylation is an important step in the initiation of smooth muscle contraction. However, MLC phosphorylation alone cannot account for all aspects of contractile regulation, suggesting the involvement of other elements. In this article we present evidence obtained from Triton X-100 detergent skinned and intact tissue which demonstrates that vascular smooth muscle contraction can be initiated by a Ca2+-dependent mechanism that does not require prior MLC phosphorylation. We show that Ca2+ can initiate contractions supported by cytidine triphosphate (CTP) and that these contractions are inhibited by calmodulin antagonists, suggesting a Ca2+–calmodulin dependence of force distinct from that for MLC phosphorylation. Evidence is presented to demonstrate that carotid medial fibers contain a mitogen-activated protein (MAP) kinase which is activated by Ca2+ and may catalyze caldesmon phosphorylation. Based in part on our results and those of other investigators, we propose that direct Ca2+–calmodulin binding to caldesmon or phosphorylation of caldesmon by a Ca2+-dependent MAP kinase disinhibits caldesmon. Disinhibition of caldesmon allows an inherent basal level of actin-activated myosin ATPase activity to be expressed. The result is the slow development of force.Key words: mitogen-activated protein kinase, caldesmon, Triton X-100, detergent-skinned fibers, cytidine triphosphate, calmodulin.


2002 ◽  
Vol 367 (2) ◽  
pp. 517-524 ◽  
Author(s):  
Jing Ti DENG ◽  
Cindy SUTHERLAND ◽  
David L. BRAUTIGAN ◽  
Masumi ETO ◽  
Michael P. WALSH

Integrin-linked kinase (ILK) has been implicated in Ca2+- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr→Ala mutation at Thr38 of CPI-17 and Thr57 of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr38 of CPI-17 or Thr57 of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr38 or Thr57 respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr38Ala and PHI-1-Thr57Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca2+ sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr38Ala and PHI-1-Thr57Ala treated with ILK in the presence of adenosine 5′-[γ-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.


2003 ◽  
Vol 284 (4) ◽  
pp. H1182-H1189 ◽  
Author(s):  
Hwee Teoh ◽  
Mary Zacour ◽  
Avraham D. Wener ◽  
Lakshman Gunaratnam ◽  
Michael E. Ward

We hypothesized that increased myofibrillar type 1 protein phosphatase (PP1) catalytic activity contributes to impaired aortic smooth muscle contraction after hypoxia. Our results show that inhibition of PP1 activity with microcystin-LR (50 nmol/l) or okadaic acid (100 nmol/l) increased phenylephrine- and KCl-induced contraction to a greater extent in aortic rings from rats exposed to hypoxia (10% O2) for 48 h than in rings from normoxic animals. PP1 inhibition also restored the level of phosphorylation of the 20-kDa myosin light chain (LC20) during maximal phenylephrine-induced contraction to that observed in the normoxic control group. Myofibrillar PP1 activity was greater in aortas from rats exposed to hypoxia than in normoxic rats ( P < 0.05). Levels of the protein myosin phosphatase-targeting subunit 1 (MYPT1) that mediates myofibrillar localization of PP1 activity were increased in aortas from hypoxic rats (193 ± 28% of the normoxic control value, P < 0.05) and in human aortic smooth muscle cells after hypoxic (1% O2) incubation (182 ± 18% of the normoxic control value, P < 0.05). Aortic levels of myosin light chain kinase were similar in normoxic and hypoxic groups. In conclusion, after hypoxia, increased MYPT1 protein and myofibrillar PP1 activity impair aortic vasoreactivity through enhanced dephosphorylation of LC20.


1993 ◽  
Vol 265 (5) ◽  
pp. C1319-C1324 ◽  
Author(s):  
H. Itoh ◽  
A. Shimomura ◽  
S. Okubo ◽  
K. Ichikawa ◽  
M. Ito ◽  
...  

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.


1984 ◽  
Vol 401 (1) ◽  
pp. 107-109 ◽  
Author(s):  
M. Gagelmann ◽  
U. Mrwa ◽  
S. Bostrom ◽  
J. C. R�egg ◽  
D. Hartshorne

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