Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells.
Phosphorylation of myosin light chain (MLC) and contraction of differentiated smooth muscle cells in vascular walls are regulated by Ca2+ -dependent activation of MLC kinase, and by Rho-kinase- or protein-kinases-C-dependent inhibition of MLC phosphatase (MLCP). We examined regulatory pathways for MLC kinase and MLCP in cultured vascular smooth muscle cells (VSMCs), and for isometric force generation of VSMCs reconstituted in collagen fibers. Protein levels of RhoA, Rho-kinase and MYPT1 (a regulatory subunit of MLCP) were upregulated in cultured VSMCs, whereas a MLCP inhibitor protein, CPI-17, was downregulated. Endothelin-1 evoked a steady rise in levels of Ca2+, MLC phosphorylation and the contractile force of VSMCs, whereas angiotensin-II induced transient signals. Also, Thr853 phosphorylation of MYPT1 occurred in response to stimuli, but neither agonist induced phosphorylation of MYPT1 at Thr696. Unlike fresh aortic tissues, removal of Ca2+ or addition of voltage-dependent Ca2+ -channel blocker did not inhibit contractions of reconstituted VSMC fibers induced by agonists or even high concentrations of extracellular K+ ions. Inhibitors of Ins(1,4,5)P3-receptor and Rho-kinase antagonized agonist-induced or high-K+ -induced contraction in both reconstituted fibers and fresh tissues. These results indicate that both Ins(1,4,5)P3-induced Ca2+ release and Rho-kinase-induced MYPT1 phosphorylation at Thr853 play pivotal roles in MLC phosphorylation of cultured VSMCs where either Ca2+ -influx or CPI-17-MLCP signaling is downregulated.
Woodsome
Terence P
TP
Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472, USA.
Polzin
Atsuko
A
Kitazawa
Kazuyo
K
Eto
Masumi
M
Kitazawa
Toshio
T
D000804:Angiotensin II / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007537:Isometric Contraction / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010656:Phenylephrine / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D054645:Protein Phosphatase 1; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D014662:Vasoconstrictor Agents / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D002352:Carrier Proteins; D019332:Endothelin-1; D005456:Fluorescent Dyes; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D014662:Vasoconstrictor Agents; D014966:Xanthenes; D000804:Angiotensin II; D010656:Phenylephrine; C059715:Fluo-3; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; D002118:Calcium