The role of myosin light chain kinase-dependent phosphorylation of myosin light chain in phorbol ester-induced contraction of rabbit aorta.
We investigated the role of 20 kDa myosin light chain (MLC20) phosphorylation in contractions following protein kinase C (PKC) activation by 12-deoxyphorbol-13-isobutyrate (DPB) in rabbit aortae. DPB induced a sustained contraction and phosphorylation of MLC20 independent of a change in cytosolic Ca2+ ([Ca2+]i). Phosphorylation on Ser19 of MLC20, which is a target site of MLC kinase (MLCK), was 9.2 +/- 5.1% and 22.3 +/- 4.9% of the phosphorylation caused by KCl, at 5 and 30 min of application of DPB, respectively. When KCl-precontracted muscles were rinsed with Ca2+-free, EGTA solution, [Ca2+]i rapidly declined, MLC20 was dephosphorylated and the tension decreased. If DPB was present in the Ca2+-free solution, the relaxation and the dephosphorylation of either total MLC20 or Ser19 were inhibited. The phospholipase A2 inhibitor ONO-RS-082 partially antagonized the effects of DPB on the tension and the MLC20 dephosphorylation. In Ca2+-free solution, DPB induced a contraction smaller than that in normal solution without an increase in MLC20 phosphorylation, and the contraction was also sensitive to ONO-RS-082. These results suggest that a part of MLC20 phosphorylation following PKC activation is due to inhibition of MLC20 phosphatase and the phosphorylation is responsible for the contraction. Furthermore, a mechanism independent of [Ca2+]i and phosphorylation may play a significant role in the PKC-dependent contraction. The involvement arachidonic acid is suggested, not only in the inhibition of dephosphorylation but also in the Ca2+-independent regulation of contractile proteins.
Miura
M
M
Department of Veterinary Pharmacology, Faculty of Agriculture, Miyazaki University, Miyazaki 889-21 Japan.
Iwanaga
T
T
Ito
K M
KM
Seto
M
M
Sasaki
Y
Y
Ito
K
K
D000818:Animals; D001011:Aorta / Q000187:drug effects* / Q000378:metabolism*; D016718:Arachidonic Acid / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000502:physiology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011817:Rabbits; D014661:Vasoconstriction / Q000502:physiology*
D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D016718:Arachidonic Acid; D015240:Phorbol 12,13-Dibutyrate; D009219:Myosin-Light-Chain Kinase