Stimulatory effects of arachidonic acid on myosin ATPase activity and contraction of smooth muscle via myosin motor domain.
We have been searching for a mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin (Nakamura A, Xie C, Zhang Y, Gao Y, Wang HH, Ye LH, Kishi H, Okagaki T, Yoshiyama S, Hayakawa K, Ishikawa R, Kohama K. Biochem Biophys Res Commun 369: 135-143, 2008). In this article, we report that arachidonic acid (AA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (R(max)) of 6.84 +/- 0.51 relative to stimulation by the vehicle and with a half-maximal effective concentration (EC(50)) of 50.3 +/- 4.2 microM. In the presence of actin, R(max) was 1.72 +/- 0.08 and EC(50) was 26.3 +/- 2.3 microM. Our experiments with eicosanoids consisting of the AA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated, AA stimulated contraction of smooth muscle tissue with an R(max) of 1.45 +/- 0.07 and an EC(50) of 27.0 +/- 4.4 microM. In addition to the ATPase activities of the myosin, AA stimulated those of heavy meromyosin, subfragment 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AA on these preparations were about twofold. The site of AA action was indicated to be the step-releasing inorganic phosphate (P(i)) from the reaction intermediate of the myosin-ADP-P(i) complex. The enhancement of P(i) release by AA was supported by computer simulation indicating that AA docked in the actin-binding cleft of the myosin motor domain. The stimulatory effect of AA was detectable with both unphosphorylated myosin and the myosin in which RLC was fully phosphorylated. The AA effect on both myosin forms was suggested to cause excess contraction such as vasospasm.
Katayama
Takeshi
T
Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, Japan.
Watanabe
Masaru
M
Tanaka
Hideyuki
H
Hino
Mizuki
M
Miyakawa
Takuya
T
Ohki
Takashi
T
Ye
Li-Hong
LH
Xie
Ce
C
Yoshiyama
Shinji
S
Nakamura
Akio
A
Ishikawa
Ryoki
R
Tanokura
Masaru
M
Oiwa
Kazuhiro
K
Kohama
Kazuhiro
K
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D003198:Computer Simulation; D006168:Guinea Pigs; D008297:Male; D023421:Models, Animal; D009119:Muscle Contraction / Q000187:drug effects* / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D009218:Myosins / Q000187:drug effects / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D024745:Smooth Muscle Myosins / Q000378:metabolism* / Q000648:ultrastructure
D010710:Phosphates; D016718:Arachidonic Acid; D024745:Smooth Muscle Myosins; D009218:Myosins