Choi Soo-Kyoung, Ahn Duck-Sun, Lee Young-Ho
Department of Physiology, College of Medicine, BK 21 Project for Medical Sciences, Yonsei University, CPO Box 8044, Seoul 120-752, Republic of Korea.
Cardiovasc Res. 2009 May 1;82(2):324-32. doi: 10.1093/cvr/cvp054. Epub 2009 Feb 13.
Although stimulation with sphingosylphosphorylcholine (SPC) or sphingosine-1-phosphate (S1P) generally leads to similar vascular responses, the contractile patterns and their underlying signalling mechanisms are often distinct. We investigated the different reliance upon Ca2+-dependent and Ca2+-sensitizing mechanisms of constriction in response to SPC or S1P in coronary arteries.
Contractile responses, changes in [Ca2+]i, and phosphorylation of myosin light chain phosphatase-targeting subunit (MYPT1) were measured. SPC induced a concentration-dependent sustained contraction. S1P evoked a rapid rise in force (initial transient), which was followed by a secondary sustained force. In the absence of extracellular Ca2+, the concentration dependency of constriction to SPC was shifted to the right, but with no change in maximum force, whereas S1P-induced contraction was significantly blunted. Cyclopiazonic acid (CPA) significantly decreased the initial transient force induced by S1P. In isolated single cells, S1P markedly increased [Ca2+]i, whereas only a modest elevation was noted with SPC. The S1P-induced elevation of [Ca2+]i was abolished by pre-treatment with CPA and was significantly reduced in the absence of extracellular Ca2+. In beta-escin-permeabilized strips, SPC augmented pCa 6.3-induced force; this was significantly inhibited by fasudil hydrochloride. S1P induced little or no augmentation of pCa 6.3-induced force. In intact arteries, SPC-induced contraction was completely inhibited by fasudil hydrochloride. Fasudil hydrochloride had no effect on the initial transient force induced by S1P but significantly inhibited the secondary sustained force. SPC induced a several-fold increase in Thr696 and Thr853 phosphorylation of MYPT1, but S1P did not affect phosphorylation of MYPT1.
Our results suggest that constriction of coronary arteries in response to the bioactive lipid S1P or SPC occurs by distinct signalling pathways. Activation of the RhoA/RhoA-associated kinase pathway and subsequent phosphorylation of MYPT1 play a key role in SPC-induced coronary contraction, whereas elevation of [Ca2+]i is crucial for S1P-induced coronary constriction.
尽管用鞘氨醇磷酸胆碱（SPC）或1-磷酸鞘氨醇（S1P）刺激通常会导致相似的血管反应，但收缩模式及其潜在的信号传导机制往往不同。我们研究了冠状动脉对SPC或S1P收缩反应中对钙依赖性和钙敏化机制的不同依赖情况。
测量收缩反应、细胞内钙离子浓度（[Ca2+]i）的变化以及肌球蛋白轻链磷酸酶靶向亚基（MYPT1）的磷酸化情况。SPC诱导浓度依赖性的持续收缩。S1P引起快速的力上升（初始瞬态），随后是二次持续力。在无细胞外钙离子的情况下，SPC收缩的浓度依赖性向右移动，但最大力无变化，而S1P诱导的收缩明显减弱。环匹阿尼酸（CPA）显著降低S1P诱导的初始瞬态力。在分离的单细胞中，S1P显著增加[Ca2+]i，而SPC仅引起适度升高。S1P诱导的[Ca2+]i升高在CPA预处理后被消除，且在无细胞外钙离子时显著降低。在β-七叶皂苷渗透的条带中，SPC增强了pCa 6.3诱导的力；这被盐酸法舒地尔显著抑制。S1P对pCa 6.3诱导的力几乎没有增强作用。在完整动脉中，盐酸法舒地尔完全抑制SPC诱导的收缩。盐酸法舒地尔对S1P诱导的初始瞬态力无影响，但显著抑制二次持续力。SPC诱导MYPT1的Thr696和Thr853磷酸化增加数倍，但S1P不影响MYPT1的磷酸化。
我们的结果表明，冠状动脉对生物活性脂质S1P或SPC的收缩是通过不同的信号通路发生的。RhoA/RhoA相关激酶途径的激活以及随后MYPT1的磷酸化在SPC诱导的冠状动脉收缩中起关键作用，而[Ca2+]i的升高对S1P诱导的冠状动脉收缩至关重要。
