Comparison of contractile mechanisms of sphingosylphosphorylcholine and sphingosine-1-phosphate in rabbit coronary artery

Abstract

[한글]

Sphingomyelin의 대사물질인 sphingosylphosphorylcholine (SPC)과 sphingosine-1-phosphat (S1P)는 여러 혈관, 특히 관상동맥과 기저동맥에서 혈관 수축을 유발한다고 알려져 있다. SPC와 S1P가 유사한 혈관 반응을 유발한다고 알려져 왔지만 그 수축 양상과 그에 관련된 기전은 종종 다르게 나타난다. 따라서 본 연구에서는 SPC와 S1P에 의한 수축 양상의 차이와 그에 관련된 기전에 대하여 알아보고자 하였다. 정상 혈관과 막 투과도가 증가된 혈관에서 수축반응을 기록하였으며 세포 내 Ca2+ 농도의 변화와 MYPT1의 인산화를 측정하였다.SPC 는 점진적으로 증가하여 유지되는 수축반응을 유발하였고, S1P는 초기에 빠르게 증가하였다가 감소하여 유지되는 수축반응을 유발하였다. SPC에 의한 수축은 외부 Ca2+이 존재할 때나 외부 Ca2+이 제거된 상태에서 비슷하게 나타났으나, S1P에 의한 수축은 외부 Ca2+이 존재할 때 보다 외부 Ca2+이 제거된 상태에서 더 작게 나타났다. SERCA의 억제제인 cyclopiazonic acid (CPA)는 SPC에 의한 수축반응을 변화시키지 않았으나, S1P에 의해 유발된 전반부의 일시적인 수축반응은 유의하게 감소시켰다. 분리된 단일세포에서 SPC는 세포 내 Ca2+ 농도를 증가시켰으나 그 효과는 미미하였고, S1P는 세포 내 Ca2+ 농도를 유의하게 증가시켰다. 외부 Ca2+이 없을 때 S1P에 의한 세포 내 Ca2+ 농도의 증가는 유의하게 억제되었으나, 전반부의 일시적인 세포 내 Ca2+ 농도의 증가는 여전히 남아 있었으며 이것은 CPA 처리에 의해서 현저히 소실되었다.

[영문]It has been suggested that sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) induce contraction in various vascular tissues, especially coronary and basilar arteries. Although SPC or S1P generally leads to similar vascular responses, the contractile patterns and their underlying signaling mechanisms are often distinct. Therefore, in this study, we investigated the difference of patterns between SPC- and S1P-induced contraction and their underlying signaling mechanisms. Contractile responses in intact and permeabilized strips, changes in intracellular Ca2+ concentration ([Ca2+]i) and phosphorylation of myosin light chain phosphatase targeting subunit (MYPT1) were measured.SPC induced a gradual and 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+, SPC-induced contraction was similar to that in the presence of extracellular Ca2+ but S1P-induced contraction was smaller than that in the presence of extracellular Ca2+. Cyclopiazonic acid (CPA), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, did not change in SPC-induced contraction but significantly decreased the initial transient force induced by S1P. In isolated single cells, SPC slightly increased [Ca2+]i but S1P significantly increased [Ca2+]i. In the absence of extracellular Ca2+, S1P-induced increase in [Ca2+]i was significantly inhibited, which was significantly inhibited by pretreatment of CPA. At constant Ca2+ levels using -escin permeabilized strips, SPC induced augmentation of pCa6.3-induced force but this force was significantly inhibited by fasudil hydrochloride, ROCK inhibitor. S1P induced little or no augmentation of force induced by pCa6.3. 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 several folds increase in Thr696 and Thr853 phosphorylation of MYPT1 but S1P did not affect phosphorylation of MYPT1.Our results suggest that SPC-induced contraction shows gradual and sustained patterns but S1P-induced contraction shows initial transient and secondary sustained patterns. Furthermore, activation of RhoA/RhoA-associated kinase pathway and phosphorylation of MYPT1 may play key mechanisms for SPC-induced contraction but elevation of [Ca2+]i may play a key mechanism for S1P-induced contraction.