Division of Experimental Vascular Research, Institute of Clinical Science in Lund, Lund University, Lund, Sweden.
PLoS One. 2012;7(3):e33008. doi: 10.1371/journal.pone.0033008. Epub 2012 Mar 7.
Cigarette smoking is a strong cardiovascular risk factor and endothelin (ET) receptors are related to coronary artery diseases. The present study established an in vivo secondhand smoke (SHS) exposure model and investigated the hypothesis that cigarette smoke induces ET receptor upregulation in rat coronary arteries and its possible underlying mechanisms.
METHODOLOGY/PRINCIPAL FINDINGS: Rats were exposed to SHS for 200 min daily for 8 weeks. The coronary arteries were isolated and examined. The vasoconstriction was studied by a sensitive myograph. The expression of mRNA and protein for receptors was examined by real-time PCR, Western blot and immunofluorescence. Compared to fresh air exposure, SHS increased contractile responses mediated by endothelin type A (ET(A)) and type B (ET(B)) receptors in coronary arteries. In parallel, the expression of mRNA and protein for ET(A) and ET(B) receptors of smoke exposed rats were higher than that of animals exposed to fresh air, suggesting that SHS upregulates ET(A) and ET(B) receptors in coronary arteries in vivo. Immunofluorescence staining showed that the enhanced receptor expression was localized to the smooth muscle cells of coronary arteries. The protein levels of phosphorylated (p)-Raf-1 and p-ERK1/2 in smoke exposed rats were significantly higher than in control rats, demonstrating that SHS induces the activation of the Raf/ERK/MAPK pathway. Treatment with Raf-1 inhibitor GW5074 suppressed SHS-induced enhanced contraction mediated by ET(A) receptors, and inhibited the elevated mRNA and protein levels of ET(A) and ET(B) receptors caused by SHS. The results of correlation and regression analysis showed that phosphorylation of Raf and ERK1/2 were independent determinants to affect protein expression of ET(B) and ET(A) receptors.
CONCLUSIONS/SIGNIFICANCE: Cigarette smoke upregulates ET(B) and ET(A) receptors in rat coronary artery, which is associated with the activation of the Raf/ERK/MAPK pathway.
吸烟是心血管疾病的一个重要危险因素，内皮素（ET）受体与冠状动脉疾病有关。本研究建立了一种体内二手烟（SHS）暴露模型，并提出了假设，即香烟烟雾会导致大鼠冠状动脉中 ET 受体上调及其可能的潜在机制。
方法/主要发现：大鼠每天暴露于 SHS 200 分钟，持续 8 周。分离并检查冠状动脉。通过灵敏的肌描记器研究血管收缩。通过实时 PCR、Western blot 和免疫荧光法检查受体的 mRNA 和蛋白表达。与新鲜空气暴露相比，SHS 增加了内皮素 A（ET(A)）和 B（ET(B)）受体介导的冠状动脉收缩反应。同时，暴露于烟雾的大鼠 ET(A)和 ET(B)受体的 mRNA 和蛋白表达高于暴露于新鲜空气的动物，表明 SHS 在体内上调了冠状动脉中的 ET(A)和 ET(B)受体。免疫荧光染色显示，增强的受体表达定位于冠状动脉的平滑肌细胞。暴露于烟雾的大鼠中磷酸化（p）-Raf-1 和 p-ERK1/2 的蛋白水平明显高于对照组大鼠，表明 SHS 诱导 Raf/ERK/MAPK 通路的激活。用 Raf-1 抑制剂 GW5074 处理可抑制 SHS 诱导的 ET(A)受体介导的增强收缩，并抑制 SHS 引起的 ET(A)和 ET(B)受体的 mRNA 和蛋白水平升高。相关性和回归分析的结果表明，Raf 和 ERK1/2 的磷酸化是影响 ET(B)和 ET(A)受体蛋白表达的独立决定因素。
结论/意义：香烟烟雾会上调大鼠冠状动脉中的 ET(B)和 ET(A)受体，这与 Raf/ERK/MAPK 通路的激活有关。
