Lu Tong, Ye Dan, Wang Xiaoli, Seubert John M, Graves Joan P, Bradbury J Alyce, Zeldin Darryl C, Lee Hon-Chi
Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
J Physiol. 2006 Sep 1;575(Pt 2):627-44. doi: 10.1113/jphysiol.2006.113985. Epub 2006 Jun 22.
We have reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP) epoxygenase metabolites of arachidonic acid (AA), are potent sarcolemmal ATP-sensitive K+ (KATP) channel activators. However, activation of cardiac and vascular KATP channels by endogenously produced EETs under physiological intracellular conditions has not been demonstrated and direct comparison of the mechanisms whereby EETs activate the KATP channels in cardiac myocytes versus vascular smooth muscle cells has not been made. In this study, we examined the effects of AA on KATP channels in freshly isolated cardiac myocytes from rats, wild-type (WT) and transgenic mice overexpressing CYP2J2 cDNA, and mesenteric arterial smooth muscle cells from rats. We also compared the activation of cardiac and vascular KATP channels by extracellularly and intracellularly applied 11,12-EET. We found that 1 microm AA enhanced KATP channel activities in both cardiac and vascular smooth muscle cells, and the AA effects were inhibited by preincubation with CYP epoxygenase inhibitors. Baseline cardiac KATP current densities in CYP2J2 transgenic mice were 190% higher than those of WT mice, and both were reduced to similar levels by CYP epoxygenase inhibition. Western blot analysis showed that expression of Kir6.2 and SUR2A was similar between WT and CYP2J2 transgenic hearts. 11,12-EET (5 microm) applied intracellularly enhanced the KATP currents by 850% in cardiac myocytes, but had no effect in vascular smooth muscle cells. In contrast, 11,12-EET (5 microm) applied extracellularly increased KATP currents by 520% in mesenteric arterial smooth muscle cells, but by only 209% in cardiac myocytes. Preincubation with 100 microm m-iodobenzylguanidine or 5 microm myristoylated PKI amide did not alter the activation of cardiac KATP channels by 5 microm 11,12-EET, but significantly inhibited activation of vascular KATP channels. Moreover, EET only enhanced the inward component of cardiac KATP currents, but activated both the inward and outward components of vascular KATP currents. Our results indicate that endogenously derived CYP metabolites of AA potently activate cardiac and vascular KATP channels. EETs regulate cardiac electrophysiology and vascular tone by KATP channel activation, albeit through different mechanisms: the cardiac KATP channels are directly activated by EETs, whereas activation of the vascular KATP channels by EETs is protein kinase A dependent.
我们曾报道，环氧二十碳三烯酸（EETs）是花生四烯酸（AA）经细胞色素P450（CYP）环氧酶代谢产生的产物，是强效的肌膜ATP敏感性钾离子（KATP）通道激活剂。然而，在生理细胞内条件下内源性产生的EETs对心脏和血管KATP通道的激活作用尚未得到证实，且尚未对EETs激活心肌细胞与血管平滑肌细胞中KATP通道的机制进行直接比较。在本研究中，我们检测了AA对来自大鼠、野生型（WT）和过表达CYP2J2 cDNA的转基因小鼠的新鲜分离心肌细胞以及大鼠肠系膜动脉平滑肌细胞中KATP通道的影响。我们还比较了细胞外和细胞内应用11,12 - EET对心脏和血管KATP通道的激活作用。我们发现1 μM AA增强了心肌细胞和血管平滑肌细胞中的KATP通道活性，且AA的作用可被与CYP环氧酶抑制剂预孵育所抑制。CYP2J2转基因小鼠的基线心脏KATP电流密度比WT小鼠高190%，且二者在CYP环氧酶抑制后均降至相似水平。蛋白质印迹分析显示，WT和CYP2J2转基因心脏中Kir6.2和SUR2A的表达相似。细胞内应用11,12 - EET（5 μM）可使心肌细胞中的KATP电流增强850%，但对血管平滑肌细胞无作用。相反，细胞外应用11,12 - EET（5 μM）可使肠系膜动脉平滑肌细胞中的KATP电流增加520%，但在心肌细胞中仅增加209%。用100 μM间碘苄胍或5 μM肉豆蔻酰化PKI酰胺预孵育不会改变5 μM 11,12 - EET对心脏KATP通道的激活作用，但会显著抑制血管KATP通道的激活。此外，EET仅增强心脏KATP电流的内向成分，但激活血管KATP电流的内向和外向成分。我们的结果表明，AA内源性衍生的CYP代谢产物可有效激活心脏和血管KATP通道。EETs通过激活KATP通道调节心脏电生理和血管张力，尽管通过不同机制：心脏KATP通道由EETs直接激活，而EETs对血管KATP通道的激活是蛋白激酶A依赖性的。
