Endothelial Lipoxygenase Metabolites and Vascular Tone

描述(申请人提供)：内皮细胞释放一氧化氮、前列腺素I2和内皮衍生超极化因子(EDHF)，降低血管张力，对抗血管收缩。缺血性心脏病、心力衰竭和高血压与血管收缩有关，这部分归因于内皮依赖性血管收缩的减少。恢复内皮功能的治疗方法代表着对心血管疾病有希望的新疗法，对健康有重大影响。我们鉴定了花生四烯酸的15-脂氧合酶(LO)代谢产物11(R)，12(S)，15(S)-三羟基二十碳三烯酸(11，12，15-Theta)，它是EDHF。它部分地通过激活阿帕明敏感、小电导、钙激活钾(SKCa)通道和超极化的平滑肌细胞(SMCs)来松弛动脉。它还通过抑制RhoA的激活发挥作用。11，12，15-Theta介导部分内皮依赖性舒张乙酰胆碱(ACh)，增加Flow和AA，并拮抗血管紧张剂的作用。我们将验证这一假设，即11，12，15-Theta通过一种独特的机制组合介导ACh和AA的松弛：(1)激活异源三聚体鸟嘌呤核苷酸结合蛋白(G蛋白)，开放SKCa通道，导致SMC超极化；(2)抑制小GTP酶RhoA的激活，降低对照蛋白的钙敏感性。这些研究表明，11，12，15-Theta是一种EDHF和内皮来源的钙(Ca)脱敏剂。11(R)，12(S)，15(S)-Theta增加GTP335S与兔动脉膜的结合，涉及G蛋白偶联结合部位/受体。利用生化和分子方法，我们将鉴定与11，12，15-Theta作用相偶联的G蛋白(S)。我们将利用膜片钳和生物测定的方法，研究G蛋白在11，12，15-Theta对SKCa通道活动、超极化和扩张的调节中的作用。这些研究将为11，12，15-Theta的分子作用机制和血管SKCa通道活性的调节提供新的见解。去甲肾上腺素和血管紧张素等激动剂通过激活使收缩蛋白对钙敏感的RhoA-Rho激酶级联反应来促进收缩。11，12，15-Theta减少RhoA活性形式RhoA-GTP的形成。这些研究将验证这一假说，即11，12，15-Theta通过抑制RhoA激活，导致Rho激酶活性降低，增加肌球蛋白轻链磷酸酶活性，减少磷酸化肌球蛋白轻链，从而导致松弛。这些研究将了解11，12，15-Theta对SMC和动脉中RhoA活性的影响，并确定RhoA抑制对11，12，15-Theta松弛的作用。G蛋白在11，12，15-Theta抑制RhoA中的作用将被确定。这些研究将确定内皮衍生松弛因子11，12，15-Theta的两种新的作用机制，并提供增强内皮功能的新治疗方法。

DESCRIPTION (provided by applicant): Endothelial cells release nitric oxide, prostaglandin I2 and endothelium-derived hyperpolarizing factor (EDHF) that reduce vascular tone and counteract vasoconstriction. Ischemic heart disease, heart failure and hyperten- sion are associated with vasoconstriction that is attributed, in part, to reductions in endothelium-dependent di- lation. Therapeutic approaches restoring endothelial function represent promising new treatments for cardio- vascular diseases with a major impact on health. We have identified a 15-lipoxygenase (LO) metabolite of ara- chidonic acid, 11(R),12(S),15(S)-trihydroxy-eicosatrienoic acid (11,12,15-THETA), that acts as an EDHF. It relax arteries, in part, by activating apamin-sensitive, small conductance, calcium-activated potassium (SKCa) channels and hyperpolarizing smooth muscle cells (SMCs). It also acts by inhibition of RhoA activation. 11,12,15-THETA mediates a portion of the endothelium-dependent relaxations to acetylcholine (ACh), in- creases in flow and AA and opposes the action of vasoconstrictors. We will test the hypothesis that 11,12,15- THETA mediates relaxations to ACh and AA by a unique combination of mechanisms: (1) activating a hetero- trimeric guanine nucleotide binding protein (G protein) that opens SKCa channels resulting in hyperpolarization of SMCs and (2) inhibiting the activation of the small GTPase RhoA reducing the calcium sensitivity of contrac- tile proteins. These studies will indicate that 11,12,15-THETA is an EDHF and an endothelium-derived calcium (Ca) desensitizer. 11(R),12(S),15(S)-THETA increases GTP335S binding to rabbit arterial membranes implicat- ing a G protein-coupled binding site/receptor. Using biochemical and molecular methods, we will identify the G protein(s) that is coupled to 11,12,15-THETA action. Using patch clamp and bioassay methods, we will char- acterize the role of the G protein in the regulation of SKCa channel activity, hyperpolarization and dilation by 11,12,15-THETA. These studies will provide new insights into the molecular mechanism of action of 11,12,15- THETA and the regulation of vascular SKCa channel activity. Agonists such as norepinephrine and angiotensin promote constriction by activating the RhoA-Rho kinase cascade that sensitizes contractile proteins to Ca. 11,12,15-THETA decreases the formation of the active form of RhoA, RhoA-GTP. These studies will test the hypothesis that 11,12,15-THETA causes relaxation by inhibiting RhoA activation resulting in decreased Rho kinase activity increasing myosin light chain phosphatase activity and decreasing phosphorylated myosin light chain. These studies will access the effect of 11,12,15-THETA on RhoA activity in SMCs and arteries and de- termine the contribution of RhoA inhibition to the relaxations to 11,12,15-THETA. The role of a G protein in RhoA inhibition by 11,12,15-THETA will be determined. These studies will identify two new mechanisms of action for the endothelium-derived relaxing factor 11,12,15-THETA and provide a new therapeutic approach to enhancing endothelial function.