Role of 20-HETE in SAH

这个项目从一开始就专注于花生四烯酸(AA)的细胞色素P450(CYP)代谢产物在控制脑血管张力中的作用，以及这些系统与大脑中其他血管活性物质的相互作用。 我们发现AA在脑动脉中被细胞色素P4A酶代谢产生20-羟基二十碳四烯酸(20-HETE)。20-HETE是一种有效的脑动脉收缩剂，通过激活PKC来降低钙激活的K+通道的开放概率。TT还可通过激活Rho来增加收缩装置对钙的敏感性 激活剂。血管平滑肌(VSM)中20-HETE的形成受血管紧张素II、内皮素和5-羟色胺的刺激，而被NO、CO和超氧阴离子自由基抑制。20-HETE形成的抑制剂在体外阻断了脑小动脉对跨壁压力升高的肌源性反应，并在体内阻断了脑血流的自动调节。 20-HETE也在调节脑血管对血管扩张剂(NO和CO)和血管收缩药(AII、内皮素、5-羟色胺)的反应中发挥重要作用。在初步研究中，我们建立了一种新的灵敏的方法，利用LC/MS测定脑脊液中20-HETE的浓度，并产生了20-HETE的合成和作用的特异性抑制剂。 我们发现蛛网膜下腔出血(SAIL)后大鼠脑脊液中20-HETE的水平升高，20-HETE合成的抑制剂可以预防SAH后脑血流量(CBF)的急剧下降，并能逆转迟发性血管痉挛。然而，SAH后触发20-HETE释放的因素，合成20-HETE和 20-HETE参与SAH后急性和/或迟发性血管痉挛的机制尚不清楚。先前的研究表明，蛛网膜下腔出血后会释放血红蛋白。这可能有助于脑血管系统中20-HETE的产生突然增加，因为血红蛋白与NO和CO结合，这两者都在张力上抑制20-HETE的形成。 HETE.刺激20-HETE形成的AA和血管紧张剂(内皮素和5-羟色胺)也在航行后通过凝血释放。因此，这一建议将检验这一假说，即20-HETE的产生和释放的增加有助于SAH后CBF的初始下降，以及反馈上调CYP4A和4F酶的表达以及20-HETE的转录增加有助于迟发性血管痉挛的后期发展。具体目标1将检查凝血释放的一些因子(5-羟色胺、内皮素和 大鼠蛛网膜下腔出血后脑脊液中20-HETE合成和/或释放的细胞类型(VSM、血小板、PMN、淋巴细胞和单核细胞)。具体目标2将探索20-HETE导致SAH后大鼠脑血流量急剧下降的一些机制。具体目标3将检测SAH后大鼠脑实质和脑血管中CYP4A和4F酶的表达以及20-HETE产生的时间进程，并将确定20-HETE的形成或作用的抑制剂是否可以预防或逆转迟发性血管痉挛。总体而言，这些翻译研究是我们先前工作的自然延伸，目的是探索20-HETE在控制脑血管张力中的作用，并可能为减少SAH后迟发性脑血管痉挛的发展提供新的见解。

This program project from its inception has focused on the role of cytochrome P450 (CYP) metabolites of arachidonic acid (AA) in the control of cerebral vascular tone and the interaction of these systems with other vasoactive mediators in the brain. We found that AA is metabolized by CYP4A enzymes in cerebral arteries to produce 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a potent constrictor of cerebral arteries that reduces the open state probability of Ca2+-activated K+ channels through activation of PKC. tt also increases the sensmvlty of the contractile apparatus to Ca by activating Rho kinase. The formation of 20-HETE in vascular smooth muscle (VSM) is stimulated by angiotensin II, endothelin and serotonin and is inhibited by NO, CO and superoxide radicals. Inhibitors of the formation of 20-HETE block the myogenic response of cerebral arterioles to elevations in transmural pressure in vitro and autoregulation of cerebral blood flow in vivo. 20-HETE also plays an important role in modulating the cerebral vascular responses to vasodilators (NO and CO) and vasoconstrictors (AII, endothelin, 5-HT). In preliminary studies, we developed a new and sensitive method to measure the concentration of 20-HETE in CSF using LC/MS and generated specific inhibitors of the synthesis and actions of 20-HETE. We found that the levels of 20-HETE in CSF increases in rats following subarachnoid hemorrhage (SAIl) and that inhibitors of the synthesis of 20-HETE prevent the acute fall in cerebral blood flow (CBF) following SAH and can reverse delayed vasospasm. However, the factors that trigger the release of 20-HETE following SAH, the cells that synthesize 20-HETE and the mechanisms by which 20-HETE contribute to acute and/or delayed vasospasm following SAH are unknown. Previous studies indicated that hemoglobin is released following SAH. This likely contributes to an abrupt increase in the production of 20-HETE in the cerebral vasculature since hemoglobin binds NO and CO which both tonically inhibit the formation of 20- HETE. AA and vasoconstrictors (endothelin and 5-HT) that stimulate the formation of 20-HETE are also released by clotting blood after SAIl. Thus, this proposal will test the hypothesis that an elevation in the production and release of 20- HETE contributes to the initial fall in CBF following SAH and that feedback upregulation of the expression of the CYP4A and 4F enzymes and increased tbrmation of 20-HETE contributes to the later development of delayed vasospasm. Specific Aim 1 will examine some of the factors released by clotting blood (5-HT, endothelin and oxyhemoglobin) and the cell types (VSM, platelets, PMNs, lymphocytes and monocytes) that increase the synthesis and/or release of 20-HETE in CSF following SAH in rats. Specific Aim 2 will explore some of the mechanisms by which 20-HETE contributes to the acute fall in CBF following SAH in rats. Specific Aim 3 will examine the time course of changes in expression of the CYP4A and 4F enzymes and the production of 20-HETE in the brain parenchyma and cerebral vasculature following SAH in rats and will determine whether inhibitors of the formation or actions of 20-HETE can prevent or reverse delayed vasospasm. Overall, these translational studies are a natural extension of our previous work to explore the role of 20- HETE in the control of cerebral vascular tone and may provide new insights to reduce the development of delayed cerebral vasospasm following SAH.