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.
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