Localized increases in neural activity lead to localized increases in cerebral blood flow. Many potential mediators of this functioning coupling have been investigated over the past century, yet the precise mechanism of functional hyperemia remains unclear. We propose that the excitatory neurotransmitter, glutamate, stimulates the production of epoxyeicosatrienoic acids (EETs) from arachidonic acid by the expoxygenase activity of cytochrome P450 2C11 in astrocytes. The cerebral microcirculation is completely surrounded by astrocyte processes, and astrocyte-derived EETs may act as intraparenchymal, paracrine vasodilators. In support of this hypothesis, previous work has shown that a) cultured astrocytes express cytochrome P450 2C11, b) P450 epoxygenase inhibitors and antisense oligonucleotides of 2C11 inhibit glutamate stimulation of EET production in cultured astrocytes and glutamate-evoked cerebral vasodilation in vivo, and c) submicrocellular concentration of EETs increase K+ currents in cerebral vascular smooth muscle of intraparenchymal microvessels. In the present proposal, we will determine if the action of glutamate on EET production in astrocyte culture is mediated by specific glutamate receptor subtypes and if actions of these receptor subtypes generates a transferable substance capable of hyperpolarizing parenchymal smooth muscle. Parallel studies will be done in vivo in rat brain with microdialysis to determine if action of different types of glutamate receptor causes increased EET release and local increases in tissue blood flow, both of which are blocked by different types of epoxygenase inhibitors nd by chronic administration of antisense oligonucleotides. We will also determine if increased expression of cytochrome P450 2C11 augments EETs-dependent vasodilation to glutamate receptor activation. Physiological activation of blood flow in the whisker barrel cortex by vibrissal stimulation has been reported to be partially attenuated by nitric oxide synthase (NOS) inhibitors by some, but not all investigators. We will investigate the role of the epoxygenase pathway in this functional hyperemic response by using pharmacologic probes and molecular strategies to decrease and increase expression of 2C11. Lastly, in mice deficient in the neuronal NOS isoform, the cortical blood flow response to vibrissal stimulation is fully intact and not inhibited by nitroarginine thereby suggesting up regulation of another pathway. We will determine if cytochrome P450 2C11 epoxygenase activity represents this up-regulated pathway. These interdisciplinary studies will utilizing a variety of molecular, biochemical and physiological techniques to delineate the importance of a novel pathway in astrocytes linking neuronal activity to microcirculatory blood flow regulation.
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