The brain relies almost exclusively on oxidative metabolism to support neural activity and is therefore critically dependent upon adequate blood flow. The hypotheses to be tested in this competing renewal are that astrocytes sense neural activity via spillover of excitatory transmitters and release dilatory metabolites to adjacent capillaries, thereby increasing distribution of blood flow to active neurons, and secondarily, that epoxygenases metabolites of astrocytes are mitogenic and endothelial cells. We have isolated, cloned and sequenced a cytochrome P450 gene of the 2C family of epoxygenases which catalyzes formation of vasodilatory epoxyeicosatrienoic acids (EETs) from arachidonic acid (AA). EETs increase K+ channel activity to hyperpolarize and relax arteriolar smooth muscle, and are released from astrocytes stimulated by glutamate. Inhibition of EETs formation by site directed molecular or pharmacological mechanisms prevents increase in nutritive blood flow measured by laser-Doppler flowmetry.
The specific aims of this grant are three-fold. (1) We will determine the ability of astrocyte-released EETs to inhibit normal autoregulatory mechanisms caused by vasoconstrictors, shunting blood flow by opposing the depolarization of cerebral arterioles at physiological pressures. Nitric oxide (NO) also hyperpolarizes arterial muscle by mechanisms which include increasing outward K+ current, thus may amplify the hyperpolarizing and dilator action of EETs; therefore, the additional contribution of NO to countering autoregulatory mechanisms in cerebral vessels will be examined. (2) To begin to investigate the cellular and ionic mechanisms behind EETs induced modulation of autoregulation, we will examine the capacity of astrocyte- produced EETs to modulate K+ and Ca2+ channel activity and [Ca2+]i in cerebrovascular smooth muscle and endothelial cells. (3) Preliminary data show that EETs also initiate mitogenic activity of capillary endothelial cells and stimulate tube formation in co-culture with astrocytes, providing a mechanism to increase capillary endothelial cells and stimulate tube formation in co-culture with astrocytes, providing a mechanism to increase capillary density in areas of high neural activity. Therefore, the signal transduction cascade responsible for cellular growth by EETs increased from astrocytes will be examined by a combination of cellular, molecular, genetic and in vitro techniques to test the hypothesis set forth above. The physiological significance of these phenomenon cannot be over-stated. While many mechanisms are described involving formation of dilatory paracrine substances in the metabolic control of nutritive cerebral blood flow, we have gathered a wealth of data supporting a role for astrocytes and their ability to metabolize AA to vasoactive epoxides. Our investigations will provide vital new information defining mechanisms by which epoxygenase metabolites of astrocytes inhibit autoregulatory vasoconstriction of cerebral arteries.
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