This Project will examine interactions between nitric oxide (NO) and 20- HETE in the regulation of cerebral blood flow, since recent work has indicated that both systems play central but opposing roles in the control of cerebral vascular tone. Specifically, we will examine the hypothesis that NO inhibits the formation of the potent vasoconstrictor 20-HETE in vascular smooth muscle cells and this inhibitory action contributes to the ability of NO to activate K+ channels and dilate the cerebral arterioles. This hypothesis is based on our preliminary data indicating that: 1) NO directly inhibits the synthesis of 20-HETE; 2) the vasodilator response to NO in small cerebral arterioles of the rat appears to be largely cGMP independent; and 3) that preventing the fall in 20-HETE levels in cerebral arterioles attenuates the activation of K+ channels and vasodilatory response to NO in small cerebral arterioles attenuates the activation of K+ channels and vasodilatory response to NO. In the proposed studies, the effects of various concentrations of NO to stimulate guanylyl cyclase and inhibit the production of 20-HETE will be directly studied in cerebral arterioles and vascular smooth muscle cells isolated from these vessels. We will identify the P4504A isoforms expressed in the cerebral vasculature of rats using RT-PCR with isoform specific primers and visible light spectroscopy will be utilized to study the binding of NO to the corresponding recombinant P4504A proteins expressed using a baculovirus system and Sf9 cells. The effects of NO on K+ channel activity in cerebral vascular smooth muscle cells will be characterized using patch-clamp techniques, and the relative contribution of changes in 20-HETE versus cGMP levels to these responses will be determined by altering intracellular levels of 20-HETE in the presence or absence of inhibitors of guanylyl cyclase and cGMP-dependent protein kinase. Parallel studies will be performed in isolated, perfused cerebral arterioles to determine the contribution of changes in the production of 20-HETE versus cGMP on the effects of NO to alter membrane potential and vascular tone. The significance of 20-HETE in mediating NO-induced changes in cerebral blood flow will be evaluated using laser Doppler flowmetry in anesthetized rats treated with NO-donors, synthase inhibitors, and stimuli that promote the synthesis of NO before and after blockade of the P4504A pathway with a variety of inhibitors and receptor antagonists that we have recently developed. Given the emerging importance of 20-HETE and NO to the control of cerebral blood flow, there are compelling reasons to obtain a better understanding of the cellular and ionic mechanisms by which these systems interact to regulate cerebral vascular tone.
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