application) The underlying mechanisms promoting increases in cerebral blood flow during hypoxia, hypoglycemia and hypercapnia are not fully understood. One endogenous vasodilating factor that appears to be involved is nitric oxide. The proposed experiments are designed to identify the mechanisms of nitric oxide related cerebrovasodilation and the site (endothelium or perivascular) where that nitric oxide is synthesized. For these studies, in anesthetized rats, a closed cranial window intravital microscopy system will be used. This permits the measurement of diameter changes in pial arterioles and the administration of agents to the brain surface. To examine the role of nitric oxide in the vasodilatory responses to hypoxia or hypoglycemia, of increasing severity, the nitric oxide synthase inhibitor (L-NNA) will be suffused. In controls, L-NNA will be replaced by its inactive enantiomer, D-NNA. Furthermore, in hypoxia and hypoglycemia, the role of nitric oxide will be evaluated in relation to other known nitric oxide independent vasodilating stimuli, adenosine release or beta adrenoceptor activation. To corroborate the cGMP role at the arteriolar level, putative guanylate cyclase or cGMP-dependent protein kinase inhibitors will be suffused. The mechanisms through which nitric oxide promotes cerebrovasodilation in hypoxia, hypoglycemia and hypercapnia will be divided into 2 general classes: permissive and active elements. For permissive, L-NNA will be co-suffused with a nitric oxide donor (SNAP) or a cGMP analog or a cAMP phosphodiesterase inhibitor. To test whether the permissive action of NO/cGMP relates to K-channels, additional experiments will include NOS inhibitor and cGMP analog co-suffusions with selective K+ channel blockers. Potential mechanisms leading to increased NO generation (active factors) will be examined. These include studies concerning the role of excitatory amino acid release. The nitric oxide production sites to be evaluated are endothelium (form hypoxia and hypoglycemia) and perivascular structures. For endothelium, the light/dye method will be used. For perivascular studies, vasodilatory responses will be assessed in the presence of an inhibitor of neuronal activity, tetrodotoxin or a novel inhibitor of the neuronal nitric oxide synthase (ARL 17477AR). These experiments should increase our understanding of the mechanisms involved in the vasodilatory responses of perturbations of clinic interest like hypoxia, hypoglycemia and hypercapnia and enhance our knowledge concerning the role of nitric oxide in cerebral vasodilatory function.
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