: This research program is an extension of a project regarding the role of NO in the control of cerebral vasodilation at the arteriolar level. The studies in this competing renewal application will address control mechanisms occurring chiefly in cerebral vascular smooth muscle (VSM) at the level of cyclic GMP and cyclic AMP. A basic premise of the project is that the cyclic nucleotide pathways do not function as separate entities, but rather interact through a process generically labeled as """"""""crosstalk."""""""" The central hypothesis of this proposal is that, through crosstalk regulation in VSM, each cyclic nucleotide is capable of regulating the vasodilating actions of its counterpart via interactions with phosphodiesterases (PDEs). The project will address two basic hypotheses. First, crosstalk control by one cyclic nucleotide, at the PDE level, occurs by reducing the hydrolysis of its counterpart. Second, the reduction in hydrolytic function may occur by a direct action of the cyclic nucleotide or its kinase on the PDE itself or via promoting a kinase-related subcellular sequestration of the PDE away from the bulk pool of cyclic nucleotides residing in the cytosol.
Three specific aims will be addressed, based upon the 3 principal PDEs found in cerebral VSM-the cAMP-""""""""preferring"""""""" PDE3, the cAMP-specific PDE4, and the cGMP-specific PDE5. Primary increases in endogenous cAMP and cGMP production will be elicited by the adenylyl and guanylyl cyclase activators, forskolin and YC-1, respectively. Two experimental models will used in these studies: the established closed cranial window, intravital microscopy system designed for monitoring pial arteriolar diameter changes in anesthetized rats and primary cultures of rat pial vascular smooth muscle. Both models will be used to test the first hypothesis. Those studies will involve the use of carefully chosen selective enzyme inhibitors and activators. In most cases, those selectivities have been validated by us. In addition, confirmation of the ability of one cyclic nucleotide to influence the content of the other, in the presence of these pharmacologic manipulations, will be obtained using the culture system. Sequestration issues will be addressed using pial VSM cells and high-resolution immunofluoresence detection techniques. Those studies will generally involve assessments of the relative intra-nuclear vs cytosolic expression of the 3 PDEs, in the absence and the presence of increases in the levels of the counterpart cyclic nucleotide. The roles of the cyclic nucleotide kinases will also be addressed in this regard. These studies will provide important new information regarding the mechanisms controlling a physiologic process vital to normal brain function and survival-cerebral arteriolar dilation.
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