Regulation of nutritive blood flow the brain is an extraordinarily dynamic process providing substrate to meet essential oxidative substrate to the CNS, while providing hyperemic flow to metabolically active areas. Fundamental to brain blood flow regulation is the autoregulatory capacity of the cerebral microcirculation. To begin to understand this process requires a basic definition of the cellular and molecular mechanisms through which pre-capillary arterioles sense environmental conditions and initiate appropriate signal transduction events to activate or inhibit contraction of microvascular and smooth muscle. Defining homeostatic mechanisms designed to regulate nutritive cerebral blood flow (CBF) at an integrated functional level to meet physiologic demand is beyond the scope of any one laboratory. Dr. David Harder will Direct this Program, and lead Project 1. Studies in Project 1 will focus on the molecular, cellular and signal transduction mediating autoregulation of nutritive CBF in the face of a fluctuation arterial pressure. We have recently sequenced a cytochrome P450 (P450) omegahydroxylase gene within isolated pre-capillary arteriolar muscle cells which codes for an enzyme catalyzing formation of 20-HETE from arachidonic acid (AA). Inhibition of 20-HETE formation abolishes the normal, nearly perfect, autoregulation of laser-Doppler blood flow to elevation of arterial pressure in the rat parietal cortex. Project 2 will be lead by Dr. Richard Roman, and will define the interactions between nitric oxide (NO) and P450 generated 20-HETE formation. In the regulation of nutritive CBF NO appears to bind directly to the heme moiety of, and inhibit P450 enzyme activity. Dr. roman provides compelling data for the hypothesis that the cGMP independent mechanisms of action of NO is to inhibit P450 4A omega-hydroxylase activity and block endogenous production of 20-HETE one of the most potent vasoactive agents yet identified. Project 3 will be lead by Dr. Raymond Koehler (Johns Hopkins University School of Medicine) which defines the role of astrocytic P450 epoxygenase activity in functional hyperemia in the brain. We have sequenced a P450 2C11 cDNA from astrocytes which does for a protein generating dilatory epoxyeicositrienoic acids (EETs). Glutamate induces release of EETs and regulates 2C11 gene expression. Dr. Koehler and his collaborators at Johns Hopkins have the necessary expertise to define the physiological significance of the role of astrocyte derived EETs in dampening pressure-dependent activation of the pre-capillary circulation and mediating functional hyperemia. The large scope, common theme and application of multiple state-of-the-art techniques necessitates the programmatic initiative between the Medical College of Wisconsin and Johns Hopkins University proposed in this application. This is a resubmission and has been substantially revised along the line suggested by the scientific review study sections.
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