The research proposed is designed to address the unifying hypothesis that endothelial-derived prostacyclin permits newborn cerebral microvascular smooth muscle to dilate in response to specific stimuli. To test this hypothesis, four specific aims will be pursued using newborn pigs: l) Determine, in vivo, the ability of prostacyclin to restore cerebral vasodilator responses inhibited by indomethacin and by endothelial injury. 2) Examine the relationship between the ability of prostacyclin to restore vasodilation and changes in cyclic nucleotide metabolism. 3) Characterize, in vivo, the relationship between cerebral microvascular endothelial cell prostacyclin synthesis and smooth muscle cAMP in response to increased CO2. 4) Investigate the mechanisms by which prostacyclin modifies cerebral microvascular smooth muscle responses to hypercapnia. To accomplish these aims, techniques allowing investigation of intact cerebral microcirculation and primary culture of cells from newborn pig brain will be employed. Such research will be unique by studying the intact newborn cerebral circulation, isolated components that contribute to control of that circulation, and cocultures of these components to investigate mechanisms of communication. Cranial windows allow observation of cerebral microcirculation, collection of cortical periarachnoid fluid, selective endothelial damage, in vivo, and topical application of treatments and putative permissive agonists and inhibitors. Piglet cerebral microvascular endothelial and smooth muscle cells in primary culture will be used to study the effects of endothelial prostacyclin on smooth muscle cAMP responses to CO2. Effects of stable prostacyclin analogs on cerebral microvascular smooth muscle cAMP responses to CO2 will be evaluated. cAMP produced in response to CO2 by endothelial and vascular smooth muscle cells grown in isolation and together will be measured. To examine the relative contributions of intracellular and extracellular pH to the smooth muscle response to hypercapnia, using piglet cerebral microvascular smooth muscle cells in culture, we will measure cAMP production in response to decreases of pH-i with constant pH-e and in response to reduction of pH-e while maintaining pH-i. Selected potential cellular messenger pathways for involvement in the permissive role of prostacyclin in the microvascular smooth muscle response to hypercapnia will be evaluated. The effect of CO2 on iloprost receptor binding, the contributions of protein phosphorylation to the permissive role of prostacyclin and the kinases and substrates involved will be studied. Since disorders of cerebral circulation are major causes of morbidity and mortality in neonates and can result in lifelong disabilities in survivors better understanding of factors controlling newborn cerebral hemodynamics is badly needed.
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