Blood flow is regulated very differently in the immature and mature brain, and such variations contribute significantly to higher frequencies of intracranial hemorrhage, vasospasm, and cerebral infarcts in neonates, particularly those born prematurely. The studies proposed in this application are designed to identify reasons for these variations and focus on age-related differences in the contractile properties of cerebral arteries. The proposed experiments extend recent research indicating that cerebrovascular contractility is governed in large part by the sensitivity of contractile proteins to calcium. Calcium sensitivity, in turn, varies significantly with postnatal age, and is governed by two main families of mechanisms: thick-filament regulation and thin-filament regulation. The proposed studies address the main hypothesis that postnatal maturation simultaneously decreases the cerebrovascular importance of thin-filament regulation and increases the importance of thick-filament regulation. This main hypothesis, in turn, has two corollaries, each of which proposes mechanisms whereby cerebrovascular contractile protein function is altered: 1) maturation enhances agonist-induced stimulation of thick-filament regulation through increases in myosin light-chain kinase and myosin light-chain phosphatase abundance and activity; and 2) maturation depresses agonist-induced stimulation of thin-filament regulation by increasing the abundance and activity of regulatory thin-filament proteins, including caldesmon and HSP27. To address this hypothesis and its corollaries, we will conduct experiments using carotid and cerebral arteries from newborn lambs and adult sheep to: 1) measure myofilament Ca2+ sensitivity and the simultaneous relations between force, cytosolic calcium concentration, and myosin light-chain phosphorylation in intact artery responses to graded concentrations of potassium and 5HT, and in permeabilized artery responses to varying buffered [Ca2+]; 2) measure the abundance of myosin light-chain kinase and the effects of 5HT and graded concentrations of calcium on myosin light-chain kinase activity, in situ; 3) measure the abundance of myosin light-chain phosphatase and the effects of 5HT and rho-kinase inhibitors on myosin light-chain phosphatase activity, in situ; and 4) determine the effects of ERK and p38 kinase inhibitors on the temporal relations between serotonin-induced myosin light-chain phosphorylation, contractile force, and phosphorylation of caldesmon and HSP27. Together, the results of these experiments will answer the questions: how are age-dependent changes in regulation of Ca2+ sensitivity partitioned among thick-filament and thin-filament mechanisms? In doing so, the proposed experiments will also offer a quantitative assessment of which thick-filament and thin-filament mechanisms may be involved in age-related changes in cerebrovascular contractile protein regulation, and thereby indicate the most promising future directions for research that may yield improvements in our strategies for clinical management of pre-term NICU infants.
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