MATURATION OF CEREBROVASCULAR RELAXANT MECHANISMS

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Cardiovascular homeostasis is delicately balanced between contractile and relaxant smooth muscle influences in all vascular beds. Whereas regulation of vascular contraction has been reasonably well studied in neonates, the effects of postnatal maturation on mechanisms of vasorelaxation remain understudied and poorly understood. Recent work in our laboratory strongly suggests that the hypocontractility typical of immature cerebral arteries involves an upregulation of cGMP-dependent attenuation of vascular tone that cannot be explained simply by elevated cGMP concentrations. Thus, we propose that the ability of Protein Kinase Gto elicit cerebral vasodilatation is upregulated in immature cerebral arteries. Toaddress this core hypothesis, five specific aims are proposed. Aim #1 will employ Western blots, immunohistochemistry, and kinetic measurements of enzyme activity to test the hypothesis that maturation modulates the relative abundance, distribution, and activity of Protein Kinase G (PKG). Aim #2 will utilize autoradiographic measurements of receptor density, agonist affinity and assays for IPS content to test the idea that maturation alters the ability of cGMP/PKG to influence G-protein receptor-mediated IPS mobilization. Aim #3 will use measurements of IPS receptor density and binding affinity, calcium store size, and IPS-induced calcium release to explore the idea that maturation alters the ability of PKG to attenuate IPS-mediated calcium release. Aim #4 tests the idea that maturation alters cGMP-dependent modulation of thick filament reactivity, as indicated by shifts in the relations between cytosolic calcium and myosin light chain phosphorylation. For aim #4 the time courses of myosin light chain phosphorylation induced by potassium will be measured via urea gels in samples from both intact and permeabilized arteries treated with cGMP and/or inhibitors of myosin phosphatase. Aim #5 tests the hypothesis that maturation alters the ability of cGMP/PKG to modulate thin filament reactivity, as indicated by shifts in the relations between the extent of myosin light chain phosphorylation and contractile force. For aim #5, myosin light chain phosphorylation will be measured together with contractile force and the relative abundances and phosphorylation states of the potential thin-filament regulatory proteins HSP27 and HSP20. Quantitative integration of the results of these experiments will enable an unprecedented evaluation of the sites of action of postnatal maturation on cGMP- mediated pathways of vasorelaxation, and will provide a unique assessment of the relative importance of each main group of PKG targets for overall cerebrovascular homeostasis in the fetus, neonate and adult. This approach should also identify which cGMP-dependent mechanisms may be most amenable to therapeutic pharmacological manipulation in the critically ill neonate.