The transition from fetal to adult life involves dramatic changes in vascular reactivity, particularly in cerebral arteries where both the capacity to contract and the ability to relax improve simultaneously during postnatal life. The rates and patterns of these maturational changes, in turn, are highly susceptible to environmental stresses such as chronic hypoxia, which can potently depress pharmacomechanical coupling through multiple simultaneous effects. Whereas it is clear that chronic hypoxia alters agonist-induced calcium mobilization and myofilament calcium sensitivity, the mechanisms that mediate hypoxia' s effects on vascular reactivity remain unclear. Hypoxia-induced changes in endothelial production and release of NO contribute to the overall cerebrovascular effects of hypoxia, but major changes in reactivity to NO are also involved. Similarly, changes in cGMP metabolism contribute to the cerebrovascular effects of chronic hypoxia, but again, major changes in the mechanisms coupling cGMP to vasorelaxation are an essential component of the cerebrovascular adaptation to chronic hypoxia. The single most important effector of vasorelaxation downstream of cGMP is Protein Kinase G (PKG), which in cerebral arteries mediates almost all vasodilator effects of cGMP. Despite the central importance of PKG, its role in cerebrovascular function has been largely ignored, particularly in immature cerebral arteries. Virtually nothing is known of the effects of chronic hypoxia on cerebrovascular PKG function in any artery type or age. In light of these deficits, and the strong potential for PKG to play a key role in cerebrovascular adaptation to chronic hypoxia, the proposed studies focus on the general mechanisms involved in PKG-mediated vasorelaxation, and how these are modulated by maturation and chronic hypoxia. The general hypothesis addressed by these studies is that chronic hypoxia selectively enhances the ability of Protein Kinase G to elicit cerebral vasodilatation in an age-dependent and artery-specific manner. This main hypothesis, in turn, has four main corollaries, each of which proposes that hypoxia influences a mechanism whereby Protein Kinase G modulates pharmacomechanical coupling: 1) PKG modulates coupling between activation of cell surface receptors and synthesis of the second messenger IP3; 2) PKG alters the ability of second messengers such as IP3 to elicit calcium entry and/or release; 3) PKG influences thick filament reactivity, as indicated by the relation between cytosolic calcium and the extent of myosin light chain phosphorylation; and 4) PKG differentially enhances thin filament reactivity, as indicated by the relation between myosin fight chain phosphorylation and the production of contractile force. To evaluate the main hypothesis and its corollaries, we will conduct experiments designed to: 1. Quantify the distribution, abundance, and activity of cerebrovascular PKG isoforms 1alpha and 1beta using immunohistochemical, immunoblotting, and PKG activity measurements; 2. Quantify the effect of PKG activation on the coupling efficiency between receptor activation and IP3 production using functional measurements of agonist affinity and simultaneous IP3 accumulation; 3. Determine the effect of PKG activation on calcium entry and release using fluorometric measurements of cytosolic and organellar calcium together with microautoradiographic measurements of IP3 receptor density and binding affinity; 4. Determine the effect of PKG activation on thick filament reactivity as indicated by the relation between cytosolic calcium and myosin light chain phosphorylation, measured using fluorometric measurements of cytosolic calcium together with immunoblotting of phospho- and dephospho-myosin light chain; and 5. Determine the effect of PKG activation on thin filament reactivity as indicated by the relation between the extent of myosin fight chain phosphorylation, measured using immunoblotting of phospho- and dephospho-myosin light chain, and force development measured in arterial rings. To address the effects of perinatal maturation on the function of Protein Kinase G, we will conduct these experiments in both term fetuses and non-pregnant adults. To define the importance of arterial size and type, all experiments will be conducted in a series of arteries including the common carotid, basilar, posterior communicating, and middle cerebral arteries. Finally, to enable assessment of the role of changes in Protein Kinase G function associated with hypoxic acclimatization, parallel studies will be carried out in normoxic animals and in animals acclimatized to high altitude hypoxia. Together, the results of these experiments will enable an unprecedented assessment of the mechanisms whereby maturation and hypoxic acclimatization modulate the cerebrovascular role of Protein Kinase G.
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