Alpha-2 adrenergic receptors (alpha2R) play a critical role in mediating the influence of the sympathetic nervous on vascular smooth muscle, especially in the microvasculature. This role demands that the alpha2R coupling mechanism be able to integrate extrinsic neuronal activity with local needs in order to achieve optimal vascular tone and blood flow. This project will investigate the nature of the alpha2R coupling pathway, such as G proteins and effectors which are involved, and will examine how the efficacy and character of coupling is modulated. Coupling will be studied using both contraction and biochemical endpoints in isolated vascular tissue (rabbit saphenous vein) and GTP binding and biochemical endpoints in cultured cells (primarily PC12 cells) expressing a cloned alpha2R subtype. Previous work has established the ability of unoccupied alpha2R to cause a significant level of G protein activation, termed precoupling,and the regulation of precoupling is an important focus of the project. Since preliminary data indicated that precoupling can involve both vasoconstrictictor and vasodilator pathways, depending upon protein kinase C (PKC) activation, we will further investigate the mechanism of its control. In follow-up to earlier radioligand binding and GTP binding studies in isolated membranes, the influence of cytoplasmic Na+ levels on agonist efficacy will be examined, as well as its interplay with H+ levels. While the alpha2R coupling pathway is recognized to be highly dependent upon extracellular Ca2+, this dependence may reflect its essential role in providing for activation of PKC and for facilitating the activity of phospholipases D and A . The ability of alpha2R to stimulate these phospholipases under varied levels of Ca2+ will also be investigated. Preliminary data has indicated an essential role of tyrosine kinase activation in alpha2R vascular responses, but not in alpha1R responses. Since receptor-induced PLD activation has been shown in other tissues to require tyrosine kinase activity, we will determine whether this is the basis for the kinase in vascular smooth muscle as well. An integrated working hypothesis is presented which has the potential to provide a mechanistic basis for the observed adaptability of alpha2R responsiveness. Since micro-vascular dysfunction is thought to play an important role in several forms of hypertension, as well as in other disorders, the improved understanding provided by these studies will have both physiologic and pathophysiologic significance.
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