The long-term goal of this proposal is to understand the molecular basis for physiological effects elicited by epinephrine via alpha-adrenergic receptors of the pharmacological alpha-2-subtype. We will address this long-term goal by focusing on the manner in which sodium ion (Na+) influences alpha-2-receptor-agonist interactions and alpha-2-receptor-induced function.
The specific aims of the present proposal are to 1) purify the alpha-2-receptor to homogeneity, 2) evaluate whether the effects of Na+ on the purified alpha-2-receptor are conveyed via the betagamma heterodimer of the GTP-binding protein heterotrimer (Gi: alphabetagamma) which modulates alpha-2-receptor functions or, alternatively, via a Na+/H+ antiporter, 3) reconstitute the alpha-2-adrenergic receptor with the putative Na+-effector component in lipid vesicle preparations to test the conclusions drawn in #2 and 4) utilize permeabilized platelets to determine whether Gi represents the interface between the alpha-2-receptor and the Na+/H+ exchange mechanism and/or phospholipase A-2 activity postulated to be involved in epinephrine-provoked platelet secretion. Alpha-2-receptors are one of a number of hormone and neurotransmitter receptor populations which are linked to inhibition of adenylate cyclase activity. A number of lines of evidence suggest that the resultant decreases in cAMP levels are not sufficient to """"""""signal"""""""" a physiological effect, but that other important biochemical changes must also be evoked by these receptor populations. Receptors which can inhibit cAMP accumulation are all known to be modulated by Na+. Our studies, which focus on the basis for this role of Na+, should provide fundamental new insights into how alpha-2-adrenergic receptors elicit their physiological effects, in particular, and what are the alternate signaling mechanisms utilized by receptors linked to inhibition of adenylate cyclase activity, in general. Our model system for assessment of alpha-2-receptor-provoked function, i.e. epinephrine-induced platelet aggregation and secretion, will be of particular significance in understanding normal and pathological hemostasis, since platelet plug formation is necessary to prevent excessive blood loss but, when occurring inappropriately, forms thrombi that can precipitate strokes or myocardial infarction.
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