The goal of this work is to elucidate the cellular and molecular mechanisms that modulate alpha-1 adrenergic receptor (alpha-1 AR) mediated responses in vascular smooth muscle cells. We will focus on mechanisms that are likely to regulate 1) receptor number and cellular distribution (synthesis, degradation, cellular trafficking and post- translational modification), and 2) receptor coupling to second messenger pathways.
Specific Aim I elucidates the characteristics and mechanisms of the cellular trafficking of alpha-1 AR (internalization, externalization, degradation), and the effects of second messengers and agonist on these processes.
Specific Aim II evaluates the functional consequences of alpha-1 AR glycosylation by determining the effects of glycosidase and glycosylation inhibitors on agonist-receptor interactions, the ability to activate second messenger systems, and cellular receptor trafficking.
Specific Aim III assesses the mechanism of alpha-1 AR mRNA regulation (transcriptional, post-transcriptional, protein synthesis dependence), and the role of ionic and chemical second messengers (Ca++, cAMP, PK-C, pH) in regulating alpha-1 AR mRNA levels.
Specific Aim I V addresses the mechanism of acute agonist-induced desensitization (the roles of PK-C and receptor sequestration) and evaluates the hypothesis that there are two subtypes of alpha-1 AR that exhibit differential second messenger coupling, and possibly, differential regulation. Experiments will be performed in cultured vascular smooth muscle cells. Alpha-1 adrenergic receptor number, cellular distribution and agonist binding properties will be determined with [3H]-prazosin in intact, broken and fractionated cells with both equilibrium and non-equilibrium assays. The regulation of alpha-1 receptor mRNA levels will be evaluated using a DNA probe. The relationships between receptor properties (number, distribution, agonist affinity) and functional responses will be assessed in parallel experiments with measurements of Ca++ flux, phospholipid turnover (IP3, DAG, choline release) and arachidonic acid production. These studies should improve our understanding of the physiology of alpha-1 adrenergic control of vascular tone, and may provide insight into disease states that are characterized by abnormal vascular responsiveness to catecholamines.
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