Norepinephrine and epinephrine are important neurotransmitters in the central nervous system, and alpha-2 adrenergic receptors, which are coupled to the inhibition of adenylate cyclase, are widely distributed. Clinically, alpha-2 adrenergic agonists such as clonidine and guanabenz are used to treat hypertension as well as several other disorders. While clonidine is thought to act centrally to control blood pressure, its mechanism of action is not well understood. The therapeutic effects of alpha-2 adrenergic agonists often are evident only after chronic treatment, suggesting the involvement of adaptive changes. Thus, a better understanding of the regulation and the mechanism of action of alpha-2 adrenergic receptors, their coupling to and their regulation of adenylate cyclase has a clear potential for the development of better drug therapies. The overall goal of the proposed research is to understand the molecular basis for the effects of preincubation of cells with an alpha-2 adrenergic agonist on subsequent alpha-2 adrenergic receptor binding and the activity of the enzyme adenylate cyclase. There are two main effects to be studied. The first is the down-regulation of receptor number and desensitization of the cyclic AMP response. Second, we have recently observed that alpha-2 agonist pretreatment results in a 20-fold increase in forskolin stimulated cyclic AMP synthesis, which we call """"""""sensitization"""""""". For both sets of studies, the model system to be used is the HT29 cell line. Each effect will be characterized and then its molecular mechanism investigated. For the first effect, the hypothesis to be tested is that the mechanism of alpha-2 adrenergic receptor desensitization: uncoupling; receptor internalization; and receptor loss. Our initial studies indicate that """"""""sensitization"""""""" is not mediated through cyclic AMP. Thus, other mechanisms which have been suggested for alpha-2 adrenergic receptor action, as well as other second messenger systems, will be investigated. Additional experiments are aimed at investigating the possible molecular alterations in the adenylate cyclase system which cause the increased cyclic AMP production seen with sensitization.
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