Alpha1-Adrenergic receptors (alpha1-AR) play an important role in mediating the effects of catecholamines on the physiology, phenotype and growth of vascular smooth muscle cells (VSMC). We recently found that agonist-induced down-regulation of alpha1B-AR mRNA in VSMC from the rabbit aorta (RbSMC) is due to a post-transcriptional, protein kinase C-mediated decrease in the mRNA stability. Three subtypes of the alpha1-AR (alpha1B, alpha1C and alpha1D) have been cloned, and appear to be heterogeneous with regard to tissue distribution and coupling to second messengers and physiologic responses. There is uncertainty about the relationship between the 3 cloned subtypes and pharmacologically-defined subtypes, and there is no information about the role of the alpha1-AR subtypes in mediating the effects of alpha-adrenergic agonists on growth in VSMC. The goals of this proposal are: 1) to identify the cis-elements that determine the susceptibility of alpha1B-AR mRNA to phorbol ester-induced destabilization, 2) to demonstrate and characterize a phorbol ester- stimulated binding factor for alpha1B-AR mRNA, and 3) to elucidate the coupling of the cloned alpha1B and alpha1C-AR subtypes to second messengers and growth in VSMC.
In SPECIFIC AIM I we will test the hypothesis that phorbol ester-induced destabilization of alpha1B-AR mRNA requires the presence of cis-acting structural element(s) in the mRNA by testing for the ability of phorbol esters to destabilize wild-type and modified alpha1B-AR mRNA transcripts expressed in cultured cells.
In SPECIFIC AIM II we will test the hypothesis that alpha1B-AR mRNA is destabilized by the phorbol ester-induced binding of a cell-specific, trans-acting factor by using RNA gel shift and ultraviolet cross-linking assays to demonstrate phorbol ester-induced binding of a factor in RbSMC cytosol to in vitro-transcribed rabbit alpha1B-AR mRNA. In SPECIFIC III we will use both pharmacologic methods and subtype-specific antisense oligonucleotides directed against alpha1B and alpha1C-AR mRNA to test the hypothesis that these alpha1-AR subtypes are coupled to distinct second messenger pathways and exert cooperate effects on VSMC growth (protein and DNA synthesis) and growth-related signalling pathways (activation of mitogen-activated protein kinase, and induction of c-fos and transforming growth factor-beta1 mRNAs).
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