The long-term goals of this proposal ar to study the mechanisms by which the level of a specific receptor for serotonin, the 5-HT2 receptor, are regulated in the smooth muscle cell of the uterus. Preliminary evidence, obtained in our laboratory, has indicated that the levels of messenger RNA for this receptor (the only serotonin receptor detectable in this cell type) are positively regulated by serotonin. Myometrial cells depleted of serotonin contain massively lower levels of 5-HT2 receptor mRNA; re-addition of serotonin to these cells results in a progressive increase in 5-HT2 receptor message over a ten- to twelve hour time course. Selective 5-HT2 receptor agonists fully substitute for serotonin in up-regulating 5-HT2 receptor mRNA, whereas selective 5-HT2 receptor antagonists completely prevent the serotonin-dependent increase in 5-HT2 receptor mRNA in the myometrial smooth muscle cell. Agonists and antagonists of receptors other than the 5-HT2 subtype (e.g., 5-HT1 and 5-HT3) have no effect on the levels of 5-HT2 receptor mRNA in the cells. Thus, the serotonin-dependent regulation of the 5-HT2 message appears to require the presence of the 5-HT2 receptor itself. In our proposed studies, genomic clones will be obtained which contain the 5'-flanking (promoter) region of the 5-HT2 receptor gene; 2-3 kb of this region will be sequenced, and utilized to examine the mechanism by which the gene is activated in the myometrial smooth muscle cell. Myometrial smooth muscle cells will be transfected with known sequences contained in the promoter region, coupled to a reporter gene (bacterial chloramphenicol acetyl transferase), and the sequence required for serotonin-dependent regulation of the receptor will be determined. This information, together with the results of ongoing cell biological studies, will enable us to obtain insight into the mechanisms by which this unique, agonist- dependent, receptor modulation occurs. We will combine this information with studies on the regulation of the 5-HT2 receptor, in both cultured myometrial smooth muscle cells and the in vivo pregnant uterus, assessed both by classical ligand binding and molecular biological techniques in order to better understand the role of serotonin in the physiology of the uterus. In addition, these studies should shed new light on how the mammalian uterus prepares itself for normal parturition and the massive involution that occurs during the post-partum period.
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