REGULATION OF 5-HT2 RECEPTOR IN UTERINE SMOOTH MUSCLE

子宫平滑肌中5-HT2受体的调节

• 批准号: 2202328
• 负责人: JOHN J JEFFREY
• 金额: $ 10.54万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2202328
• 关键词: cell membrane; gene expression; genetic enhancer element; genetic promoter element; genetic regulation; genetic transcription; hormone regulation /control mechanism; laboratory rat; messenger RNA; molecular cloning; nucleic acid hybridization; nucleic acid sequence; plaque assay; polymerase chain reaction; postpartum; pregnancy; serotonin; serotonin receptor; smooth muscle; southern blotting; transfection; uterus

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.

本提案的长期目标是研究 5-羟色胺的特异性受体5-HT 2受体的水平， 在子宫平滑肌细胞中调节。 初步证据， 在我们的实验室中获得的，已经表明信使的水平 这种受体的RNA（在这种细胞中唯一可检测到的血清素受体）。 细胞类型）受到血清素的积极调节。 子宫肌层细胞 缺乏血清素的人，5-HT 2受体的水平大大降低， 5-羟色胺重新加入这些细胞导致进行性的 在10 - 12小时时间内5-HT 2受体信息增加 当然了 选择性5-HT 2受体激动剂完全替代5-羟色胺 在上调5-HT 2受体mRNA，而选择性5-HT 2受体 拮抗剂完全阻止了5-HT 2依赖性的增加 受体mRNA的表达。 激动剂和 5-HT 2亚型以外的受体的拮抗剂（例如，5-HT 1和 5-HT_3）对细胞内5-HT_2受体mRNA水平无影响。 因此，5-HT 2信息的降钙素依赖性调节似乎 需要5-HT 2受体本身的存在。 在我们提出的 研究中，将获得含有5 '-侧翼的基因组克隆， 5-HT 2受体基因的启动子区域;该区域的2-3 kb将与5-HT 2受体基因的启动子区域结合。 被测序，并用于检查基因的机制， 在子宫肌层平滑肌细胞中被激活。 子宫平滑肌 细胞将用包含在启动子中的已知序列转染 区域，与报告基因（细菌氯霉素乙酰基）偶联 转移酶），以及依赖于降钙素的 将确定受体的调节。 这些信息， 加上正在进行的细胞生物学研究的结果， 让我们深入了解这种独特的激动剂- 发生依赖性的受体调节。 我们将联合收割机 随着对5-HT 2受体调节的研究，在两种培养的 子宫肌层平滑肌细胞和体内妊娠子宫， 通过经典的配体结合和分子生物学技术， 为了更好地了解血清素在生理学中的作用， 子宫 此外，这些研究应该揭示新的光如何 哺乳动物的子宫为正常分娩做好准备， 在产后期间发生的退化。