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Functional analysis of antidiuretic hormone receptor using V1a knockout mice and invention of new diuretics.

V1a基因敲除小鼠抗利尿激素受体功能分析及新型利尿剂的发明。

基本信息

批准号：
17590833
负责人：
NONOGUCHI Hiroshi
金额：
$ 2.24万
依托单位：
Kumamoto University Graduate School of Medical Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Vasopressin V(1a) and V(2) receptors (V(1a)R and V(2)R, respectively) distribute in the collecting duct of the kidney. Although the function of V(2)R mediating the antidiuretic effect of AVP has been investigated in detail, the role of V(la)R in the collecting ducts has not been elucidated. In the present study, we have investigated the role of the V(la)R pathway in V(2)R promoter activity. We cloned the 5'-flanking region of rat V(2)R (rV(2)R) and investigated rV(2)R promoter activity in the LLC-PK(1) cell line transfected to express rat V(la)R (rV(la)R) dominantly (LLC-PK(1)/rV(la)R). AVP induced a transient increase, followed by a sustained decrease, of rV(2)R promoter activity in these cells. This AVP-induced decrease of rV(2)R promoter activity was inhibited by V(1a)R, but not V(2)R, antagonist. PMA mimicked this decrease of rV(2)R promoter activity. On the contrary, cpt-cAMP increased rV(2)1t promoter activity. These PMA-and cpt-cAMP-induced effects were not observed on the deletion segment of the 5'-flanking region lacking CAAT and SP1 sites. In conclusion, 1) expression of the V(2)R is downregulated via the V(la)R pathway in LLC-PK(1)/rV(1a)R cells, and 2) expression of the V(2)R is downregulated by the PMA-induced PKC pathway and upregulated by the cAMP-PKA pathway. These opposite effects of PKC and PKA appear to be regulated by the same promoter region of CAAT and SP1 (published in Am J Physiol.)We also investigated the mechanisms of downregulation of V2 receptor in dehydration. 0ur results indicated that increased production of prostaglandin in renal medulla play a key role for the downregulation. The urine volume of Via knockout mice was smaller than that of wild type, suggestting that Vla knockout mice show a new type of nephrogenic diabetic insipidus.

加压素V（1a）和V（2）受体（分别为V（1a）R和V（2）R）分布于肾脏集合管。虽然V（2）R介导AVP抗利尿作用的功能已被详细研究，但V（1a）R在集合管中的作用尚未阐明。在本研究中，我们研究了V（la）R途径在V（2）R启动子活性中的作用。我们克隆了大鼠V（2）R（rV（2）R）的5 '侧翼区，并在转染以显性表达大鼠V（la）R（rV（la）R）的LLC-PK（1）细胞系（LLC-PK（1）/rV（la）R）中研究了rV（2）R启动子活性。AVP诱导这些细胞中rV（2）R启动子活性的短暂增加，随后持续降低。这种AVP诱导的rV（2）R启动子活性的降低可被V（1a）R拮抗剂所抑制，但不被V（2）R拮抗剂所抑制。PMA模拟了rV（2）R启动子活性的降低。相反，cpt-cAMP增加rV（2）1 t启动子活性。在缺少CAAT和SP1位点的5 ′-侧翼区的缺失片段上未观察到PMA和cpt-cAMP诱导的效应。总之，1）LLC-PK（1）/rV（1a）R细胞中V（2）R的表达通过V（1a）R途径下调，2）V（2）R的表达通过PMA诱导的PKC途径下调，通过cAMP-PKA途径上调。PKC和PKA的这些相反作用似乎由CAAT和SP1的相同启动子区域调节（发表在Am J Physiol.）我们还研究了脱水时V2受体下调的机制。结果表明，肾髓质前列腺素生成增加在下调中起关键作用。Vla基因敲除小鼠尿量明显小于野生型小鼠，提示Vla基因敲除小鼠存在一种新的肾原性尿崩症。