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

抗利尿激素V（1a）和V(2)受体(分别为V（1a)R和V(2)R）分布于肾集管中。虽然V(2)R介导AVP抗利尿作用的功能已被详细研究，但V(la)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的5′侧区（rV(2)R），并在转染了大鼠V(la)R （rV(la)R）为主表达的LLC-PK(1)细胞系(LLC-PK（1)/rV(la)R）后，研究了rV(2)R启动子活性。AVP诱导这些细胞中rV(2)R启动子活性短暂升高，随后持续降低。avp诱导的rV(2)R启动子活性降低被V(1a)R抑制，而V(2)R拮抗剂不受抑制。PMA模拟了rV(2)R启动子活性的降低。相反，cpt-cAMP增加了rV(2)1t启动子活性。这些pma和cpt- camp诱导的效应在缺乏CAAT和SP1位点的5'侧区缺失片段上没有观察到。综上所述，1)在LLC-PK(1)/rV(1a)R细胞中，V(2)R的表达可通过V(la)R通路下调；2)V(2)R的表达可通过pma诱导的PKC通路下调，cAMP-PKA通路上调。PKC和PKA的这些相反作用似乎是由CAAT和SP1的相同启动子区域调节的（发表于Am J Physiol）。我们还研究了脱水时V2受体下调的机制。我们的研究结果表明，肾髓质中前列腺素的产生增加在下调中起关键作用。Via基因敲除小鼠的尿量小于野生型，提示Vla基因敲除小鼠出现了一种新型肾源性糖尿病尿崩症。

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.