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

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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.