Wang T T, Chen M, Lachance S, Delalandre A, Carrière S, Chan J S
University of Montreal, Maisonneuve-Rosemont Hospital, Research Center, Quebec, Canada.
Kidney Int. 1994 Sep;46(3):703-10. doi: 10.1038/ki.1994.324.
To investigate whether the expression of the renal angiotensinogen (ANG) gene is regulated by beta-adrenoceptors and the cAMP-dependent protein kinase A pathway, we introduced stably the fusion gene containing the 5'-flanking regulatory sequence of the ANG gene with a human growth hormone (hGH) gene as a reporter, pOGH (ANG N-1498/+18), into opossum kidney (OK) cells. We successfully obtained several stable transformants with a high expression of the pOGH (ANG N-1498/+18) fusion gene. One stable transformant (OK 27) that is able to maintain the expression of pOGH (ANG N-1498/+18) in culture for more than a year was used in the present study. The level of expression of the pOGH (ANG N-1498/+18) in OK 27 was evaluated by the amount of immunoreactive-hGH (IR-hGH) secreted into the culture medium. The addition of isoproterenol (10(-11) M to 10(-9) M) stimulated the expression of pOGH (ANG N-1498/+18) and increased the accumulation of intracellular cAMP. Higher concentrations of isoproterenol (that is, greater than 10(-9) M) had low or minimal effect. In contrast, the addition of 8-bromo-cAMP (8-Br-cAMP) and forskolin stimulated the expression of pOGH (ANG N-1498/+18) in a dose-dependent manner. The stimulatory effect of isoproterenol was blocked by the presence of propranolol, atenolol and ICI 118,551. The addition of ICI 118,551, however, was less effective than atenolol. Furthermore, the stimulatory effect of isoproterenol and 8-Br-cAMP on the expression of the pOGH (ANG N-1498/+18) was inhibited by the presence of Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II).(ABSTRACT TRUNCATED AT 250 WORDS)
为研究肾血管紧张素原（ANG）基因的表达是否受β-肾上腺素能受体及环磷酸腺苷（cAMP）依赖性蛋白激酶A途径调控，我们将含ANG基因5'-侧翼调控序列与人生长激素（hGH）基因作为报告基因的融合基因pOGH（ANG N-1498/+18）稳定导入负鼠肾（OK）细胞。我们成功获得了多个高表达pOGH（ANG N-1498/+18）融合基因的稳定转化株。本研究采用了一个能在培养中维持pOGH（ANG N-1498/+18）表达一年以上的稳定转化株（OK 27）。通过测定培养基中分泌的免疫反应性hGH（IR-hGH）量来评估OK 27中pOGH（ANG N-1498/+18）的表达水平。加入异丙肾上腺素（10⁻¹¹M至10⁻⁹M）刺激了pOGH（ANG N-1498/+18）的表达并增加了细胞内cAMP的积累。更高浓度的异丙肾上腺素（即大于10⁻⁹M）作用较低或极小。相反，加入8-溴-cAMP（8-Br-cAMP）和福斯可林以剂量依赖性方式刺激了pOGH（ANG N-1498/+18）的表达。异丙肾上腺素的刺激作用被普萘洛尔、阿替洛尔和ICI 118,551阻断。然而，加入ICI 118,551的效果不如阿替洛尔。此外，异丙肾上腺素和8-Br-cAMP对pOGH（ANG N-1498/+18）表达的刺激作用被Rp-cAMP（cAMP依赖性蛋白激酶A I和II的抑制剂）抑制。（摘要截短于250字）
