Griendling K K, Delafontaine P, Rittenhouse S E, Gimbrone M A, Alexander R W
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
J Biol Chem. 1987 Oct 25;262(30):14555-62.
Angiotensin II stimulates sequential phospholipase C-mediated hydrolysis of initially the polyphosphoinositides and subsequently phosphatidylinositol (PI) in cultured rat aortic smooth muscle cells resulting in biphasic, sustained formation of diacylglycerol (DG). The mechanisms underlying this delayed induction of sustained DG accumulation are unknown but may be related to cellular events including processing of the angiotensin II receptor-ligand complex. In the present study, we characterized the kinetics of angiotensin II receptor sequestration and studied the effects of interventions which interfere with receptor processing on the pattern of angiotensin II-induced DG formation and phosphoinositide hydrolysis. Conversion of the angiotensin II receptor to an acid-resistant form was temperature-dependent, with half-times of 1.5 min at 37 degrees C and 7 min at 19 degrees C. Reducing the temperature to 25 or 19 degrees C caused a marked temporal separation between the two phases of DG accumulation. There was a close temporal correlation between the effect of temperature on receptor sequestration and on sustained DG accumulation. Furthermore, phenylarsine oxide (5 min, 10 microM), which inhibited angiotensin II receptor internalization, also selectively inhibited the sustained phase of DG accumulation (81 +/- 6% inhibition). Monensin and chloroquine, which interfere with receptor processing through the lysosomal-degradative pathway, had no effect on angiotensin II-induced DG formation in these cells, suggesting that the processing event important to hormonally induced sustained DG accumulation occurs early in the internalization pathway, probably at the level of the plasma membrane. Moreover, the acid-resistant state of the angiotensin II receptor-ligand complex retained its ability to signal, since removal of the surface signal by competitive antagonism with Sar1-Ile8-angiotensin II or acid-wash only slowly reversed accumulation of DG and depression of total cell calcium. These experiments support our previous observation that the initial and sustained phases of angiotensin II-induced diacylglycerol formation in vascular smooth muscle are differentially controlled and suggest that an early event in the cellular processing of the angiotensin II-receptor complex is essential to maintenance of DG accumulation.
血管紧张素II刺激培养的大鼠主动脉平滑肌细胞中磷脂酶C介导的磷脂酰肌醇多磷酸的逐步水解，随后是磷脂酰肌醇（PI）的水解，导致二酰基甘油（DG）的双相、持续形成。这种延迟诱导持续DG积累的潜在机制尚不清楚，但可能与包括血管紧张素II受体-配体复合物加工在内的细胞事件有关。在本研究中，我们表征了血管紧张素II受体隔离的动力学，并研究了干扰受体加工的干预措施对血管紧张素II诱导的DG形成模式和磷酸肌醇水解的影响。血管紧张素II受体向耐酸形式的转化是温度依赖性的，在37℃时半衰期为1.5分钟，在19℃时为7分钟。将温度降至25或19℃会导致DG积累的两个阶段之间出现明显的时间分离。温度对受体隔离和持续DG积累的影响之间存在密切的时间相关性。此外，抑制血管紧张素II受体内化的苯砷氧化物（5分钟，10 microM）也选择性地抑制了DG积累的持续阶段（抑制率为81±6%）。莫能菌素和氯喹通过溶酶体降解途径干扰受体加工，对这些细胞中血管紧张素II诱导的DG形成没有影响，这表明对激素诱导的持续DG积累重要的加工事件发生在内化途径的早期，可能在质膜水平。此外，血管紧张素II受体-配体复合物的耐酸状态保留了其信号传导能力，因为用Sar1-Ile8-血管紧张素II竞争性拮抗或酸洗去除表面信号只会缓慢逆转DG的积累和总细胞钙的降低。这些实验支持了我们之前的观察结果，即血管紧张素II诱导的血管平滑肌中二酰基甘油形成的初始阶段和持续阶段受到不同的控制，并表明血管紧张素II受体复合物细胞加工中的早期事件对于维持DG积累至关重要。
