Imai A, Gershengorn M C
Proc Natl Acad Sci U S A. 1986 Nov;83(22):8540-4. doi: 10.1073/pnas.83.22.8540.
Stimulated inositolphospholipid turnover has been proposed to be initiated and sustained by hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which may be replenished by an enhanced flux of phosphatidylinositol (PtdIns) to PtdIns 4-phosphate (PtdIns4P) to PtdIns(4,5)P2. To determine whether there is continued hydrolysis and resynthesis of PtdIns(4,5)P2 in rat pituitary cells (GH3 cells) during stimulation by thyrotropin-releasing hormone (TRH), we investigated the turnover kinetics of the inositolphospholipids and of phosphatidic acid (PtdOH). In cells incubated with 32Pi for 1 min, TRH rapidly and persistently (for at least 30 min) enhanced the rate of 32P-labeling of PtdOH. After a lag time of 1 min, TRH markedly and persistently increased 32P-labeling of PtdIns also. In contrast, TRH caused only a transient increase in 32P-labeling of PtdIns(4,5)P2 that lasted less than 2 min. There was no rapid (before 10 min) effect of TRH on 32P-labeling of PtdIns4P. By 2 min of TRH stimulation, specific 32P radioactivity in PtdOH increased from 3.6% (control) of that in the gamma-phosphate of ATP to 15%; in PtdIns, from 0.07% to 1.3%; and in PtdIns(4,5)P2, from 3.8% to 5.4% (specific 32P radioactivity in PtdIns4P was 1.7% of that in ATP in control and TRH-stimulated cells). In cells exposed to TRH for 4 min and then to 32Pi, 32P-labeling of PtdOH and PtdIns increased, but that of PtdIns(4,5)P2 was not affected. Last, persistent turnover of PtdOH and PtdIns was not caused by initial hydrolysis of PtdIns(4,5)P2 because the turnover of PtdOH and PtdIns could be terminated by displacement of TRH from its receptor by chlordiazepoxide and restarted by reoccupying the receptors with TRH. These data demonstrate that turnover of PtdIns(4,5)P2 is stimulated only transiently, whereas turnover of PtdIns and PtdOH is stimulated persistently by TRH in GH3 cells. Hence, inositolphospholipid turnover in GH3 cells does not occur via continued hydrolysis of PtdIns(4,5)P2 accompanied by enhanced flux of PtdIns to PtdIns4P to PtdIns(4,5)P2, but there is direct and persistent hydrolysis of PtdIns. The dissociation of these actions suggests that there are separate mechanisms involved in coupling TRH-receptor complexes to stimulation of PtdIns(4,5)P2 and PtdIns hydrolysis in GH3 cells.
有研究提出，刺激引起的肌醇磷脂周转是由磷脂酰肌醇4,5 - 二磷酸[PtdIns(4,5)P2]水解启动并维持的，而磷脂酰肌醇（PtdIns）向磷脂酰肌醇4 - 磷酸（PtdIns4P）再到PtdIns(4,5)P2的通量增加可能会补充PtdIns(4,5)P2。为了确定在促甲状腺激素释放激素（TRH）刺激大鼠垂体细胞（GH3细胞）过程中，PtdIns(4,5)P2是否持续水解和再合成，我们研究了肌醇磷脂和磷脂酸（PtdOH）的周转动力学。在用32Pi孵育1分钟的细胞中，TRH迅速且持续地（至少30分钟）提高了PtdOH的32P标记率。经过1分钟的延迟期后，TRH也显著且持续地增加了PtdIns的32P标记。相比之下，TRH仅引起PtdIns(4,5)P2的32P标记短暂增加，持续时间不到2分钟。TRH在10分钟之前对PtdIns4P的32P标记没有快速影响。到TRH刺激2分钟时，PtdOH中的比32P放射性从ATPγ - 磷酸中的3.6%（对照）增加到15%；PtdIns中的从0.07%增加到1.3%；PtdIns(4,5)P2中的从3.8%增加到5.4%（对照和TRH刺激细胞中PtdIns4P的比32P放射性是ATP中的1.7%）。在暴露于TRH 4分钟然后再暴露于32Pi的细胞中，PtdOH和PtdIns的32P标记增加，但PtdIns(4,5)P2的不受影响。最后，PtdOH和PtdIns的持续周转不是由PtdIns(4,5)P2的初始水解引起的，因为通过氯氮卓将TRH从其受体上置换可终止PtdOH和PtdIns的周转，而用TRH重新占据受体可使其重新启动。这些数据表明，PtdIns(4,5)P2的周转仅被短暂刺激，而TRH在GH3细胞中持续刺激PtdIns和PtdOH的周转。因此，GH3细胞中的肌醇磷脂周转不是通过PtdIns(4,5)P2的持续水解并伴随着PtdIns向PtdIns4P再到PtdIns(4,5)P2的通量增加而发生的，而是存在PtdIns的直接且持续的水解。这些作用的解离表明，在GH3细胞中，将TRH - 受体复合物与PtdIns(4,5)P2和PtdIns水解的刺激偶联存在不同的机制。
