Batty I H, Downes C P
Department of Biochemistry, University of Dundee, Scotland, U.K.
Biochem J. 1994 Feb 1;297 ( Pt 3)(Pt 3):529-37. doi: 10.1042/bj2970529.
Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP2 availability. The potential for complex regulation of PLC and PtdIns synthase is discussed.
本文描述了1321N1细胞的培养条件，在此条件下细胞内肌醇从约20 mM降至<0.5 mM，但磷酸肌醇浓度不受影响。在用[3H]肌醇对磷脂池进行稳态标记后，研究了毒蕈碱受体激动剂卡巴胆碱（1 mM）和/或LiCl（10 mM）对这些细胞或富含肌醇的细胞中磷酸肌醇周转的影响。在富含肌醇和缺乏肌醇的细胞中，在Li+存在的情况下，卡巴胆碱刺激的磷脂酶C（PLC）活性的初始（0 - 15分钟）速率相似。随后（>30 - 60分钟），仅在缺乏肌醇的细胞中，刺激的PLC活性和[3H]磷脂酰肌醇（PtdIns）浓度急剧下降。在缺乏肌醇的细胞中，单独使用卡巴胆碱会引起[3H]肌醇、[3H]肌醇一磷酸（InsP1）、[3H]肌醇二磷酸（InsP2）、[3H]肌醇三磷酸（InsP3）和[3H]肌醇四磷酸（InsP4）浓度升高，这些浓度在90分钟内基本保持稳定，而[3H]PtdIns、[3H]磷脂酰肌醇磷酸（PtdInsP）和[3H]磷脂酰肌醇二磷酸（PtdInsP2）浓度仅分别降至对照的约82%、84%和93%。在这些细胞中存在Li+时，[3H]肌醇的刺激升高被阻止，尽管[3H]InsP1、[3H]InsP2和[3H]InsP3的积累最初（0 - 30分钟）增强，但[3H]InsP1的积累速率和[3H]多磷酸肌醇的浓度随后（>30 - 60分钟）下降，[3H]PtdIns、[3H]PtdInsP和[3H]PtdInsP2浓度分别降至对照的约39%、48%和81%。在卡巴胆碱和Li+共同存在60分钟后，与缺乏肌醇细胞的初始速率相比，刺激的PLC活性降低了约70%。这种降低的PLC活性通过[3H]肌醇（1,3,4）三磷酸（Ins(1,3,4)P3）刺激浓度的变化反映出来，但[3H]肌醇（1,4,5）三磷酸（Ins(1,4,5)P3）的刺激浓度没有变化，不过Li+对后者的影响可能已被所证明的[3H]肌醇1:2环化、4,5 - 三磷酸的同时且同等刺激积累所掩盖。这些数据表明，在高度依赖肌醇再循环的细胞中，作为肌醇单磷酸酶抑制的次要后果，Li+会选择性地损害受体介导的PLC活性，但这意味着，尽管Li+对PLC活性的减弱与指示肌醇供应受限的参数密切相关，但它不太容易归因于PtdInsP2可用性的降低。本文还讨论了PLC和磷脂酰肌醇合成酶复杂调节的可能性。
