Carruthers A M, Challiss R A, Mistry R, Saunders R, Thomsen C, Nahorski S R
Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, UK.
Mol Pharmacol. 1997 Sep;52(3):406-14. doi: 10.1124/mol.52.3.406.
The regulation of phosphoinositide hydrolysis by the type 1alpha metabotropic glutamate receptor (mGluR1alpha) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S, 3R-dicarboxylic acid resulted in a marked accumulation of [3H]inositol monophosphate (InsP1) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] mass in a time- and concentration-dependent manner. Pretreatment of BHK-mGluR1alpha cells with pertussis toxin [ 100 ng/ml, 24 hr] led to a dramatic 12-16-fold increase in the accumulation of [3H]InsP1 and a 2-fold increase in Ins(1,4,5)P3 in the absence of added agonist. Although only very low levels (</=1 microM) of L-glutamate could be detected in medium taken from control and PTX-treated cell monolayers, the PTX-elicited effect on basal [3H]InsP1 was fully reversed by preincubation of cells in the presence of glutamic-pyruvic transaminase and pyruvate, suggesting that an increased sensitivity to endogenous glutamate was responsible for the apparent agonist-independent activation of phosphoinositidase C (PIC) after PTX treatment. Consistent with this hypothesis, in the presence of glutamic-pyruvic transaminase/pyruvate, the maximal [3H]InsP1 response to quisqualate was increased by >/=75%, and the EC50 shifted leftward by 65-fold [-log EC50 values (molar), 7.26 +/- 0.23 versus 5.45 +/- 0.07; n = 4) in PTX-treated compared with control cells. In contrast, antagonist effects on agonist-stimulated [3H]InsP1 responses were similar in control and PTX-treated BHK-mGluR1alpha cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (Gi/o) and PTX-insensitive stimulatory (Gq/11) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1alpha-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.
在稳定转染的幼仓鼠肾（BHK）细胞中研究了1α型代谢型谷氨酸受体（mGluR1α）对磷酸肌醇水解的调节作用。用L-谷氨酸、quisqualate和1-氨基环戊烷-1S，3R-二羧酸孵育细胞，导致[3H]肌醇单磷酸（InsP1）和肌醇-1,4,5-三磷酸[Ins(1,4,5)P3]含量以时间和浓度依赖性方式显著积累。用百日咳毒素[100 ng/ml，24小时]预处理BHK-mGluR1α细胞，在不添加激动剂的情况下，导致[3H]InsP1积累急剧增加12 - 16倍，Ins(1,4,5)P3增加2倍。尽管在对照和PTX处理的细胞单层培养基中仅能检测到非常低水平（≤1 microM）的L-谷氨酸，但细胞在谷氨酸-丙酮酸转氨酶和丙酮酸存在下预孵育可完全逆转PTX对基础[3H]InsP1的作用，这表明对内源性谷氨酸敏感性增加是PTX处理后磷酸肌醇酶C（PIC）明显的非激动剂依赖性激活的原因。与该假设一致，在谷氨酸-丙酮酸转氨酶/丙酮酸存在下，与对照细胞相比，PTX处理的细胞对quisqualate的最大[3H]InsP1反应增加≥75%，EC50向左移动65倍[-log EC50值（摩尔），7.26±0.23对5.45±0.07；n = 4]。相反，对照和PTX处理的BHK-mGluR1α细胞中拮抗剂对激动剂刺激的[3H]InsP1反应的作用相似。mGluR激动剂浓度-效应曲线的这些变化与一种模型一致，即该受体与百日咳毒素敏感的抑制性（Gi/o）和百日咳毒素不敏感的刺激性（Gq/11）G蛋白相关联，它们均可影响PIC活性。目前的观察结果与mGluR1α介导的PIC活性的双重调节一致，这可能是控制磷酸肌醇衍生信使输出的基础。
