Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.
Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.
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在人星形细胞瘤细胞膜制剂中，卡巴胆碱对鸟嘌呤核苷酸依赖性百日咳毒素不敏感的磷酸肌醇刺激作用。
DOI：
10.1042/bj2390141
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Harden,TK
中科院分区：
文献类型：
--
作者：
Hepler,JR;Harden,TK
毒蕈碱受体激动剂在完整的1321 N1人星形细胞瘤细胞中刺激磷酸肌醇形成和Ca 2+动员的功效与它们在来自这些细胞的膜中形成GTP敏感性高亲和力结合复合物的能力相关[Evans，Hepler，Masters，Brown & Harden（1985）Biochem.J.232，751-757]。这些观察结果提示的建议，鸟嘌呤核苷酸调节蛋白用于耦合毒蕈碱受体的磷脂酶C参与磷酸肌醇水解在1321 N1细胞。肌醇磷酸（InsP）的形成是在1321 N1细胞的无细胞制剂中测量的，为这一想法提供直接支持。在从肌-[3 H]肌醇预标记的1321 N1细胞制备的洗涤膜中，鸟苷5 '-[γ-硫代]三磷酸（GTP[S]）以浓度依赖性方式（K0.5约5 μ M）增加InsP 3、InsP 2和InsP 1的形成。GTP[S]和鸟苷5′-[beta gamma-imido]三磷酸（p[NH]ppG）均刺激InsP形成，是对照组的2-3倍; GTP、GDP和GMP的有效性要低得多。毫摩尔浓度的氟化钠也刺激形成肌醇磷酸在1321 N1细胞膜制剂。在存在10 μ M GTP [S]的情况下，毒蕈碱胆碱能受体激动剂卡巴胆碱刺激（K 0.5约10 μ M）InsP的形成，高于单独使用GTP[S]所实现的。氨甲酰胆碱的作用可被阿托品完全阻断。在500 μ M-卡巴胆碱存在下，核苷酸刺激InsP形成的效力顺序为GTP[S]大于p[NH]ppG大于GTP = GDP。在GTP[S]或GTP[S]加卡巴胆碱存在下，百日咳毒素在完全ADP核糖基化和功能性抑制性鸟嘌呤核苷酸调节蛋白（抑制性鸟嘌呤核苷酸调节蛋白）的浓度下对InsP形成没有影响。这些数据是一致的想法，鸟嘌呤核苷酸调节蛋白，而不是Gi参与受体介导的刺激InsP的形成在1321 N1人星形细胞瘤细胞。
The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.