Barnett J V, Shamah S M, Lassegue B, Griendling K K, Galper J B
Department of Medicine, Brigham and Women's Hospital, Boston, MA.
Biochem J. 1990 Oct 15;271(2):437-42. doi: 10.1042/bj2710437.
These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5'-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production.
这些研究证明了在胚胎鸡心房细胞中，毒蕈碱受体与磷脂酶C活性偶联的一种新机制。在取自14日龄鸡胚心脏的心房细胞单层培养物中，氨甲酰胆碱刺激肌醇三磷酸（InsP3）、肌醇二磷酸（InsP2）和肌醇一磷酸（InsP1）依次出现，其半数有效浓度（EC50，即引起最大刺激50%的浓度）为30微摩尔。在15毫摩尔锂存在的情况下，用氨甲酰胆碱（0.1毫摩尔）处理5分钟，可使InsP3水平比基础水平最高增加47±12%，InsP2比基础水平增加108±13%，InsP1比基础水平增加42±5%。这种效应被5微摩尔阿托品阻断。用百日咳毒素（15小时；0.5纳克/毫升）孵育这些细胞，可分别抑制氨甲酰胆碱刺激的InsP3、InsP2和InsP1形成42±7%、30±3%和48±7%。百日咳毒素对所有三种肌醇磷酸抑制作用的半数抑制浓度（IC50，即引起50%抑制的浓度）为0.01纳克/毫升，在0.5纳克/毫升时的半衰期为6小时。对百日咳毒素的这种部分敏感性并非由于鸟嘌呤核苷酸结合蛋白（G蛋白）的ADP核糖基化不完全，因为在百日咳毒素存在下用[32P]NAD+孵育细胞匀浆的聚丙烯酰胺凝胶放射自显影表明，用0.5纳克/毫升百日咳毒素孵育细胞15小时可导致内源性NAD+对百日咳毒素底物的完全ADP核糖基化。在用皂角苷（10微克/毫升）通透的细胞中，0.1毫摩尔鸟苷5'-[γ-硫代]三磷酸（GTP[S]）使InsP1比基础水平增加102±【标准误】15%（平均值±标准误，n = 4），InsP2增加421±67%，InsP3增加124±33%。用0.5纳克/毫升百日咳毒素孵育细胞15小时，可使皂角苷处理细胞中GTP[S]刺激的InsP1产生减少30±10%（n = 3），InsP2产生减少45±7%（n = 4），InsP3产生减少49±6%（n = 4）。这些数据表明，在胚胎鸡心房细胞中，至少有两种独立的G蛋白，一种对百日咳毒素敏感的G蛋白和一种对百日咳毒素不敏感的G蛋白，在毒蕈碱激动剂结合与磷脂酶C激活以及肌醇磷酸产生的偶联中起作用。
