Liao C F, Schilling W P, Birnbaumer M, Birnbaumer L
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1990 Jul 5;265(19):11273-84.
The membrane signaling properties of the neuronal type-5 muscarinic acetylcholine receptor (M5 AChR) as expressed in murine L cells were studied. Recipient Ltk- cells responded to ATP acting through a P2-purinergic receptor by increasing phosphoinositide hydrolysis 2-fold but were unresponsive to 17 receptor agonists that are stimulatory in other cells. L cells expressing the M5 AChR responded to carbachol (CCh) with an approximately 20-fold increase in phospholipase C activity, mobilization of Ca2+ from endogenous stores, causing a transient peak increase in the intracellular concentration of Ca2+ ([Ca2+]i), influx of extracellular Ca2+, causing a sustained increase in [Ca2+]i dependent on extracellular Ca2+, and release of [3H]arachidonic acid from prelabeled cells, without altering resting or prostaglandin E1-elevated intracellular cAMP levels. None of the effects of the M5 AChR were inhibited by pertussis toxin. The regulation of L cell [Ca2+]i was studied further. ATP had the same effects as CCh and the two agonists acted on a shared intracellular pool of Ca2+. The peak and sustained [Ca2+]i increases were reduced by cholera toxin and forskolin, neither of which altered significantly phosphoinositide hydrolysis. This is consistent with interference with the action of inositol 1,4,5-trisphosphate (IP3) through cAMP-mediated phosphorylation and suggests a continued involvement of IP3 during the sustained phase of [Ca+]i increases. The temporal pattern of the sustained [Ca2+]i increase differed whether elicited by CCh or ATP, and was enhanced in pertussis toxin-treated cells. This is consistent with existence of a kinetic control of the sustained [Ca2+]i change by a receptor-G protein-dependent mechanism independent of the IP3 effector site(s) (e.g. pulsatile activation of phospholipase C and/or pulsatile activation of a receptor/G protein-operated plasma membrane Ca2+ channel). Thus, the non-excitable L cell may be a good model for studying [Ca2+]i regulations, as may occur in other nonexcitable cells of which established cell lines do not exist, and for studying of receptors that as yet cannot be studied in their natural environment.
对在鼠L细胞中表达的神经元型5毒蕈碱型乙酰胆碱受体（M5 AChR）的膜信号特性进行了研究。受体Ltk-细胞通过P2嘌呤能受体对ATP作出反应，使磷酸肌醇水解增加2倍，但对在其他细胞中具有刺激作用的17种受体激动剂无反应。表达M5 AChR的L细胞对卡巴胆碱（CCh）作出反应，磷脂酶C活性增加约20倍，从内源性储存库中动员Ca2+，导致细胞内Ca2+浓度（[Ca2+]i）短暂峰值增加，细胞外Ca2+内流，导致依赖细胞外Ca2+的[Ca2+]i持续增加，以及从预先标记的细胞中释放[3H]花生四烯酸，而不改变静息或前列腺素E1升高的细胞内cAMP水平。百日咳毒素不抑制M5 AChR的任何作用。进一步研究了L细胞[Ca2+]i的调节。ATP与CCh具有相同的作用，这两种激动剂作用于共同的细胞内Ca2+池。霍乱毒素和福斯可林降低了[Ca2+]i的峰值和持续增加，两者均未显著改变磷酸肌醇水解。这与通过cAMP介导的磷酸化干扰肌醇1,4,5-三磷酸（IP3）的作用一致，并表明在[Ca+]i增加的持续阶段IP3持续发挥作用。由CCh或ATP引发的[Ca2+]i持续增加的时间模式不同，并且在百日咳毒素处理的细胞中增强。这与存在一种由受体-G蛋白依赖性机制对[Ca2+]i持续变化进行动力学控制一致，该机制独立于IP3效应位点（例如磷脂酶C的脉动激活和/或受体/G蛋白操作的质膜Ca2+通道的脉动激活）。因此，非兴奋性L细胞可能是研究[Ca2+]i调节的良好模型，就像在尚无成熟细胞系的其他非兴奋性细胞中可能发生的那样，并且可用于研究尚未能在其自然环境中进行研究的受体。
