Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels
Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels
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DOI：
10.1093/jb/mvj064
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发表时间：
2006-03-01
影响因子：
2.7
通讯作者：
Saffen, D
中科院分区：
文献类型：
--
作者：
Ebihara, T;Guo, FF;Saffen, D
在广泛研究的细胞系PC 12的亚系PC 12D细胞中，毒蕈碱乙酰胆碱受体（mAChR）的激活引起Ca（2+）从细胞内储存中的快速释放和外部Ca（2+）的持续内流。暴露于毒胡萝卜素后，也观察到细胞内储存的Ca（2+）释放和持续的Ca（2+）内流，毒胡萝卜素是一种倍半萜内酯，通过不可逆地抑制内质网的Ca（2+）泵来消耗细胞内Ca（2+）库。在这项研究中，我们表明，卡巴胆碱和毒胡萝卜素空相同的细胞内Ca（2+）的商店，这些商店是一个子集的细胞内商店耗尽的Ca（2+）离子载体离子霉素。在含有2 mM细胞外Ca（2+）的培养基中，用卡巴胆碱连续刺激mAChR期间，细胞内Ca（2+）储存保持耗尽，但在用阿托品抑制mAChR后迅速重新填充。在卡巴胆碱刺激的细胞中加入阿托品可使细胞内Ca（2+）水平分两步恢复至基线水平：快速下降与Ca（2+）再摄取进入内部储存相关，延迟下降与Mn（2+）渗透性Ca（2+）通道抑制相关。几条证据表明卡巴胆碱和毒胡萝卜素通过共同的机制刺激Ca（2+）内流：（i）用毒胡萝卜素预处理阻断阿托品介导的对Ca（2+）内流的抑制，（ii）单独或一起应用卡巴胆碱和毒胡萝卜素在刺激Mn（2+）内流方面同样有效，和（iii）当Ca（2+）加入到用卡巴胆碱、毒胡萝卜素或这两种试剂预处理的细胞中时，在没有细胞外Ca（2+）的情况下，观察到相同的Ca（2+）内流速率。综上所述，这些数据表明，在PC12D细胞中激活mAChR后观察到的细胞外Ca（2+）的持续流入主要是通过激活Mn（2+）可渗透的Ca（2+）库操纵的Ca（2+）通道介导的。
Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca(2+) from intracellular stores and a sustained influx of external Ca(2+) in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca(2+) from intracellular stores and a sustained influx of Ca(2+) are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca(2+) pools by irreversibly inhibiting the Ca(2+) pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca(2+) stores, and that these stores are a subset of intracellular stores depleted by the Ca(2+) ionophore ionomycin. Intracellular Ca(2+) stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca(2+), but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca(2+) levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca(2+) into internal stores and a delayed decrease that correlates with the inhibition of a Mn(2+)-permeable Ca(2+) channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca(2+) influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca(2+) influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn(2+), and (iii) identical rates of Ca(2+) influx are observed when Ca(2+) is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca(2+). Taken together, these data suggest that the sustained influx of extracellular Ca(2+) observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn(2+)-permeable, Ca(2+) store-operated Ca(2+) channel.