Inhibition of M3 muscarinic acetylcholine receptor-mediated Ca2+ influx and intracellular Ca2+ mobilization in neuroblastoma cells by the Ca2+/calmodulin-dependent protein kinase inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-trosyl]-4-phenylpipe
Inhibition of M3 muscarinic acetylcholine receptor-mediated Ca2+ influx and intracellular Ca2+ mobilization in neuroblastoma cells by the Ca2+/calmodulin-dependent protein kinase inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-trosyl]-4-phenylpipe
复制标题
Ca2 /钙调蛋白依赖性蛋白激酶抑制剂 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-trosyl]- 抑制神经母细胞瘤细胞中 M3 毒蕈碱乙酰胆碱受体介导的 Ca2 流入和细胞内 Ca2 动员
DOI：
10.1016/s0006-2952(97)00089-0
复制
发表时间：
1997
影响因子：
5.8
通讯作者：
Aronstam,RS
中科院分区：
文献类型：
--
作者：
Puhl,HL;Raman,PS;Williams,CL;Aronstam,RS
研究了Ca 2+钙调蛋白依赖性蛋白激酶（CaM激酶; EC 2.7.1.123）在毒蕈碱乙酰胆碱受体（mAChR）产生Ca 2+信号中的作用。使用染料Fura-2在SK-N-SH人神经母细胞瘤细胞中监测由mAChR激活诱导的细胞内Ca 2+浓度（[Ca 2 +]i）的变化。SK-N-SH细胞表达M3 mAChR以及II型和IV型CaM激酶，其被CaM激酶拮抗剂KN-62（1-[N，O-双（5-异喹啉磺酰基）-N-甲基-L-酪氨酰]-4-苯基哌嗪）特异性抑制。氨甲酰胆碱（100 μM）可使胞内钙库中的Ca ~（2+）释放，引起[Ca ~（2+）] i的短暂高峰，随后[Ca ~（2+）] i的持续升高依赖于细胞外Ca ~（2+）的内流，EGTA和Ni ~（2+）可抑制[Ca ~（2+）] i的持续升高。通过将细胞与0.01至100 μM KN-62预孵育，这些mAChR诱导的Ca 2+信号在同等程度上减弱。KN-62抑制mAChR诱导的Ca 2+内流和从内部储存的动员约25- 30%，在0.51 μM时产生半数最大效应。相比之下，KN-62（25 μM）几乎完全消除了氨甲酰胆碱估计的二价阳离子通过Mn 2+渗透通道的进入，如Mn 2+淬灭Fura-2荧光所示。KN-62也几乎完全阻断25 mM K+（1C_（50）~（13）μM）去极化引起的Ca ~（2+）内流。这些结果表明，CaM激酶调节细胞内Ca 2+的动员和由mAChR激活诱导的Ca 2+内流的刺激，并表明mAChR诱导的Ca 2+内流通过Ca 2+通道发生，而不是通过KN-62抑制的电压门控钙通道或Mn 2+渗透通道。
The role of Ca2+calmodulin - dependent protein kinase (CaM kinase; EC 2.7.1.123) in the generation of Ca2+signals by muscarinic acetylcholine receptors (mAChR) was studied. Changes in intracellular Ca2+concentrations ([Ca2+]i) induced by mAChR activation were monitored in SK-N-SH human neuroblastoma cells using the dye Fura-2. SK-N-SH cells express M3mAChR, as well as CaM kinase types II and IV, which are specifically inhibited by the CaM kinase antagonist KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine). Carbamylcholine (100 μM) elicited an initial transient peak in [Ca2+]idue to mobilization of Ca2+from internal stores, followed by a sustained elevation in [Ca2+]ithat depended on the influx of extracellular Ca2+and which was inhibited by EGTA and Ni2+. These mAChR-induced Ca2+signals were diminished to an equal extent by preincubating the cells with 0.01 to 100 μM KN-62. KN-62 inhibited mAChR-induced Ca2+influx and mobilization from internal stores by about 25–30%, producing a half-maximal effect at ≈ lμM. In contrast, KN-62 (25 μM) almost completely abolished carbamylcholinestimulated entry of divalent cations through Mn2+-permeant channels, as revealed by Mn2+quenching of Fura-2 fluorescence. KN-62 also almost completely abolished Ca2+influx induced by depolarization of the cells with 25 mM K+(lC50≈ 3 μM). These results suggest that CaM kinases regulate both the mobilization of intracellular Ca2+and the stimulation of Ca2+influx that are induced by mAChR activation, and indicate that the mAChR-induced influx of Ca2+occurs through Ca2+channels other than, or in addition to, the voltage-gated calcium channels or Mn2+-permeant channels which are inhibited by KN-62.