Roberts D J, Khan N, McDonald R L, Webster N J, Peers C, Vaughan P F
Institute for Cardiovascular Research, University of Leeds, LS2 9JT, Leeds, UK.
Brain Res Mol Brain Res. 2001 Feb 19;87(1):81-91. doi: 10.1016/s0169-328x(00)00294-1.
The aim of this study was to obtain further understanding of the mechanism by which activation of muscarinic M(1) receptors inhibits K(+)-evoked noradrenaline (NA) release in the human neuroblastoma SH-SY5Y. Previous studies have found that muscarinic M(1) and M(3) receptors couple to the activation of phospholipase C in SH-SY5Y cells leading to an increase in (a) intracellular calcium (Ca(2+)) and (b) activation of protein kinase C (PKC). This study used specific inhibitors of PKC and conditions which deplete Ca(2+)(i) stores to examine the role of protein kinase C and changes in Ca(2+) in mediating the inhibition of K(+)-evoked NA release by muscarine. Our data show that pretreatment of SH-SY5Y cell layers with bisindolylmaleimide I (BIM-I) (i) failed to reverse inhibition of K(+)-evoked NA release by muscarine but (ii) did overcome the attenuation of muscarine inhibition following pretreatment with TPA. Furthermore pretreating cell layers with Ca(2+)-free Hepes buffered saline in the presence of thapsigargin, conditions which prevented muscarine induced increases in Ca(2+), failed to prevent inhibition of K(+)-evoked NA release by muscarine. The effect of muscarine on K(+)-evoked uptake of Ca(2+)(e) was examined in SH-SY5Y cells loaded with Fura-2. Muscarine inhibited Ca(2+)(e)-uptake by decreasing the rate at which Ca(2+) entered SH-SY5Y cells via voltage sensitive Ca(2+)-channels. Thus this study shows that muscarine inhibits depolarisation-evoked NA release by a mechanism which is not dependent on activation of PKC or release of Ca(2+) from internal stores.
本研究的目的是进一步了解毒蕈碱M(1)受体激活抑制人神经母细胞瘤SH-SY5Y细胞中钾离子诱发去甲肾上腺素（NA）释放的机制。先前的研究发现，毒蕈碱M(1)和M(3)受体与SH-SY5Y细胞中磷脂酶C的激活偶联，导致（a）细胞内钙（[Ca(2+)]i）增加和（b）蛋白激酶C（PKC）激活。本研究使用PKC特异性抑制剂和耗尽Ca(2+)i储备的条件，以研究蛋白激酶C的作用以及[Ca(2+)]i变化在介导毒蕈碱对钾离子诱发NA释放的抑制中的作用。我们的数据表明，用双吲哚马来酰亚胺I（BIM-I）预处理SH-SY5Y细胞层（i）未能逆转毒蕈碱对钾离子诱发NA释放的抑制作用，但（ii）确实克服了用佛波酯（TPA）预处理后毒蕈碱抑制作用的减弱。此外，在毒胡萝卜素存在的情况下，用无钙的Hepes缓冲盐水预处理细胞层，这些条件可防止毒蕈碱诱导的[Ca(2+)]i增加，但未能防止毒蕈碱对钾离子诱发NA释放的抑制作用。在加载Fura-2的SH-SY5Y细胞中研究了毒蕈碱对钾离子诱发的细胞外钙（Ca(2+)e）摄取的影响。毒蕈碱通过降低Ca(2+)通过电压敏感Ca(2+)通道进入SH-SY5Y细胞的速率来抑制Ca(2+)e摄取。因此，本研究表明，毒蕈碱通过一种不依赖于PKC激活或从内部储存释放Ca(2+)的机制抑制去极化诱发的NA释放。
