McGinnis K M, Shariat-Madar Z, Gnegy M E
Department of Pharmacology, University of Michigan School of Medicine, Ann Arbor 48109-0632, USA.
J Neurochem. 1998 Jan;70(1):139-46. doi: 10.1046/j.1471-4159.1998.70010139.x.
Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca2+ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca2+ channel blocker Ni2+. Blocking the influx of extracellular Ca2+ had no effect on carbachol-mediated increases in cytosolic CaM (168 +/- 18% of control values for carbachol treatment alone vs. 163 +/- 28% for Ni2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca2+ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 +/- 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca2+ by pretreatment with the cell-permeant Ca2+ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 +/- 37% of control without BAPTA/AM vs. 136 +/- 13% with BAPTA/AM). The effect of direct entry of extracellular Ca2+ into the cell by K+ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K+ elicited an immediate and persistent increase in intracellular free Ca2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca2+, which releases Ca2+ from intracellular stores, induced an increase in cytosolic CaM (203 +/- 30% of control). The mechanism for the CaM release may involve activation of the alpha isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K+ depolarization. These data demonstrate that release of Ca2+ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.
在人神经母细胞瘤细胞系SK-N-SH中，毒蕈碱受体激动可引起钙调蛋白（CaM）从膜组分重新分布至胞质溶胶。用离子霉素提高细胞内Ca²⁺浓度也会升高胞质CaM。本研究的目的是探讨细胞外和细胞内Ca²⁺库在毒蕈碱受体介导的SK-N-SH细胞胞质CaM增加中的作用。在负载fura-2的细胞中监测刺激介导的细胞内Ca²⁺变化，并用放射免疫分析法测定100,000g胞质溶胶和膜组分中的CaM。用非特异性Ca²⁺通道阻滞剂Ni²⁺预处理可消除SK-N-SH细胞中通常在卡巴胆碱处理时出现的细胞外Ca²⁺内流。阻断细胞外Ca²⁺内流对卡巴胆碱介导的胞质CaM增加（单独卡巴胆碱处理的对照值的168±18%，而Ni²⁺和卡巴胆碱处理为163±28%）或膜CaM减少没有影响。同样，从培养基中去除细胞外Ca²⁺也不影响卡巴胆碱介导的胞质CaM增加（对照的168±26%）。另一方面，用细胞可渗透的Ca²⁺螯合剂BAPTA/AM预处理以防止卡巴胆碱介导的细胞内游离Ca²⁺增加，确实减弱了卡巴胆碱介导的胞质CaM增加（无BAPTA/AM时为对照的221±37%，有BAPTA/AM时为136±13%）。评估了通过K⁺去极化使细胞外Ca²⁺直接进入细胞的作用。用60 mM K⁺孵育SK-N-SH细胞可引起细胞内游离Ca²⁺浓度立即且持续增加，但CaM定位没有相应改变。相反，在经毒胡萝卜素处理使细胞内Ca²⁺直接升高的细胞中，胞质CaM升高至少30分钟，而颗粒状CaM减少。此外，在无细胞外Ca²⁺的情况下用离子霉素处理，其从细胞内储存释放Ca²⁺，可诱导胞质CaM增加（对照的203±30%）。CaM释放的机制可能涉及蛋白激酶C的α同工酶的激活，毒胡萝卜素比K⁺去极化更能使该同工酶从胞质溶胶向膜的转位更显著。这些数据表明，细胞内储存释放Ca²⁺对于卡巴胆碱介导的人神经母细胞瘤SK-N-SH细胞中CaM的重新分布很重要。
