Muscarinic receptor regulation of cytoplasmic Ca2+ concentrations in human SK-N-SH neuroblastoma cells: Ca2+ requirements for phospholipase C activation.
Muscarinic receptor regulation of cytoplasmic Ca2+ concentrations in human SK-N-SH neuroblastoma cells: Ca2+ requirements for phospholipase C activation.
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发表时间：
1989-02
影响因子：
3.6
通讯作者：
Stephen K. Fisher;L. M. Domask;R. M. Roland
中科院分区：
文献类型：
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作者：
Stephen K. Fisher;L. M. Domask;R. M. Roland
在人SK-N-SH神经母细胞瘤细胞中，研究了毒蕈碱受体介导的肌醇脂质水解与Ca ~（2+）信号产生之间的关系。通过fura-2荧光测量测定的静息细胞质钙浓度[（Ca 2 +]i）为59 +/- 2 nM。在添加oxotremorine-M后，[Ca 2 +]i（293 +/- 18 nM）增加4倍，在8 μ M的激动剂浓度下获得半最大刺激，该值与先前观察到的磷酸肌醇水解增强的值相似。此外，部分毒蕈碱激动剂磷酸肌醇营业额（氨甲酰胆碱，氧-2，槟榔碱）引起相应的增加较小的[Ca 2 +]i比oxotremorine-M。EGTA的加入在2分钟内降低了基础[Ca 2 +]i，并显著降低（大于60%）激动剂诱导的[Ca 2 +]i升高幅度。将毒蕈碱激动剂添加到已经用[3 H]肌醇预标记的SK-N-SH细胞中导致肌醇单磷酸、双磷酸和三磷酸的快速（5-15秒）释放。当在与用于荧光测量的条件类似的条件下测定时，EGTA还抑制肌醇磷酸的基础和氧化震颤素-M刺激释放45- 61%。相反，离子霉素都升高[Ca 2 +]i和刺激肌醇磷酸的释放。向毛地黄皂苷透化细胞中加入Ca 2+（10 nM-2 μ M）可直接刺激标记的肌醇单磷酸、双磷酸和三磷酸的释放3-4倍，在145 nM游离Ca 2+（Ca 2 +f）下观察到半数最大效应（EC 50）。通过包含鸟苷-5-O-（3-硫代）-三磷酸（GTP γ S）或氧代震颤素-M，获得磷酸肌醇释放的进一步（6倍）钙依赖性增加。在激动剂和GTP γ S的组合存在下，发生所有三种肌醇磷酸的协同释放，在35-40 nM Ca 2 +f下观察到半最大刺激，该值类似于静止细胞中的[Ca 2 +]i。这些结果表明：（i）[Ca 2 +]i初始升高的幅度与磷酸肌醇衍生的第二信使分子的产生直接相关;（ii）磷脂酶C介导的SK-N-SH细胞中肌醇脂质的分解对生理相关Ca 2+浓度的调节特别敏感。它的结论是，在SK-N-SH细胞，无论是高于或低于基础[Ca 2 +]i的升高可以调节肌醇脂质的水解程度和随后产生的钙信号。
The relationship between muscarinic receptor-mediated inositol lipid hydrolysis and the generation of Ca2+ signals has been examined in human SK-N-SH neuroblastoma cells. The resting cytoplasmic calcium concentration [( Ca2+]i) as determined by fura-2 fluorescence measurements was 59 +/- 2 nM. Upon the addition of oxotremorine-M, there was a 4-fold increase in [Ca2+]i (293 +/- 18 nM), with half-maximal stimulation obtained at an agonist concentration of 8 microM, a value similar to that previously observed for the enhancement of phosphoinositide hydrolysis. Addition of partial muscarinic agonists for phosphoinositide turnover (bethanechol, oxo-2, and arecoline) elicited correspondingly smaller increases in [Ca2+]i than did oxotremorine-M. Inclusion of EGTA lowered the basal [Ca2+]i within 2 min and markedly reduced (greater than 60%) the magnitude of the agonist-induced rise in [Ca2+]i. Addition of muscarinic agonists to SK-N-SH cells that had been prelabeled with [3H]inositol led to the rapid (5-15 sec) release of inositol mono-, bis-, and triphosphates. When assayed under conditions similar to those employed for the fluorescence measurements, EGTA also inhibited both the basal and oxotremorine-M-stimulated release of inositol phosphates by 45-61%. Conversely, ionomycin both elevated [Ca2+]i and stimulated the release of inositol phosphates. The addition of Ca2+ (10 nM-2 microM) to digitonin-permeabilized cells directly stimulated the release of labeled inositol mono-, bis-, and trisphosphates by 3-4-fold with a half-maximal effect (EC50) observed at 145 nM free Ca2+ (Ca2+f). A further (6-fold) calcium-dependent increase in inositol phosphate release was obtained by inclusion of either guanosine-5-O-(3-thio)-trisphosphate (GTP gamma S) or oxotremorine-M. In the combined presence of agonist and GTP gamma S, a synergistic release of all three inositol phosphates occurred, with half-maximal stimulation observed at 35-40 nM Ca2+f, a value similar to the [Ca2+]i in quiescent cells. These results indicate (i) that the magnitude of the initial rise in [Ca2+]i is directly related to the production of phosphoinositide-derived second messenger molecules and (ii) that the phospholipase C-mediated breakdown of inositol lipids in SK-N-SH cells is particularly sensitive to regulation by physiologically relevant Ca2+ concentrations. It is concluded that, in SK-N-SH cells, either an elevation above or reduction below basal [Ca2+]i can modulate the extent of hydrolysis of inositol lipids and the subsequent generation of calcium signals.