Quik M, Philie J, Choremis J
Department of Pharmacology, McGill University, Montreal, Quebec, Canada.
Mol Pharmacol. 1997 Mar;51(3):499-506.
Our previous work had demonstrated stable expression of rat alpha7 alpha-bungarotoxin (alpha-BGT) binding sites by GH4C1 rat pituitary cells, a clonal line that does not endogenously express nicotinic receptors. The stably expressed alpha7 sites had similar binding affinities, pharmacological profiles, kinetic properties, and molecular size as rat brain alpha-BGT receptors, suggesting that they represent a good model system for studying receptor function. The present data show that nicotinic receptor agonists increase intracellular calcium levels ([Ca2+]i), as assessed using Fura-2, in alpha7/GH4C1 cells in a dose-dependent manner with EC50 values that correlate well with the affinity of these ligands for alpha7/GH4C, alpha-BGT receptors. Nicotinic receptor antagonists inhibited agonist-induced increases in [Ca2+]i, with IC50 values in the nanomolar to micromolar range. The nicotinic agonist-induced increase in [Ca2+]i required extracellular calcium and did not occur in the presence of CdCl2, suggesting that agonist-induced increases in [Ca2+]i are due to an influx of extracellular calcium through voltage-gated calcium channels. Preexposure of the alpha7/GH4C1 cells to 8-bromo cAMP resulted in an enhanced [Ca2+]i in response to agonist, suggesting that phosphorylation by adenylate cyclase may regulate receptor responsiveness. Interestingly, short-term preexposure (40-60 sec) of the cells to subthreshold concentrations of nicotinic agonist-enhanced receptor-stimulated calcium influx (up to 55%) while activating agonist concentrations completely blocked receptor-mediated responses. Long-term exposure of alpha7/GH4C1 cells to K+ resulted in about a 2-fold increase in alpha-BGT receptors and in agonist-evoked calcium influx. The sensitivity of these up-regulated receptors were modulated by subthreshold and activating concentrations of agonist in a manner similar to control receptors. The present results, demonstrating a biphasic regulation of alpha7 receptor-mediated calcium influx by nicotinic agonists, suggest that these receptors may play an important role in neuronal function under control and depolarizing conditions.
我们之前的研究表明，GH4C1大鼠垂体细胞能稳定表达大鼠α7α-银环蛇毒素（α-BGT）结合位点，该细胞系不内源性表达烟碱受体。稳定表达的α7位点与大鼠脑α-BGT受体具有相似的结合亲和力、药理学特征、动力学特性和分子大小，这表明它们是研究受体功能的良好模型系统。目前的数据表明，烟碱受体激动剂能以剂量依赖的方式增加α7/GH4C1细胞内的钙水平（[Ca2+]i），使用Fura-2评估时，其EC50值与这些配体对α7/GH4C、α-BGT受体的亲和力密切相关。烟碱受体拮抗剂能抑制激动剂诱导的[Ca2+]i增加，IC50值在纳摩尔到微摩尔范围内。烟碱激动剂诱导的[Ca2+]i增加需要细胞外钙，且在CdCl2存在时不发生，这表明激动剂诱导的[Ca2+]i增加是由于细胞外钙通过电压门控钙通道内流所致。将α7/GH4C1细胞预先暴露于8-溴环磷酸腺苷（8-bromo cAMP）会导致其对激动剂的反应中[Ca2+]i增强，这表明腺苷酸环化酶的磷酸化可能调节受体的反应性。有趣的是，将细胞短期预先暴露（40 - 60秒）于亚阈值浓度的烟碱激动剂会增强受体刺激的钙内流（高达55%），而激活激动剂浓度则完全阻断受体介导的反应。将α-7/GH4C1细胞长期暴露于钾离子会导致α-BGT受体增加约2倍，并使激动剂诱发的钙内流增加。这些上调受体的敏感性受到亚阈值和激活浓度激动剂的调节，其方式与对照受体相似。目前的结果表明烟碱激动剂对α7受体介导的钙内流具有双相调节作用，这表明这些受体可能在正常和去极化条件下的神经元功能中发挥重要作用。
