Studies on signal transduction mechanisms from nicotinic acetylcholine receptors to nucleus

考虑到NGF/cAMP信号和烟碱型乙酰胆碱受体(NAChR)介导的信号通路之间的相互作用，我们首先研究了NGF和cAMP对PC12细胞、PC12h细胞、表达显性阴性RAS的PC12细胞和非显性PC12细胞(PC12-WARD)nAChR表达的调节。CAMP类似物通过蛋白激酶A下调PC12h和PC12-野生细胞nAChRα3亚单位(α3mRNA)的表达水平。NGF上调PC12h细胞的α3mRNA水平，下调PC12-野生细胞的α3mRNA水平，这种下调是通过RAS-MAP激酶级联途径实现的。高K~+去极化对PC12h细胞α-3m RNA水平无影响。基于这些结果，我们推测至少有两个未知的下游因子MAPK和CREB调节PC12细胞中的α3mRNAs。接下来，我们研究了尼古丁诱导的MAPK和CREB在PC12h细胞中的磷酸化机制。α7nAChR亚基选择性拮抗剂对尼古丁诱导的ERK磷酸化无影响。L类电压敏感型钙通道拮抗剂可抑制尼古丁诱导的ERK磷酸化。这些结果表明，尼古丁诱导的α磷酸化不涉及含有ERK7的nAChRs。钙调素拮抗剂、CaM激酶抑制剂、MAP激酶抑制剂对尼古丁诱导的ERK磷酸化的抑制作用和显性抑制RAS的表达表明，CaM和RAS-MAP激酶级联反应参与了尼古丁诱导的ERK磷酸化。此外，已有研究表明，在PC12h细胞中，尼古丁诱导的CREB磷酸化大部分是由尼古丁诱导的ERK磷酸化所介导的。

Considering the crosstalk between NGF/cAMP signaling and nicotinic acetylcholine receptor (nAChR)-mediating signaling pathways, we first investigated regulation of nAChR expression by NGF and cAMP in three sublines of PC12 cells, PC12h cells, PC12 cells expressing dominant negative Ras and the parential PC12 cells (PC12-wild). cAMP analogue down-regulated level of nAChR α3 subunit mRNA (α3mRNA) in PC12h and PC12-wild cells through protein kinase A.NGF up-regulated the α3mRNA level in PC12h cells and down-regulated the α3mRNA in PC12-wild cells, which is mediated by Ras-MAP kinase cascade. Membrane depolarization with high K^+ had no effect on the α3mRNA level in PC12h cells. Based on these results, we proposed that at least two unknown downstream factors of MAP kinase (ERK) and CREB regulate α3mRNAs in PC12 cells.Next we investigated mechnisms of nicotine-induced MAP kinase (ERK) and CREB phosphorylation in PC12h cells. Nicotine-induced ERK phosphorylation was transient and its level was lower than that of ERK phosphorylation induced by high K^+ depolarization and NGF.α7 nAChR subunit-selective antagonists had no effect on nicotine-induced ERK phosphorylation. L-type voltage-sensitive calcium channel antagonists inhibited nicotine-induced ERK phosphorylation. These results suggest that no α7-containing nAChRs was involved in nicotine-induced ERK phosphorylation. Inhibition of nicotine-induced ERK phosphorylation by a calmodulin antagonist, CaM kinase inhibitor, MAP kinase kinase inhibitor and expression of dominant inhibitory Ras shows that CaM kinase and Ras-MAP kinase cascade are involved in nicotine-induced ERK phosphorylation. Furthermore, it has been suggested that most of nicotine-induced CREB phosphorylation is mediated by nicotine-induced ERK phosphorylation in PC12h cells.