Brock Martina, Nickel Ann-Christin, Madziar Beata, Blusztajn Jan Krzysztof, Berse Brygida
Department of Pathology and Laboratory Medicine, Boston University School of Medicine, 715 Albany Street, Room L-808C, Boston, MA 02118, USA.
Brain Res. 2007 May 11;1145:1-10. doi: 10.1016/j.brainres.2007.01.119. Epub 2007 Feb 2.
Synthesis, storage and release of acetylcholine (ACh) require the expression of several specialized enzymes, including choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT). Extracellular factors that regulate CHT expression and their signaling pathways remain poorly characterized. Using the NSC-19 cholinergic cell line, derived from embryonic spinal cord, we compared the effects of the second messenger cAMP on the expression of CHT and the cholinergic locus containing the ChAT and VAChT genes. Treatment of NSC-19 cells with dbcAMP and forskolin, thus increasing intracellular cAMP levels, significantly reduced CHT mRNA expression, while it upregulated ChAT/VAChT mRNA levels and ChAT activity. The cAMP-induced CHT downregulation was independent of PKA activity, as shown in treatments with the PKA inhibitor H-89. The alternative Epac-Rap pathway, when stimulated by a specific Epac activator, led to significant downregulation of CHT and ChAT, and, to a lesser extent, VAChT. In contrast, the PKA activator 6-BNZ-cAMP stimulated the expression of all three genes, but with varying concentration-dependence profiles. Our results indicate that elevations of intraneuronal cAMP concentration have differential effects on the cholinergic phenotype, depending on the involvement of different downstream effectors. Interestingly, although CHT is expressed predominantly in cholinergic cells, its regulation appears to be distinct from that of the cholinergic locus.
乙酰胆碱（ACh）的合成、储存和释放需要多种特殊酶的表达，包括胆碱乙酰转移酶（ChAT）、囊泡乙酰胆碱转运体（VAChT）和高亲和力胆碱转运体（CHT）。调节CHT表达的细胞外因子及其信号通路仍未得到充分表征。利用源自胚胎脊髓的NSC-19胆碱能细胞系，我们比较了第二信使cAMP对CHT表达以及包含ChAT和VAChT基因的胆碱能基因座表达的影响。用二丁酰环磷腺苷（dbcAMP）和福斯可林处理NSC-19细胞，从而提高细胞内cAMP水平，显著降低CHT mRNA表达，同时上调ChAT/VAChT mRNA水平和ChAT活性。如用PKA抑制剂H-89处理所示，cAMP诱导的CHT下调与PKA活性无关。当由特定的Epac激活剂刺激时，替代的Epac-Rap途径导致CHT和ChAT显著下调，对VAChT的下调程度较小。相比之下，PKA激活剂6-苄基-cAMP刺激所有三个基因的表达，但具有不同的浓度依赖性特征。我们的结果表明，神经元内cAMP浓度的升高对胆碱能表型有不同的影响，这取决于不同下游效应器的参与情况。有趣的是，尽管CHT主要在胆碱能细胞中表达，但其调节似乎与胆碱能基因座的调节不同。
