Laboratory of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
Neurochem Int. 2012 May;60(6):631-9. doi: 10.1016/j.neuint.2012.02.024. Epub 2012 Mar 2.
Complement C5a is associated primarily with inflammation. The widespread expression of its receptors, C5aR and C5L2 in neuronal cells, however, suggests additional regulatory roles for C5a in the CNS. C5aR agonist (PL37-MAP) evokes Ca(2+)-influx in GT1-7 neuronal cell line and the Ca(2+)-influx is regulated by estradiol. In the present study, we examined further the mechanism of Ca(2+)-influx and the contribution of the two estrogen receptor (ER) isotypes, ERα and ERβ, to estrogenic modulation of intracellular Ca(2+)-content. GT1-7 neurons were treated with isotype selective ER agonists for 24h then C5aR agonist evoked Ca(2+)-responses were measured by Ca(2+)-imaging. Transcriptional changes were followed by real-time PCR. We found that not only estradiol (100 pM), but the ERα selective agonist PPT (100 pM) enhanced the PL37-MAP-evoked Ca(2+)-influx (E2: 215%, PPT: 175%, compared to the PL37-MAP-evoked Ca(2+)-influx). In contrast, the ERβ selective agonist DPN (100 pM) significantly reduced the Ca(2+)-influx (32%). Attenuated Ca(2+)-response (25%) was observed in Ca-free environment and depletion of the Ca(2+)-pool by CPA eliminated the remaining elevation in the Ca(2+)-content, demonstrating that the majority of Ca(2+) originated from the extracellular compartment. L-type voltage-gated Ca(2+)-channel (L-VGCC) blocker nifedipine abolished the Ca(2+)-influx, while R-type Ca(2+)-channel blocker SNX-482 had no effect, exemplifying the predominant role of L-VGCC in this process. Acute pre-treatments (8 min) with ER agonists did not affect the evoked Ca(2+)-influx, revealing that the observed effects of estrogens were genomic. Therefore, we checked estrogenic regulation of C5a receptors and L-VGCC subunits. ER agonists increased C5aR mRNA expression, whereas they differentially regulated C5L2. Estradiol decreased transcription of Ca(v)1.3 L-VGCC subunit. Based on these results we propose that estradiol may differentially modulate C5a-induced Ca(2+)-influx via L-VGCCs in neurons depending on the expression of the two ER isotypes.
补体 C5a 主要与炎症有关。然而，其受体 C5aR 和 C5L2 在神经元细胞中的广泛表达表明 C5a 在中枢神经系统中具有额外的调节作用。C5aR 激动剂 (PL37-MAP) 可在 GT1-7 神经元细胞系中引发 Ca(2+)内流，而 Ca(2+)内流受雌二醇调节。在本研究中，我们进一步研究了 Ca(2+)内流的机制以及两种雌激素受体 (ER) 同种型 ERα 和 ERβ 对细胞内 Ca(2+)含量的雌激素调节的贡献。用同种型选择性 ER 激动剂处理 GT1-7 神经元 24 小时后，通过 Ca(2+)成像测量 C5aR 激动剂引起的 Ca(2+)反应。通过实时 PCR 跟踪转录变化。我们发现，不仅雌二醇 (100 pM)，而且 ERα 选择性激动剂 PPT(100 pM)增强了 PL37-MAP 诱导的 Ca(2+)内流 (E2：215%，PPT：175%，与 PL37-MAP 诱导的 Ca(2+)内流相比)。相比之下，ERβ 选择性激动剂 DPN(100 pM) 显着降低了 Ca(2+)内流 (32%)。在无钙环境中观察到 Ca(2+)反应减弱 (25%)，而 CPA 耗尽 Ca(2+)池消除了 Ca(2+)含量的剩余升高，表明大部分 Ca(2+)来自细胞外区室。L 型电压门控 Ca(2+)通道 (L-VGCC) 阻滞剂硝苯地平消除了 Ca(2+)内流，而 R 型 Ca(2+)通道阻滞剂 SNX-482 没有作用，证明 L-VGCC 在该过程中起主要作用。ER 激动剂的急性预处理 (8 分钟) 不会影响诱发的 Ca(2+)内流，这表明雌激素的观察到的作用是基因组的。因此，我们检查了雌激素对 C5a 受体和 L-VGCC 亚基的调节。ER 激动剂增加了 C5aR mRNA 的表达，而它们对 C5L2 则有不同的调节作用。雌二醇降低了 Ca(v)1.3 L-VGCC 亚基的转录。基于这些结果，我们提出雌二醇可能通过神经元中的 L-VGCC 以依赖于两种 ER 同种型表达的方式对 C5a 诱导的 Ca(2+)内流进行差异调节。
