Estrogen receptor α and β differentially mediate C5aR agonist evoked Ca2+-influx in neurons through L-type voltage-gated Ca2+ channels

Abstract

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.