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

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 Ca2+-influx in GT1-7 neuronal cell line and the Ca2+-influx is regulated by estradiol. In the present study, we examined further the mechanism of Ca2+-influx and the contribution of the two estrogen receptor (ER) isotypes, ERα and ERβ, to estrogenic modulation of intracellular Ca2+-content. GT1-7 neurons were treated with isotype selective ER agonists for 24 h then C5aR agonist evoked Ca2+-responses were measured by Ca2+-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 Ca2+-influx (E2: 215%, PPT: 175%, compared to the PL37-MAP-evoked Ca2+-influx). In contrast, the ERβ selective agonist DPN (100 pM) significantly reduced the Ca2+-influx (32%). Attenuated Ca2+-response (25%) was observed in Ca-free environment and depletion of the Ca2+-pool by CPA eliminated the remaining elevation in the Ca2+-content, demonstrating that the majority of Ca2+ originated from the extracellular compartment. L-type voltage-gated Ca2+-channel (L-VGCC) blocker nifedipine abolished the Ca2+-influx, while R-type Ca2+-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 Ca2+-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 Cav1.3 L-VGCC subunit. Based on these results we propose that estradiol may differentially modulate C5a-induced Ca2+-influx via L-VGCCs in neurons depending on the expression of the two ER isotypes.

► ER agonists differentially modulate the C5aR-mediated Ca2+-influx in GT1-7 neurons.
► Effect of ERα and ERβ agonists on this Ca2+-influx is genomic.
► L-type voltage-gated Ca2+-channels are involved in these processes.
► ER agonists up-regulate expression of the C5aR and differentially regulate C5L2.