Determining relaxin-3/RXFP3 activation by inhibition of forskolin induced cAMP assay

摘要

OBJECTIVE Relaxin-3 is a novel neuropeptide of the relaxin insulin family of peptides.So far,many studies have reported the role of relaxin-3/RXFP3 system in regulating feeding,stress response and cognition.In previous studies using RXFP3 stable cell lines,inhibition of adenylate cyclase by the activation of Gi/o proteins have been reported.Based on this signaling event,we have developed inhibition of forskolin induced cAMP assay to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS To detect inhibition of adenylate cyclase up on RXFP3 activation by human relaxin-3(H3relaxin),HEK-RXFP3,CHO-RXFP3,SN56 and GT1-7cells were plated in poly-L-lysine coated 24 well plates.The following day,the cells were starved in serum free cell culture media for 6h.Next,cells were treated in triplicate with serum free media(control)and H3 relaxin for 5-15 min.Then,the cells were treated with serum free media with DMSO(control)or forskolin(5-100μmol·L-1)for 15 min at 37℃ with 5%CO2.At the end of the incubation,cell culture media was discarded and the cells were lysed with 0.1mol·L-1 HCl.The cAMP concentration in each lysate was detected by ELISA(Cayman Chemicals).The data from three experiments were analysed using one way ANOVA followed by Bonferroni post hoc test or Dunnett′s post hoc test.RESULTS In CHO-RXFP3 and HEK-RXFP3 cells,10nmol·L-1 of H3 relaxin was able to significantly inhibit the forskolin(5μmol·L-1)induced cAMP levels(P<0.05).In SN56 neuronal like cell line endogenously expressing RXFP3,100nmol·L-1 H3 relaxin was able to significantly reduce forskolin(3μmol·L-1)induced cAMP(P<0.05).However,in wild type HEK293 Tand CHO-K1 cells,10n mol·L-1 H3 relaxin was not able to significantly reduce the forskolin 22(5μmol·L-1)induced cAMP levels.In GT1-7 mouse hypothalamic cells endogenously expressing RXFP3,100nmol·L-1 H3 relaxin and 5or 3μmol·L-1 forskolin,was able toa significantly increase cAMP levels(P<0.05).CONCLUSION Inhibition of forskolin induced cAMP assay can be used to detect Gi/o mediated cAMP inhibition related signaling events due to RXFP3 activation by its agonists in CHO-RXFP3,HEK-RXFP3 and SN56 cell lines.