Ca2+ participates in α1B-adrenoceptor-mediated cAMP response in HEK293 cells

Aim: To investigate the alpha(1B)-adrenoceptor (alpha(1B)-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. Methods: Full-length cDNA encoding alpha(1B)-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and alpha(1B)-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. Results: Under agonist stimulation, alpha(1B)-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin (PTX) had little effect on basal cAMP accumulation as well as norepirephrine (NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin (CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdCl2 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59% +/- 0.47% to 1.00% +/- 0.31%, 0.78% +/- 0.23%, and 0.90% +/- 0.40%, respectively. Conclusion: By coupling with a PTX-insensitive G protein, alpha(1B)-AR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC), thereby increasing the cAMP production in HEK293 cell lines.