Acta Pharmacologica Sinica 2005 January; 26 (1): 77-84; doi: 10.1111/j.1745-7254.2005.00018.x

Aim: To investigate the α_1B-adrenoceptor (α_1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells.

Methods Full-length cDNA encoding α_1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and α_1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromato-graphy, respectively.

Results: Under agonist stimulation, α_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 norepinephrine (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, α_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.