Ca2+ participates in alpha1B-adrenoceptor-mediated cAMP response in HEK293 cells.
OBJECTIVE
To investigate the alpha1B-adrenoceptor (alpha1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells.


METHODS
Full-length cDNA encoding alpha1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and alpha1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively.


RESULTS
Under agonist stimulation, alpha1B-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.


CONCLUSIONS
By coupling with a PTX-insensitive G protein, alpha1BAR 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.
Song
Yao
Y
Institute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing 100083, China.
Li
Yun-fang
YF
Dong
Er-dan
ED
Han
Qi-de
QD
Zhang
You-yi
YY
D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology*; D009638:Norepinephrine / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
C547716:ADRA1B protein, human; D002147:Calmodulin; D018076:DNA, Complementary; D018340:Receptors, Adrenergic, alpha-1; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D002118:Calcium; D009638:Norepinephrine