Cell proliferation and Ca(2+)-calmodulin dependent protein kinase activation mediated by alpha 1A- and alpha 1B-adrenergic receptor in HEK293 cells.
OBJECTIVE
To examine the ability of alpha 1-AR subtypes on proliferation and Ca(2+)-calmodulin dependent protein kinase (CCDPK, formerly called MAPK) activation in transfected human embryo kidney 293 (HEK293) cells.


METHODS
pREP8/alpha 1A-AR, pREP4/alpha 1B-AR, and pREP9/alpha 1D-AR were transfected, respectively, into HEK293 cells by calcium phosphate precipitation. The expression of alpha 1-AR was detected by radioligand binding assays. DNA synthesis was measured by [3H]thymidine incorporation. CCDPK activity was determined by immunoprecipitation method and myelin basic protein was used as substrate.


RESULTS
Three clonal HEK293 cell lines stably expressing alpha 1A- or alpha 1B- or alpha 1D-AR were chosen and characterized by radioligand binding assay with receptor densities of about 0.6 nmol.g-1. Treatment with norepinephrine (NE) in the presence of propranolol for 24 h increased DNA synthesis in HEK293/alpha 1A- or HEK293/alpha 1B-AR cells concentration-dependently, with EC50 values of 48.8 nmol.L-1 (95% confidence limits 9.7-246 nmol.L-1) and 8.4 nmol.L-1 (95% confidence limits 2.1-32.9 nmol.L-1), respectively. The increase of DNA synthesis induced by NE 10 mumol.L-1 was 201% +/- 28% and 269% +/- 44% of basal, and the activation of CCDPK was 171% +/- 84% and 292% +/- 92% of basal in HEK293/alpha 1A-AR and HEK293/alpha 1B-AR cells, respectively. Preincubation with prazosin completely abolished NE-induced CCDPK activation in HEK293/alpha 1A- and alpha 1B-AR cells. Those changes were not found in HEK293/alpha 1D-AR cells.


CONCLUSIONS
The activation of alpha 1A- or alpha 1B-AR but not alpha 1D-AR induces cell proliferation.
Institute of Vascular Medicine, The Third Hospital, Beijing Medical University, Beijing 100083, China.
B B
BB
Gao
B L
BL
Lei
Y Y
YY
Zhang
Q D
QD
Han
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney / Q000166:cytology*; D009638:Norepinephrine / Q000494:pharmacology*; D018340:Receptors, Adrenergic, alpha-1 / Q000096:biosynthesis*; D014162:Transfection
C547713:ADRA1A protein, human; C547716:ADRA1B protein, human; C547719:ADRA1D protein, human; D000316:Adrenergic alpha-Agonists; D018340:Receptors, Adrenergic, alpha-1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009638:Norepinephrine