Aim

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.

Conclusion

The activation of alpha 1A-or alpha 1B-AR but not alpha 1D-AR induces cell proliferation.