Vol. 292, Issue 1, 387-393, January 2000

Endogenously Generated Nitric Oxide by Nitric-Oxide Synthase Gene Transfer Inhibits Cellular Proliferation¹

Yoshikazu Maeda, Uichi Ikeda, Ken-ichi Oya, Masahisa Shimpo, Shuichi Ueno, Koji Okada, Toshikazu Saito, Hiroyuki Mano, Keiya Ozawa and Kazuyuki Shimada

Departments of Cardiology (Y.M., U.I., Ken.O., M.S., S.U., K.S.), Endocrinology and Metabolism (Ko.O., T.S.), and Molecular Biology (H.M., Kei.O.), Institute of Hematology, Jichi Medical School, Tochigi, Japan.

We investigated whether endothelial nitrite oxide synthase (NOS) gene transfer inhibited cellular proliferation. Endothelial NOS and endothelin type A receptor genes were transferred into 293 cells, a human embryonic kidney cell line, by calcium-phosphate coprecipitation. The cytosolic free Ca²⁺ levels ([Ca²⁺]_i) of transfected cells were estimated with fura-2 fluorescence. Thymidine incorporation was increased by endothelin-1 in type A receptor-transfected cells. The endothelial NOS gene transfer did not affect endothelin-1-induced increase in [Ca²⁺]_i of type A receptor-transfected cells, but markedly inhibited mitogen-activated protein kinase and c-fos promoter activities. The endothelial NOS gene transfer also inhibited thymidine incorporation into type A receptor-transfected cells in response to endothelin-1, which was abolished in the presence of the NOS inhibitor N^G-monomethyl-L-arginine acetate. The endothelin-1-induced increase in cell number was significantly suppressed by endothelial NOS gene transfer as well as by the mitogen-activated protein kinase inhibitor PD98059. These results indicate that endothelial NOS gene transfer inhibits cellular proliferation via inhibition of the mitogen-activated protein kinase cascade.