Guanabenz-mediated inactivation and enhanced proteolytic degradation of neuronal nitric-oxide synthase

Guanabenz, a metabolism-based irreversible inactivator of neuronal nitric-o xide synthase (nNOS) in vitro, causes the loss of immunodetectable nNOS in vivo. This process is selective in that the slowly reversible inhibitor NG- nitro-L-arginine did not decrease the levels of nNOS in vivo. To better und erstand the mechanism for the loss of nNOS protein in vivo we have investig ated the effects of guanabenz and N-G-nitro-L-arginine in HEB: 293 cells st ably transfected with the enzyme. me show here that guanabenz, but not N-G- nitro-L-arginine, caused the inactivation and loss of nNOS protein in the H ER 293 cells. in studies with cycloheximide or in pulse-chase experiments w ith [S-35]methionine, we demonstrate that the loss of nNOS was due in large part to enhanced proteolysis of the protein with the half-life decreasing by one-half from 20 to 10 h. Other metabolism-based irreversible inactivato rs to nNOS, N-G-methyl-L-arginine, and N-5-(1-iminoethyl)-L-ornithine, but not the reversible inhibitor 7-nitroindazole (7-NI), caused a similar decre ase in the half-life of nNOS, Proteasomal inhibitors, lactacystin, Cbz-leuc ine-leucine-leucinal, and N-acetyl-leucine-leucine-norleucinal, but not the lysosomal protease inhibitor leupeptin, were found to effectively inhibit the proteolytic degradation of nNOS, Thus Fee have shown for the first time that the irreversible inactivators of nNOS, perhaps through covalent alter ation of the enzyme, enhance the proteolytic turnover of the enzyme by a me chanism involving the proteasome.