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

Guanabenz, a metabolism-based irreversible inactivator of neuronal nitric-oxide synthase (nNOS) in vitro, causes the loss of immunodetectable nNOS in vivo. This process is selective in that the slowly reversible inhibitor N(G)-nitro-L-arginine did not decrease the levels of nNOS in vivo. To better understand the mechanism for the loss of nNOS protein in vivo, we have investigated the effects of guanabenz and N(G)-nitro-L-arginine in HEK 293 cells stably transfected with the enzyme. We show here that guanabenz, but not N(G)-nitro-L-arginine, caused the inactivation and loss of nNOS protein in the HEK 293 cells. In studies with cycloheximide or in pulse-chase experiments with [(35)S]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 inactivators 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 decrease in the half-life of nNOS. Proteasomal inhibitors, lactacystin, Cbz-leucine-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 we have shown for the first time that the irreversible inactivators of nNOS, perhaps through covalent alteration of the enzyme, enhance the proteolytic turnover of the enzyme by a mechanism involving the proteasome.