Publications : 2000

Noguchi S, Jianmongkol S Bender AT, Kamada Y, Demady DR, Osawa Y. 2000. Guanabenz-mediated inactivation and enhanced proteolytic degradation of neuronal nitric oxide synthase. J Biol Chem 275(4):2376–2380.


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 andN 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 [35S]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, andN 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, andN-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.

Nitric-oxide synthases (NOS)1 are cytochrome P450-like hemoprotein enzymes that catalyze the conversion ofL-arginine to citrulline and nitric oxide (1-4). Nitric oxide is a signaling molecule that is involved in a variety of physiological processes, including neurotransmission, vasorelaxation, platelet aggregation, and penile erection as well as in a variety of pathological conditions including septic shock, reperfusion injury, arthritis, atherosclerosis, diabetes, and graft rejection (5-8). It has been noted (9) that because nitric oxide is not stored, released, or inactivated after synaptic release by conventional regulatory mechanisms the biosynthetic regulation of the enzyme is of great importance. For the neuronal isoform the Ca2+-mediated activation is of prime importance. However, the factors that regulate proteolytic degradation of the enzyme have not been investigated. One approach that has been successfully utilized for the study of the proteolytic turnover of other P450 cytochromes is the use of suicide inactivators (10-13). We wished to utilize this approach for the study of NOS turnover.

Suicide inactivators are chemically inert molecules that mimic the natural substrate of the enzyme and become metabolized to a highly reactive intermediate that can covalently alter important active site entities, resulting in inactivation of the enzyme (14). In effect, the compound causes the enzyme to catalyze its own demise. Because the compound must not only have affinity for the active site but also must be metabolized in a manner to generate a reactive intermediate that is in close proximity to an important active site entity, these agents have the potential to be highly specific in vivo. In addition, they react with the form of the enzyme that is engaged in catalysis and thus are useful mechanistic probes into the nature of the bioactivation reaction. In some cases, these agents covalently alter P450 cytochromes in a manner that enhances their proteolysis and turnover (10-1315-17).

Guanabenz, a clinically used antihypertensive agent with a guanidino moiety, was recently shown, with the use of brain and penile cytosol, to be a metabolism-based inactivator of neuronal nitric-oxide synthase (nNOS) (18). Moreover, the treatment of rats with guanabenz was found to cause not only a decrease in activity, but also a concomitant loss of immunodetectable nNOS protein. To further understand the molecular mechanisms responsible for the loss of nNOS protein in vivo, we chose to model the effects of guanabenz with the use of HEK 293 cells stably transfected with nNOS. In the current study, we have shown that guanabenz causes the enhanced proteolytic turnover of nNOS. Suicide inactivators of nNOS, such asN G-methyl-L-arginine orN 5-(1-iminoethyl)-L-ornithine, also caused the enhanced proteolytic degradation of the enzyme, whereas the slowly reversible inhibitorN G-nitro-L-arginine or the reversible inhibitor 7-NI did not. Studies with protease inhibitors indicate that the proteasome is responsible, in part, for recognition of the inactivated protein. Thus, the steady state levels of nNOS may be regulated by a selective proteolytic process, which can be studied by the use of metabolism-based inactivators.