Abstract
It is established that guanabenz inhibits neuronal nitric-oxide (NO) synthase (nNOS) and causes the enhanced proteasomal degradation of nNOS in vivo. Although the time- and NADPH-dependent inhibition of nNOS has been reported in studies where guanabenz was incubated with crude cytosolic preparations of nNOS, the exact mechanism for inhibition is not known. Moreover, even less is known about how the inhibition of nNOS triggers its proteasomal degradation. In the current study, we show, with the use of purified nNOS, that guanabenz treatment leads to the oxidation of tetrahydrobiopterin and formation of a pterin-depleted nNOS, which is not able to form NO. With the use of 14C-labeled guanabenz, we were unable to detect any guanabenz metabolites or guanabenz-nNOS adducts, indicating that reactive intermediates of guanabenz probably do not play a role in the inhibition. Superoxide dismutase, however, prevents the guanabenz-mediated oxidation of tetrahydrobiopterin and inhibition of nNOS, suggesting the role of superoxide as an intermediate. Studies in rats show that administration of tetrahydrobiopterin prevents the inhibition and loss of penile nNOS due to guanabenz, indicating that the loss of tetrahydrobiopterin plays a major role in the effects of guanabenz in vivo. Our findings are consistent with the destabilization and enhanced degradation of nNOS found after tetrahydrobiopterin depletion. These studies suggest that drug-mediated destabilization and subsequent enhanced degradation of protein targets will likely be an important toxicological consideration.
Footnotes
-
This work was supported in part by the National Institutes of Health Grants GM077430 and ES08365. E.R.L. is a trainee under Pharmacological Sciences Training Program GM07767 from the National Institutes of Health. A.Y.D. is a trainee under Minority Supplement to ES08365.
-
A.Y.D. and G.J.J. contributed equally to this work.
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.106.009951.
-
ABBREVIATIONS: NOS, nitric-oxide synthase; nNOS, neuronal nitric oxide-synthase; BH4, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin); NO, nitric oxide; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography; BH2, 7,8-dihydrobiopterin; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; KI, potassium iodide; CHIP, C terminus of Hsc-70 interacting protein; SOD, superoxide dismutase; CAT, catalase.
-
↵1 Current affiliation: Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
-
↵2 Current affiliation: Department of OB/GYN, Okayama University Medical School, Okayama City, Japan.
-
↵3 Current affiliation: The Dow Chemical Company, Midland, Michigan.
- Received February 22, 2006.
- Accepted May 22, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|