Presence of excess tetrahydrobiopterin during nitric oxide production from inducible nitric oxide synthase in LPS-treated rat aorta

Tetrahydrobiopterin (BH4) is one of the cofactors of nitric oxide synthase (NOS), and the synthesis of BH4 is induced as well as inducible NOS (iNOS) by lipopolysaccharide (LPS) and/or cytokines. BH4 has a protective effect against the cytotoxicity induced by nitric oxide (NO) and/or reactive oxygen species in various types of cells. The purpose of this study was to examine whether or not an excess of BH4 is present during the production of NO by iNOS in LPS-treated de-endothelialized rat aorta. Addition of LPS (10 microg/ml) to the aorta bath solution caused L-arginine (L-Arg)-induced relaxation from 1.5 hr after the addition of LPS in de-endothelialized rat aorta pre-contracted with 30 mM KCl. The L-Arg-induced relaxation was prevented by NOS inhibitors. BH4 content also increased from 3 hr after the addition of LPS. mRNAs of iNOS and GTP cyclohydrolase I (GTPCH), a rate-limiting enzyme of BH4 synthesis, were increased from 1.5 hr after addition of LPS. Although the expression of iNOS and GTPCH mRNAs was observed in the media, the expression levels in the media were much lower than those in the adventitia. Ten millimolar 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTPCH, strongly reduced L-Arg-induced relaxation, and decreased BH4 content to below the basal level in LPS-treated aorta, whereas 0.5 mM DAHP reduced the LPS-induced increase in BH4 content to the basal level but did not affect L-Arg-induced relaxation. The inhibition of L-Arg-induced relaxation by 10 mM DAHP was overcome by the addition of BH4 (10 microM). These results suggest that although BH4 is essential for NO production from iNOS, the increase in BH4 content above the basal level is not needed for eliciting L-Arg-induced relaxation by the treatment with LPS. Thus, an excess amount of BH4 may be synthesized during NO production by iNOS in LPS-treated rat aorta.