Differential inhibition of constitutive and inducible nitric oxide synthase in vascular endothelial cells by analogues of tetrahydrobiopterin

In the vasculature, a physiologic production of nitric oxide (NO) is maintained by endothelial nitric oxide synthase (eNOS). Induction of inducible nitric oxide synthase (iNOS) under inflammatory conditions (e.g. septic shock) resulting in high levels of nitric oxide (NO) is believed to be partly responsible for the pathophysiologic changes in the vascular system that occur under inflammatory conditions (e.g. septic shock). Both NOS isoforms are dependent on the obligatory cofactor tetrahydrobiopterin (BH4). We investigated the selectivity and potency of the BH4 analogues 4-amino-BH4 and 5-methyl-BH4 in inhibiting eNOS and iNOS in a murine vascular endothelial cell (MVEC) model expressing either eNOS or iNOS under physiologic and inflammatory conditions, respectively. Exogenous BH4 and its precursor sepiapterin both enhanced physiologic eNOS activity in resting MVEC, while 4-amino-BH4 slightly inhibited eNOS. 5-methyl-BH4 did not have any effect on eNOS. BH4, sepiapterin, and 5-methyl-BH4 had no effect on iNOS in inflammatory activated MVEC. In contrast, 4-amino-BH4 selectively inhibited iNOS with a potency comparable to the unselective NOS inhibitor Nomega-monomethyl-L-arginine (L-NMMA). The present study demonstrates that 4-amino-BH4 selectively and potently inhibits iNOS in vascular endothelial cells, while its effect on eNOS is minimal. The selective inhibition of iNOS is a promising strategy for the treatment of inflammatory conditions with high output of NO. Further in vivo studies are required to determine whether inhibition of NO production by analogues of BH4 offers any advantage compared to inhibition by L-arginine analogues.