Bromocriptine is a strong inhibitor of brain nitric oxide synthase: possible consequences for the origin of its therapeutic effects.

The ergot alkaloid bromocriptine (BKT) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (NOS) (IC50 = 10 +/- 2 microM) whereas it was poorly active towards inducible macrophage NOS (IC50 > 100 microM). BKT affects the activation of NOS by calmodulin, as it not only inhibits L-arginine oxidation to NO and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by neuronal NOS. These results suggest that BKT could exert some of its therapeutic effects by interfering with the NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

Strong inhibition of neuronal nitric oxide synthase by the calmodulin antagonist and anti-estrogen drug tamoxifen.

The anti-estrogen drug tamoxifen (TMX) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (nNOS) (IC50 = 2 +/- 0.5 microM), whereas it was inactive toward inducible macrophage NOS (IC50 > 100 microM). TMX affected the activation of NOS by calmodulin, as it not only inhibited L-arginine oxidation to nitric oxide and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by nNOS. These results suggest that TMX could exert some of its biological effects by interfering with constitutive NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

Formation of nitric oxide synthase-iron(II) nitrosoalkane complexes: severe restriction of access to the iron(II) site in the presence of tetrahydrobiopterin.

Nitric oxide synthases (NOS) are heme proteins, closely related to cytochromes P450, that catalyze oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline. To get further insight into their active site, we have studied the ability of recombinant mouse inducible NOS (iNOS) and rat brain neuronal NOS (nNOS), and of their oxygenase domains (iNOSoxy and nNOSoxy), to form Fe(II)-nitrosoalkane complexes. In the absence of BH4, iNOSoxy, nNOSoxy, and full-length iNOS readily form complexes characterized by Soret peaks around 448 nm, after reaction with various nitroalkanes and sodium dithionite. These complexes displayed physicochemical characteristics very similar to those of previously reported microsomal cytochrome P450-Fe(II)-nitrosoalkane complexes: (i) a Soret peak around 450 nm, (ii) a clear stability in the presence of CO, and (iii) a fast destruction upon oxidation of the iron by ferricyanide. Thus, in the absence of l-Arg and BH4, NOSs Fe(II) appear to be largely opened to even large R-NO ligands with R = cyclohexyl or p-Cl-C6H4-CH2CH(CH3) for instance, in a manner similar to microsomal P450s Fe(II). As expected, the presence of l-Arg inhibits the formation of NOSs Fe(II)-RNO complexes. More surprisingly, the presence of BH4 also strongly inhibits the formation of the NOSs Fe(II) complexes even with the smallest nitrosoalkane ligand, CH3NO (IC50 values of 0.5 and 4 microM for nNOSoxy and iNOSoxy, respectively). Accordingly, recombinant full-length nNOS containing BH4 and l-Arg is completely unable to form Fe(II)-nitrosoalkane complexes, even with CH3NO. These results suggest that, in the absence of l-Arg and BH4, the distal pocket of NOSs Fe(II) is largely opened even to bulky ligands, in a manner similar to that of microsomal cytochromes P450. On the contrary, the distal heme pocket of iNOS and nNOS seems to be closed after binding of l-Arg and BH4, particularly in the Fe(II) state. This results in a highly restricted access for Fe(II) ligands, except very small ones such as CO, NO, and O2. Such effects of BH4 in controlling the size of the distal heme pocket of NOS Fe(II) correspond to a new role of biopterins in biological systems.