C60-Fullerene monomalonate adducts selectively inactivate neuronal nitric oxide synthase by uncoupling the formation of reactive oxygen intermediates from nitric oxide production.

C(60)-Fullerene monomalonate adducts inactivate selectively the neuronal nitric oxide synthase isoform in a manner completely preventable by the concurrent presence of superoxide dismutase and catalase. This inactivation is time-, fullerene concentration-, and turnover-dependent and is not reversible by dilution. The di(carboxypropan-3-ol)methano-[60]-fullerene (diol adduct) has no effect on NADPH consumption by nNOS as measured in the absence of arginine substrate, but dramatically increases NADPH consumption in the presence of arginine. This fullerene-enhanced NADPH consumption is linked to oxygen as electron acceptor and is accompanied by the increased production of hydrogen peroxide. These effects of fullerene monomalonate adducts are unique to the nNOS isoform and are not observed using either the iNOS or the eNOS isoform. The inhibitory effects of fullerene monomalonate adducts are unaltered and insurmountable by increased concentrations of arginine, tetrahydrobiopterin, or calmodulin. These observations indicate that fullerene monomalonate adducts uncouple in the presence of arginine the formation of reactive oxygen intermediates from NO production by nNOS. These reactive oxygen intermediates dissociate from the enzyme and, acting from solution, inactivate NOS NO forming activity.

Inhibition of nitric oxide synthase isoforms by tris-malonyl-C(60)-fullerene adducts.

C(3)-tris-malonyl-C(60)-fullerene and D(3)-tris-malonyl-C(60)-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citrulline formation by C(3)-tris-malonyl-C(60)-fullerene occurs with IC(50) values of 24, 17, and 123 microM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 microM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 microM C(3)-tris-malonyl-C(60)-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C(3)-tris-malonyl-C(60)-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC(50) value of 22 microM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 microM. The inhibition of nNOS activity by C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of product formation but did not alter the EC(50) value for activation by calmodulin. C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K(m) for l-arginine substrate or the EC(50) value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C(3)-tris-malonyl-C(60)-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C(3)-tris-malonyl-C(60)-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface.

Cellular and enzymatic studies of N(omega)-propyl-l-arginine and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea as reversible, slowly dissociating inhibitors selective for the neuronal nitric oxide synthase isoform.

N(omega)propyl-l-arginine (NPA) and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea (TFMPITU) inhibit selectively the neuronal nitric oxide (NO) synthase (nNOS) isoform. In the presence of Ca(2+) and calmodulin (CaM), NPA and TFMPITU produce a time- and concentration-dependent suppression of nNOS catalyzed NO formation. This suppression of activity occurs by a first order kinetic process as revealed from linear Kitz-Wilson plots but does not depend on catalytic turnover since it occurs in the absence of NADPH. Following full suppression of NO synthetic activity by either NPA or TFMPITU, NO synthesis can be restored slowly by excess arginine or by dilution, indicating that the effects of these agents are reversible. This behavior is consistent with a dissociation of NPA and TFMPITU from nNOS slowed by a conformational transition produced by Ca(2+) CaM-binding. NPA and TFMPITU bind to nNOS rapidly producing a heme-substrate interaction as revealed by difference spectrophotometry. At physiological conditions (100 microM extracellular arginine), NPA and TFMPITU inhibit Ca(2+)-dependent NO formation by GH(3) pituitary cells with IC(50) values of 19 and 47 microM, respectively, but require millimolar concentrations to inhibit NO formation by cytokine-induced RAW 264.7 murine macrophages. The inhibition of NO formation by these agents in GH(3) cells is rapidly reversible and not due to suppression of cellular arginine uptake.