Cellular and Enzymatic Studies of Nω-Propyl-[formula omitted]-Arginine and S-Ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea as Reversible, Slowly Dissociating Inhibitors Selective for the Neuronal Nitric Oxide Synthase Isoform

Abstract

Nωpropyl-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 Ca2+ 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 Ca2+ CaM-binding. NPA and TFMPITU bind to nNOS rapidly producing a heme–substrate interaction as revealed by difference spectrophotometry. At physiological conditions (100 μM extracellular arginine), NPA and TFMPITU inhibit Ca2+-dependent NO formation by GH3 pituitary cells with IC50 values of 19 and 47 μM, 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 GH3 cells is rapidly reversible and not due to suppression of cellular arginine uptake.