Vol. 58, Issue 5, 967-975, November 2000

Structure-Related Inhibition of Calmodulin-Dependent Neuronal Nitric-Oxide Synthase Activity by Melatonin and Synthetic Kynurenines

Josefa León, Manuel Macías, Germaine Escames, Encarna Camacho, Huda Khaldy, Miguel Martín, Antonio Espinosa, Miguel A. Gallo, and Darío Acuña-Castroviejo

Departamento de Fisiología, Instituto de Biotecnología (J.L., Ma.M., G.E., H.K., Mi.M., D.A.-C.) and Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia (E.C., A.E., M.A.G.) Universidad de Granada, Granada, Spain

We recently described that melatonin and some kynurenines modulate the N-methyl-D-aspartate-dependent excitatory response in rat striatal neurons, an effect that could be related to their inhibition of nNOS. In this report, we studied the effect of melatonin and these kynurenines on nNOS activity in both rat striatal homogenate and purified rat brain nNOS. In homogenates of rat striatum, melatonin inhibits nNOS activity, whereas synthetic kynurenines act in a structure-related manner. Kynurenines carrying an NH₂ group in their benzenic ring (NH₂-kynurenines) inhibit nNOS activity more strongly than melatonin itself. However, kynurenines lacking the NH₂ group or with this group blocked do not affect enzyme activity. Kinetic analysis shows that melatonin and NH₂-kynurenines behave as noncompetitive inhibitors of nNOS. Using purified rat brain nNOS, we show that the inhibitory effect of melatonin and NH₂-kynurenines on the enzyme activity diminishes with increasing amounts of calmodulin in the incubation medium. However, changes in other nNOS cofactors such as FAD or H₄-biopterin, do not modify the drugs' response. These data suggest that calmodulin may be involved in the nNOS inhibition by these compounds. Studies with urea-polyacrylamide gel electrophoresis further support an interaction between melatonin and NH₂-kynurenines, but not kynurenines lacking the NH₂ group, with Ca²⁺-calmodulin yielding Ca²⁺-calmodulin-drug complexes that prevent nNOS activation. The results show that calmodulin is a target involved in the intracellular effects of melatonin and some melatonin-related kynurenines that may account, at least in part, for the neuroprotective properties of these compounds.