Structure-related inhibition of calmodulin-dependent neuronal nitric-oxidesynthase activity by melatonin and synthetic kynurenines

We recently described that melatonin and some kynurenines modulate the N-me thyl-D-aspartate-dependent excitatory response in rat striatal neurons, an effect that could be related to their inhibition of nNOS. In this report, w e studied the effect of melatonin and these kynurenines on nNOS activity in both rat striatal homogenate and purified rat brain nNOS. In homogenates o f rat striatum, melatonin inhibits nNOS activity, whereas synthetic kynuren ines act in a structure-related manner. Kynurenines carrying an NH2 group i n 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 th at melatonin and NH2-kynurenines behave as noncompetitive inhibitors of nNO S. Using purified rat brain nNOS, we show that the inhibitory effect of mel atonin and NH2-kynurenines on the enzyme activity diminishes with increasin g amounts of calmodulin in the incubation medium. However, changes in other nNOS cofactors such as FAD or H-4-biopterin, do not modify the drugs' resp onse. These data suggest that calmodulin may be involved in the nNOS inhibi tion by these compounds. Studies with urea-polyacrylamide gel electrophores is further support an interaction between melatonin and NH2-kynurenines, bu t not kynurenines lacking the NH2 group, with Ca2+-calmodulin yielding Ca2-calmodulin-drug complexes that prevent nNOS activation. The results show t hat calmodulin is a target involved in the intracellular effects of melaton in and some melatonin-related kynurenines that may account, at least in par t, for the neuroprotective properties of these compounds.