847 by different doses of glutamate suggests two mechanisms with opposite effects: 1) a time-dependent negative feedback induced by physiological concentrations of glutamate that limits nNOS activation and precludes the overproduction of NO; and 2) a pathological stimulation by high concentrations of glutamate that leads to unregulated nNOS activation and production of toxic levels of NO. These mechanisms may share pathways, respectively, with NMDA receptor-induced forms of synaptic plasticity and excitotoxicity. nNOS 1 is an enzyme expressed in brain which catalyzes the conversion of arginine to citrulline and NO (1-4), the latter a novel diffusible second messenger with multiple physiologic and pathologic effects (3, 5-7). One regulator of nNOS is the NMDAR, a tetrameric cation channel consisting of NR1 and NR2 subunits which is targeted to excitatory synapses where it functions in neural plasticity (8). Stimulation of the NMDAR by glutamate and glycine induces the influx of Ca 2ϩ through the receptor pore, thereby activating Ca 2ϩ -dependent NMDAR functions (9 -12). PSD95, a scaffolding protein, binds both the NMDAR and nNOS at excitatory synapses and assembles them into a macromolecular signaling complex in which nNOS is under NMDAR control (13-19). Suppression of PSD95 expression blocks NMDAR and Ca 2ϩ -dependent nNOS activation, and uncoupling of the NMDAR from PSD95 suppresses NMDAR signaling (14,20).Transient elevations in intracellular [Ca 2ϩ ] following NMDAR activation stimulate nNOS by promoting the binding of Ca 2ϩ -calmodulin (Ca 2ϩ -CaM). In addition, it has been shown that the activity of nNOS undergoes complex regulation by phosphorylation (21-23). Of particular note, the protein kinase CaMKII phosphorylates recombinant nNOS at Ser 847 , which reduces nNOS activity by inhibiting the binding of Ca 2ϩ -CaM (23, 24). However, the NMDAR-induced mechanism of regulation of nNOS by phosphorylation at specific residues remains largely unknown.We have shown previously that mutations at the apex of the pore of the NMDAR NR1 subunit which block Ca 2ϩ entry through the channel reduce NMDAR-dependent excitotoxicity in heterologous cells and neurons (25). Because Ca 2ϩ -dependent activation of nNOS by the NMDAR has been linked to NMDAR excitotoxic effects (6, 7, 20, 26 -30), we have also analyzed the NMDAR-mediated mechanism of modulation of phosphorylation of nNOS. We have shown that after excitotoxic activation of the NMDAR in cultured primary cortical neurons, nNOS undergoes an overall dephosphorylation by a pathway dependent on the phosphatases calcineurin and PP1/PP2A (30).It is well established that the effectiveness of synapses and even the viability of the neuron can be altered by NMDAR-dependent activity that can be achieved by various patterns of stimulation. Here, we analyze the effects of NMDAR activation on the level of phosphorylation of nNOS at Ser 847 , a modification implicated in the regulation of nNOS. We show that the NMDAR induces a novel bidirectional control of phosphorylation of nNOS...

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