Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca 2ϩ fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 M) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 M glutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca 2ϩ -calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for the in vivo control of nNOS.

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...

Glucose transporter 3 (GLUT3) is the main facilitative glucose transporter in neurons. Glucose provides neurons with a critical energy source for neuronal activity. However, the mechanism by which neuronal activity controls glucose influx via GLUT3 is unknown. We investigated the influence of synaptic stimulation on GLUT3 surface expression and glucose import in primary cultured cortical and hippocampal neurons. Synaptic activity increased surface expression of GLUT3 leading to an elevation of intracellular glucose. The effect was blocked by N-Methyl-D-Aspartate receptor (NMDAR) and neuronal nitric oxide synthase (nNOS) inhibition. The Akt Inhibitor, Akt-I blocked NMDAR-induced GLUT3 surface expression while a nNOS-phosphomimetic mutant (S1412D) enhanced GLUT3 expression at cell surface. These results suggest that NMDAR/Akt-dependent nNOS phosphorylation is coupled to GLUT3 trafficking. We demonstrated that activation of cGMP-dependent protein kinase (cGK) increased the surface expression of GLUT3, which was repressed by Rp-8-pCPT-cGMPS, a potent cell permeable inhibitor of cGKs. These studies characterize the molecular basis for activity dependent increases in surface GLUT3 after stimulation of the NMDARs. NMDAR-induced increase in surface GLUT3 represents a novel pathway for control of energy supply during neuronal activity that is critical for maintaining glucose homeostasis during neuronal transmission.

Nitric oxide (NO) generated by neuronal NO synthase (nNOS) initiates penile erection, but has not been thought to participate in the sustained erection required for normal sexual performance. We now show that cAMP-dependent phosphorylation of nNOS mediates erectile physiology, including sustained erection. nNOS is phosphorylated by cAMP-dependent protein kinase (PKA) at serine(S)1412. Electrical stimulation of the penile innervation increases S1412 phosphorylation that is blocked by PKA inhibitors but not by PI3-kinase/Akt inhibitors. Stimulation of cAMP formation by forskolin also activates nNOS phosphorylation. Sustained penile erection elicited by either intracavernous forskolin injection, or augmented by forskolin during cavernous nerve electrical stimulation, is prevented by the NOS inhibitor L-NAME or in nNOS-deleted mice. Thus, nNOS mediates both initiation and maintenance of penile erection, implying unique approaches for treating erectile dysfunction.gasotransmitter | smooth muscle relaxation | endothelial NOS | cyclic GMP | phosphoantibody N itric oxide (NO) is well established as a mediator of penile erection (1, 2). Neuronal NO synthase (nNOS) is highly localized to the penile innervation (3, 4). Electrical stimulation of the cavernous nerve (CN) to the penis elicits penile erection, which is abolished with NOS inhibitors and markedly reduced in nNOS-α-deleted mice (nNOSα −/− ) (5-7). Neuronal depolarizationinduced production of NO reflects calcium entry activating calmodulin associated with nNOS to stimulate NO formation (8,9). This activation is short-lived and thus is believed to account only for the initiation of penile erection (10, 11). Sessa and colleagues (12) established that increased blood flow and associated shear stress, acting via PI3-kinase, augments the activity of the serine protein kinase Akt, which phosphorylates vascular endothelial NOS (eNOS) at serine(S)1179, causing prolonged NO formation at resting intracellular calcium levels (13). The increased penile blood flow through cavernosal vessels initiated by nNOS activation similarly stimulates penile Akt to phosphorylate eNOS, thereby promoting sustained maximal penile erection (14).nNOS possesses a phosphorylation consensus sequence at S1412 that closely resembles the sequence surrounding eNOS-S1179, and is thought to be a target for Akt in some systems (15-21). We wondered whether nNOS phosphorylation at S1412 might regulate penile erection. Using a highly selective antibody for phospho-S1412-nNOS (P-nNOS) we report electrically stimulated nNOS phosphorylation via cAMP-dependent protein kinase (PKA) and not through Akt. P-nNOS activity contributes to sustained erection in concert with phospho-eNOS stimulation, and the neuronal and endothelial NOS activities are independently regulated by separate signaling cascades. We propose a model integrating the posttranslational phospho-stimulation of normal erectile physiology. ResultsElectrical Stimulation of Penile Innervation Augments Phosphorylation of nNOS. We developed a C-terminal ...

In previous studies, we have shown that the PCFI-I mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCFI-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. polypeptide is the only TFIIIC subunit that can be photocross-linked to the region footprinted by TFIIIB. This subunit is a likely candidate to mediate the recruitment of TFIIIB. Similarly, the 95-and 55-kDa subunits may interact with the A block, and the 138-kDa subunit may interact with the B block. To help resolve the roles of individual TFIIIC subunits in transcription complex assembly, a major effort to clone these genes has been under way. So far, this has been achieved for the 95-, 131-, and 138-kDa polypeptides (23,26,29,34).Studies with yeast and human cells indicate that TFIIIB is a multisubunit factor (20,24,35,36). Detailed characterization of the yeast factor suggests that it comprises three components: the TATA-binding protein (TBP), a 70-kDa TFIIB-like polypeptide (TFIIIB70), and a 90-kDa polypeptide (TFIIIB90). These three polypeptides are stably associated under most conditions and copurify as a complex with TFIIIB activity over numerous columns. However, by using strong cation exchangers, specifically MonoS, TFIIIB activity has been separated into two fractions, designated TFIIIB ' and TFIIIB" (18). By photocross-linking, these two fractions were found to contain the TFIIIB70 and TFIIIBgo polypeptides, respectively, which had been identified previously in less pure TFIIIB fractions with this technique (3). Following the demonstration of a universal role for TBP in eukaryotic transcription (7, 32), TBP was identified as a component of TFIIIB and traced by Western blotting (immunoblotting) to the TFIIIB' fraction (20). This result together with the cloning of the gene for TFIIIB70 (5,6,25) has permitted the demonstration that these proteins are the only TFIIIB components in the TFIIIB' fraction. The two proteins expressed in bacteria can replace the TFIIIB' fraction in a reconstituted transcription system (20). The TFIIIB" fraction has not been purified to homogeneity. However, TFIIIB90 may be the only TFIIIB subunit in this fraction, since TFIIIB" transcription activity can be provided by sodium dodecyl sulfate (SDS) gel-eluted and renatured proteins in the 90-kDa size range (20