No abstract

N-methyl-D-aspartate (NMDA) glutamate receptor-mediated increases in intracellular calcium are thought to play a critical role in synaptic plasticity. The mechanisms by which changes in cytoplasmic calcium transmit the glutamate signal to the nucleus, which is ultimately important for long-lasting neuronal responses, are poorly understood. We show that NMDA receptor stimulation leads to activation of p21 ras (Ras) through generation of nitric oxide (NO) via neuronal NO synthase. The competitive NO synthase inhibitor, L-nitroarginine methyl ester, prevents Ras activation elicited by NMDA and this effect is competitively reversed by the NO synthase substrate, L-arginine. NMDA receptor stimulation fails to activate Ras in neuronal cultures from mice lacking neuronal NO synthase. NMDA-induced Ras activation occurs through a cGMP-independent pathway as 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a potent and selective inhibitor of guanylyl cyclase, has no effect on NMDA receptor-induced activation of Ras, and the cellpermeable cGMP analog, 8Br-cGMP, does not activate Ras. Furthermore, NO directly activates immunoprecipitated Ras from neurons. NMDA also elicits tyrosine phosphorylation of extracellular signal-regulated kinases, a downstream effector pathway of Ras, through a NO͞non-cGMP dependent mechanism, thus supporting the physiologic relevance of endogenous NO regulation of Ras. These results suggest that Ras is a physiologic target of endogenously produced NO and indicates a signaling pathway for NMDA receptor activation that may be important for long-lasting neuronal responses.Neuronal survival, differentiation, and plasticity involve signal transduction cascades that occur in large part through activation of p21 ras (Ras) (1-4). Ras is highly expressed in the developing and adult nervous systems and plays an important classical role in mediating growth factor responses (1-4). Recent studies suggest that increases in intracellular calcium also can activate Ras (5-10). Activation of Ras by increases in cytoplasmic calcium levels may play critical roles in mediating and͞or modulating activity-induced changes such as neuronal differentiation, synaptic strength, and neuronal survival, in part, through activation of extracellular signal-regulated kinases (Erks) (5-12). The molecular mechanisms by which changes in intracellular calcium levels in neurons activate Ras is not known. However, recent studies in tumor cell lines suggest the existence of multiple pathways that could be important in generating calcium-mediated activation of Ras, including Src, Ras-GRF, PYK2, and epidermal growth factor receptor (5-10). Despite the identification of these calciumdependent pathways to Ras, none of these pathways have been directly demonstrated to mediate calcium-dependent Ras activation in neurons (5-10).Nitric oxide (NO) is an important messenger molecule with many diverse actions in the nervous, vascular, and immune systems (13)(14)(15). NO is produced in a calcium͞calmodulin-dependent fashion from L-argin...

In skeletal muscle, neuronal nitric oxide synthase (nNOS) is anchored to the sarcolemma via the dystrophin-glycoprotein complex. When dystrophin is absent, as in Duchenne muscular dystrophy patients and in mdx mice, nNOS is mislocalized to the interior of the muscle fiber where it continues to produce nitric oxide. This has led to the hypothesis that free radical toxicity from mislocalized nNOS may contribute to mdx muscle pathology. To test this hypothesis directly, we generated mice devoid of both nNOS and dystrophin. Overall, the nNOS-dystrophin null mice maintained the dystrophic characteristics of mdx mice. We evaluated the mice for several features of the dystrophic phenotype, including membrane damage and muscle morphology. Removal of nNOS did not alter the extent of sarcolemma damage, which is a hallmark of the dystrophic phenotype. Furthermore, muscle from nNOS-dystrophin null mice maintain the histological features of mdx pathology. Our results demonstrate that relocalization of nNOS to the cytosol does not contribute significantly to mdx pathogenesis.