The generation of nitric oxide and its roles in neurotransmission and neurotoxicity.

The N-methyl-D-aspartate (NMDA) receptor plays a key role in synaptic plasticity and is thought to underlie memory, learning and development of the nervous system. The NMDA receptor is a ligand-gated ion channel complex that contains distinct recognition sites for endogenous and exogenous ligands, including glutamate, glycine, Mg2+, Zn2+ and noncompetitive blockers such as MK-801. In the central nervous system, nitric oxide (NO) is produced in some neurons following activation of excitatory amino acids receptors, particularly those of the NMDA receptor. Nitric oxide is synthesized from a L-arginine by the cytoplasmic enzyme nitric oxide synthase (NOS) which is a calcium dependent enzyme, and this pathway is inhibited by the analogues of L-arginine such as NG-monomethyl-L-arginine (L-NMMA) and is augmented by NMDA receptor activation. Activation of the NMDA receptor results in the elevation of intracellular calcium ([Ca2+]i) which in turn activates NOS via the calcium-calmodulin complex. Nitric oxide is not a classical neurotransmitter in the central nervous system since it is not released by exocytosis and does not interact with a receptor protein but rather diffuses rapidly across the membrane and binds with the iron in heme-containing proteins. Nitric oxide can serve as both an oxidizing and reducing agent. It has strong affinity for heme proteins such as guanylyl cyclase, but there is evident that NO may have a regulatory role by oxidizing sulfhydryl groups of non-heme proteins such as those on the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-modulated palmitoylation of endothelial nitric oxide synthase.

The nitric oxide synthases (NOS) comprise a family of enzymes which differ in primary structure, biological roles, subcellular distribution, and post-translational modifications. The endothelial nitric oxide synthase (ec-NOS) is unique among the NOS isoforms in being modified by N-terminal myristoylation, which is necessary for its targeting to the endothelial cell membrane. The subcellular localization of the ecNOS, but not enzyme myristoylation, is dynamically regulated by agonists such as bradykinin, which promote ecNOS translocation from membrane to cytosol, as well as enhancing enzyme phosphorylation. Using transiently transfected endothelial cells, we now show that a myristoylation-deficient mutant ecNOS undergoes phosphorylation despite restriction to the cytosol, suggesting that phosphorylation may be a consequence rather than a cause of ecNOS translocation. We therefore explored whether other post-translational modifications might regulate ecNOS targeting and now report that ecNOS is reversibly palmitoylated. Biosynthetic labeling of endothelial cells with [3H]palmitic acid followed by immunoprecipitation of ecNOS revealed that the enzyme is palmitoylated; the label is released by hydroxylamine, consistent with formation of a fatty acyl thioester, and authentic palmitate can be recovered from labeled ecNOS following acid hydrolysis. Importantly, pulse-chase experiments in endothelial cells biosynthetically labeled with [3H]palmitate show that bradykinin treatment promotes ecNOS depalmitoylation. We conclude that ecNOS palmitoylation is dynamically regulated by bradykinin and propose that depalmitoylation of the enzyme may result in its cytosolic translocation and subsequent phosphorylation.