Endothelial nitric oxide synthase (eNOS) is active only as a homodimer. Recent data has demonstrated that exogenous NO can act as an inhibitor of eNOS activity both in intact animals and vascular endothelial cells. However, the exact mechanism by which NO exerts its inhibitory action is unclear. Our initial experiments in bovine aortic endothelial cells indicated that exogenous NO decreased NOS activity with an associated decrease in eNOS dimer levels. We then undertook a series of studies to investigate the mechanism of dimer disruption. Exposure of purified human eNOS protein to NO donors or calcium-mediated activation of the enzyme resulted in a shift in eNOS from a predominantly dimeric to a predominantly monomeric enzyme. Further studies indicated that endogenous NOS activity or NO exposure caused S-nitrosylation of eNOS and that the presence of the thioredoxin and thioredoxin reductase system could significantly protect eNOS dimer levels and prevent the resultant monomerization and loss of activity. Further, exogenous NO treatment caused zinc tetrathiolate cluster destruction at the dimer interface. To further determine whether S-nitrosylation within this region could explain the effect of NO on eNOS, we purified a C99A eNOS mutant enzyme lacking the tetrathiolate cluster and analyzed its oligomeric state. This enzyme was predominantly monomeric, implicating a role for the tetrathiolate cluster in dimer maintenance and stability. Therefore, this study links the inhibitory action of NO with the destruction of zinc tetrathiolate cluster at the dimeric interface through S-nitrosylation of the cysteine residues. N itric oxide (NO) is produced from L-arginine and oxygen by nitric oxide synthases (NOS) (EC. 1.14.13.39) (1). Three isoforms of NOS are known. Constitutive forms are present in endothelial cells [endothelial NOS (eNOS)] and neurons (neuronal NOS), and a third inducible isoform is present in macrophages (inducible NOS) (2-4). Dimerization is an absolute requirement for catalytic activity in all three NOS isoforms (5-7). NOS isoforms have two domains, the N-terminal oxygenase domain and the C-terminal reductase domain. The oxygenase domain has the binding site for tetrahydrobiopterin (BH 4 ), heme, and the substrate L-arginine and the reductase domain binds FAD, FMN, and NADPH. NO is produced in the oxygenase domain by the oxidation of arginine in a two-step reaction that generates 9).Our previous studies indicate that exogenous NO exposure inhibits eNOS activity (10, 11). However, the mechanism for the inhibition remains unclear. NO gas and NO donors have the potential to induce S-nitrosylation of proteins (12). We hypothesized that the inhibitory action on NO on eNOS could be acting by means of S-nitrosylation, with a resultant alteration in eNOS dimeric structure and activity. To test this hypothesis, we analyzed the nitrosylating effect of endogenous and exogenous NO on eNOS oligomeric state and catalytic activity. Enzyme activation or the addition of exogenous NO produced an inhibitory effect on t...

The structural phenotype of neural connections in the auditory brainstem is sculpted by spontaneous and stimulus-induced neural activities during development. However, functional and molecular mechanisms of spontaneous action potentials (SAPs) in the developing cochlea are unknown. Additionally, it is unclear how regenerating hair cells establish their neural ranking in the constellation of neurons in the brainstem. We have demonstrated that a transient Ca 2؉ current produced by the Cav3.1 channel is expressed early in development to initiate spontaneous Ca 2؉ spikes. Ca 2ϩ currents ͉ cochlea ͉ hearing ͉ spontaneous activity

Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken ␤-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor ␣-difluoromethylornithine (DFMO). Within 2-3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes for ornithine decarboxylase and S-adenosylmethionine decarboxylase, which are enzymes needed for the synthesis of putrescine, spermidine, and spermine, are lethal at early stages of embryonic development (1, 2). There is convincing evidence that the formation of hypusine in eIF5A, which requires spermidine as a precursor, is essential for eukaryotes (3). However, the function(s) of spermine is not so well established. Yeast mutants with inactivated spermine synthase grow at a normal rate (4). Mammalian cells in culture also grow normally in the presence of inhibitors of spermine synthase (5) or after inactivation of the spermine synthase gene (SpmS) (6 -8