Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin,respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.     

Contribution of bradykinin and nitric oxide to AT2 receptor-mediated differentiation in PC12 W cells

We investigated the effect of angiotensin II on intracellular cyclic GMP content and neurite outgrowth as an indicator of cell differentiation in PC12 W cells. Neurite outgrowth was examined by phase-contrast microscopy. Outgrown neurites were classified as small, medium and large, and were expressed as neurites per 100 cells. Angiotensin II (10-7 m) increased the outgrowth of medium and large neurites by mean +/- SEM 20.2 +/- 2.3 and 6.6 +/- 1.4 compared with 1.66 +/- 0.5 and 0.1 +/- 0.06 neurites per 100 cells in control. Cellular cyclic GMP content increased by 50-250% with angiotensin II at concentrations of 10-6-10-4 m. Both blockade of AT2 receptors and of nitric oxide synthase markedly reduced angiotensin II-induced neurite outgrowth and cyclic GMP production. In contrast, B2 receptor blockade had no effect or even increased these angiotensin II effects. Sodium nitroprusside and 8-bromo-cyclic GMP both mimicked the effects of angiotensin II on cell differentiation. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both angiotensin II and 8-bromo-cyclic GMP. Our results demonstrate that angiotensin II can stimulate cell differentiation in PC12 W cells by nitric oxide-related and cyclic GMP-dependent mechanisms. The effects of angiotensin II on cell differentiation and cyclic GMP production were mediated via the AT2 receptor and further enhanced by bradykinin B2 receptor blockade.     

Proteasome activation and nNOS down-regulation in neuroblastoma cells expressing a Cu,Zn superoxide dismutase mutant involved in familial ALS

Reactive oxygen and nitrogen species have emerged as predominant effectors of neurodegeneration. We demonstrated that expression of the fully active G93A Cu,Zn superoxide dismutase mutant in neuroblastoma cells is associated with an increased level of oxidatively modified proteins, in terms of carbonylated residues. A parallel increase in proteasome activity was detected and this was mandatory in order to assure cell viability. In fact, proteasome inhibition by lactacystin or MG132 resulted in programmed cell death. Nitrosative stress was not involved in the oxidative unbalance, as a decrease in neuronal nitric oxide production and down-regulation of neuronal nitric oxide synthase (nNOS) level were detected. The nNOS down-regulation was correlated to increased proteolytic degradation by proteasome, because comparable levels of nNOS were detected in G93A and parental cells upon treatment with lactacystin. The altered rate of proteolysis observed in G93A cells was specific for nNOS as Cu,Zn superoxide dismutase (Cu,Zn SOD) degradation by proteasome was influenced neither by its mutation nor by increased proteasome activity. Treatment with the antioxidant 5,5'-dimethyl-1-pyrroline N-oxide resulted in inhibition of protein oxidation and decrease in proteasome activity to the basal levels. Overall these results confirm the pro-oxidant activity of G93A Cu,Zn SOD mutant and, at the same time, suggest a cross-talk between reactive oxygen and nitrogen species via the proteasome pathway.     

Nitric oxide production by arsenite

Arsenic can either enhance or reduce nitric oxide (NO) production, depending on the type of cell, the species and dose of arsenical tested. The mechanisms of how arsenic increases or decreases NO production remain unclear. Because NO is associated with many pathological conditions, it is conceivable that in those arsenic-target tissues, the NO production may be upregulated by continuous arsenic exposure, and a prolonged over-production of NO may cause inflammation hence a pathological condition. A prolonged interference with the normal physiological level of NO may also play a role in the initiation, promotion, and progression of arsenic-related human cancers. Suppression of NO production has been shown to reduce arsenite-induced oxidative DNA damage, inhibition of pyrimidine dimer excision, and micronuclei. However, a completely reliable story on how NO is involved in arsenic-related human disease is still lacking.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Rapid increase in endothelial nitric oxide production by bradykinin is mediated by protein kinase A signaling pathway

Bradykinin (BK) acutely increases endothelial nitric oxide (NO) production by activating endothelial NO synthase (eNOS), and this increase is in part correlated with enhanced phosphorylation/dephosphorylation of eNOS by several protein kinases and phosphatases. However, the signaling mechanisms producing this increase are still controversial. In an attempt to delineate the acute effect of BK on endothelial NO production, confluent bovine aortic endothelial cells were incubated with BK, and NO production was measured by NO-specific chemiluminescence. Significant increase in NO levels was detected as early as 1 min after BK treatment, with concomitant increase in the phosphorylation of Ser(1179) (bovine sequence) site of eNOS (eNOS-Ser(1179)). This acute effect of BK on both increases was blocked only by treatment of protein kinase A inhibitor H-89, but not by the inhibitors of calmodulin-dependent kinase II and protein kinase B, suggesting that the rapid increase in NO production by BK is mediated by the PKA-dependent phosphorylation of eNOS-Ser(1179).     

Lipopolysaccharide down-regulates inducible nitric oxide synthase expression in swine heart in vivo

Studies of the regulation of iNOS expression have provided many contradictory results. Comparing iNOS expression profile between cell types or organs of the same animal under the same experimental conditions may provide an explanation for these conflicting results. We have examined iNOS mRNA and protein expression in heart and liver of the same group of pigs. We found that there is a sharp difference in iNOS expression between heart and liver. The iNOS mRNA and protein was constitutively expressed in the heart at high level, but was not detectable in the liver of the same control animal. Lipopolysaccharide (LPS, 100 microg/kg, i.v.) caused a marked iNOS induction in the liver, but significantly down-regulated iNOS expression in the heart. This differential iNOS expression appears to be physiologically relevant, since LPS and the iNOS inhibitor, S-methylisothiourea, exerted different effects on hepatic and myocardial blood flow. Our data demonstrate a fundamental difference in iNOS regulation in the heart and liver of swine, and may explain the contradictory data on the regulation of iNOS expression.     

Cyclooxygenase 2 inhibits SAPK activation in neuronal apoptosis

Cyclooxygenase 2 (COX-2) expressed in cultured neuronal PC12 cells under inducible promoter protects cells from trophic withdrawal apoptosis. Stimulation of SAPK is thought to play a significant role in initiation of PC12 cell death. We have therefore examined whether COX-2 expression inhibits trophic withdrawal-mediated activation of SAPK. SAPK activity increased during the first 6h after NGF removal in mock-transfected PC12 cells. COX-2 expression attenuated the increase of SAPK, as detected by Western blot analysis with phosphorylation state specific anti-SAPK antibodies and by SAPK activity assays. We propose that COX-2 attenuated SAPK activation by preventing activation of nNOS, which occurs, as we have shown before, via COX-2-mediated expression of dynein light chain (DLC). Activation of SAPK in neuronal cell death was attenuated by DLC expression. These observations support a role for NO production and SAPK activation in the neuronal death mechanisms.