S-nitroso-N-acetyl-DL-penicillamine Research Articles

Nitric oxide plays a dual role in the oxidative injury of cultured rat microglia but not astroglia

Nitric oxide (NO) and oxidative stress caused by reactive oxygen species (ROS) accumulation are two important factors that lead to the progression of human neurological diseases. NO can be detrimental or protective to neurons under oxidative toxicity; however, in the case of brain exposure to oxidative stress, in addition to neurons, the existence of glia may also be disturbed by toxic ROS. The influence NO will have on ROS-mediated glial injury remains unclear. Here, we examined the effects of NO on cell viability under oxidative stress induced by hydrogen peroxide (H2O2) in rat primary mixed glia cultures, as well as pure astroglia and microglia cultures. We found that in mixed glia cultures, both H2O2 and NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) elicited cell death in a concentration-dependent manner. Combinations of H2O2 and SNAP at sublytic concentrations were sufficient to damage mixed glia, and sublytic concentrations of SNAP could reduce the insults that resulted from toxic H2O2. Furthermore, in microglia or astroglia, sublytic concentrations of H2O2 were toxic when combined with SNAP, and the potency was increased with an increased SNAP concentration. In microglia but not astroglia, a toxic H2O2-induced apoptotic injury was attenuated by a sublytic level of SNAP. H2O2 at toxic levels activated p38 mitogen-activated protein kinases (MAPK) and p53 pathways and increased DNA double strand breaks (DSBs) in microglia, whereas the rescue exerted by sublytic SNAP against toxic H2O2 occurred via the activation of both Akt and extracellular-signal-regulated kinase (ERK) cascades and decreased DNA DSBs. Moreover, a sublytic concentration of SNAP induced both heat shock protein 70 and heme oxygenase-1, which may be involved in decreasing the susceptibility of microglia to H2O2 toxicity. These results suggest that NO exhibits a concentration-dependent dual action of weakening or enhancing oxidative injury in mixed glia, particularly microglia.

β-Adrenergic receptor antagonists inhibit vasculogenesis of embryonic stem cells by downregulation of nitric oxide generation and interference with VEGF signalling.

The β-adrenoceptor antagonist Propranolol has been successfully used to treat infantile hemangioma. However, its mechanism of action is so far unknown. The hypothesis of this research was that β-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific β-adrenergic receptor (β-adrenoceptor) antagonist Propranolol, the β1-adrenoceptor-specific antagonist Atenolol and the β2-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three β-blockers dose-dependently downregulated formation of capillary structures in ES cell-derived embryoid bodies and decreased the expression of the vascular cell markers CD31 and VE-cadherin. Furthermore, β-blockers downregulated the expression of fibroblast growth factor-2 (FGF-2), hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor 165 (VEGF165), VEGF receptor 2 (VEGF-R2) and phospho VEGF-R2, as well as neuropilin 1 (NRP1) and plexin-B1 which are essential modulators of embryonic angiogenesis with additional roles in vessel remodelling and arteriogenesis. Under conditions of β-adrenoceptor inhibition, the endogenous generation of nitric oxide (NO) as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) was decreased in embryoid bodies, whereas an increase in NO generation was observed with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Consequently, vasculogenesis of ES cells was restored upon treatment of differentiating ES cells with β-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that β-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.

Relationship between nitric oxide- and calcium-dependent signal transduction pathways in growth hormone release from dispersed goldfish pituitary cells

Nitric oxide (NO) and Ca2+ are two of the many intracellular signal transduction pathways mediating the control of growth hormone (GH) secretion from somatotropes by neuroendocrine factors. We have previously shown that the NO donor sodium nitroprusside (SNP) elicits Ca2+ signals in identified goldfish somatotropes. In this study, we examined the relationships between NO- and Ca2+-dependent signal transduction mechanisms in GH secretion from primary cultures of dispersed goldfish pituitary cells. Morphologically identified goldfish somatotropes stained positively for an NO-sensitive dye indicating they may be a source of NO production. In 2h static incubation experiments, GH release responses to the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were attenuated by CoCl2, nifedipine, verapamil, TMB-8, BHQ, and KN62. In column perifusion experiments, the ability of SNP to induce GH release was impaired in the presence of TMB-8, BHQ, caffeine, and thapsigargin, but not ryanodine. Caffeine-elicited GH secretion was not affected by the NO scavenger PTIO. These results suggest that NO-stimulated GH release is dependent on extracellular Ca2+ availability and voltage-sensitive Ca2+ channels, as well as intracellular Ca2+ store(s) that possess BHQ- and/or thapsigargin-inhibited sarcoplasmic/endoplasmic reticulum Ca2+-ATPases, as well as TMB-8- and/or caffeine-sensitive, but not ryanodine-sensitive, Ca2+-release channels. Calmodulin kinase-II also likely participates in NO-elicited GH secretion but caffeine-induced GH release is not upstream of NO production. These findings provide insights into how NO actions many integrate with Ca2+-dependent signalling mechanisms in goldfish somatotropes and how such interactions may participate in the GH-releasing actions of regulators that utilize both NO- and Ca2+-dependent transduction pathways.

Excess nitric oxide impairs liver X receptor α‐ATP‐binding cassette transporter A1‐dependent cholesterol efflux in macrophage foam cells (671.1)

Excess nitric oxide (NO) promotes the progression of atherosclerosis by increasing the oxidation of low‐density lipoprotein (LDL) and inflammatory responses. However, little is known about the impact of NO and its underlying molecular mechanism on lipid metabolism of macrophage foam cells. Treatment with S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP), an NO donor, exacerbated oxidized LDL (oxLDL)‐induced cholesterol accumulation in macrophages, because of reduced efficacy of cholesterol efflux. In addition, SNAP decreased the protein level of ATP‐binding cassette transporter A1 (ABCA1) without affecting scavenger receptor type A (SR‐A), CD36, ABCG1 or SR‐B1 levels. This SNAP‐mediated downregulation of ABCA1 was mainly through the effect of NO but not peroxynitrite. Furthermore, the SNAP‐downregulated ABCA1 was due to the decrease in the liver X receptor α (LXRα)‐dependent transcriptional regulation. Moreover, genetic deletion of inducible NOS increased the serum capacity of reverse cholesterol efflux and protein expression of LXRα, ABCA1 and SR‐BI in aortas and retarded atherosclerosis in apolipoprotein E‐deficient mice. Our findings provide new insights in the pro‐atherogenic effect of excess NO on cholesterol metabolism in macrophages.

Interactions betweenN-Ethylmaleimide-Sensitive Factor and GluR2 in the Nucleus Accumbens Contribute to the Expression of Locomotor Sensitization to Cocaine

Many studies have reported a withdrawal-dependent increase in synaptic AMPA receptor (AMPAR) levels in the nucleus accumbens (NAc) of cocaine-sensitized rats; however, the exact relationship between the expression of sensitization and altered AMPAR surface expression in the NAc has not yet been investigated. We demonstrated that the expression of behavioral sensitization was negatively controlled by N-ethylmaleimide-sensitive factor (NSF)-GluR2 interactions in the NAc. The upregulation of NSF-GluR2 interactions, which may be resulted by the increase in NSF S-nitrosylation after withdrawal from cocaine, was associated with the changes in the expression of behavioral sensitization. Disruption of NSF-GluR2 interactions in the NAc with a specific peptide, TAT-pep-R845A, increased the locomotor response of rats to cocaine by decreasing GluR2 surface insertion. In contrast, prevention of GluR2-containing AMPARs removal from synapses with Pep2-EVKI attenuated the expression of behavioral sensitization. Similarly, treatment with the nitric oxide donor, S-Nitroso-N-acetyl-DL-penicillamine (SNAP), attenuated the expression of locomotor sensitization by promoting GluR2 surface expression. This effect was mediated by the binding of S-nitrosylated NSF to GluR2, which promoted the surface expression of AMPARs. Noticeably, exogenous injection of SNAP into NAc also attenuated the expression of cocaine-induced conditioned place preference. Thus, these results indicate that increased NSF-GluR2 interactions in the NAc after withdrawal from cocaine attenuated the expression of behavioral sensitization and serves as a negative regulatory mechanism in drug-exposed individuals.

Cytoprotection by the NO-Donor SNAP Against Ischemia/Reoxygenation Injury in Mouse Embryonic Stem Cell-Derived Cardiomyocytes

Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation conditions. Here we tested the cytoprotective effect of the NO-donor SNAP and its downstream cellular pathway. Mouse ESC-derived cardiomyocytes were subjected to 150-min simulated ischemia (SI) followed by 120-min reoxygenation or corresponding non-ischemic conditions. The following treatments were applied during SI or normoxia: the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), the protein kinase G (PKG) inhibitor, the KATP channel blocker glibenclamide, the particulate guanylate cyclase activator brain type natriuretic peptide (BNP), and a non-specific NO synthase inhibitor (N-Nitro-L-arginine, L-NNA) alone or in different combinations. Viability of cells was assayed by propidium iodide staining. SNAP attenuated SI-induced cell death in a concentration-dependent manner, and this protection was attenuated by inhibition of either PKG or KATP channels. However, SI-induced cell death was not affected by BNP or by L-NNA. We conclude that SNAP protects mESC-derived cardiomyocytes against SI/R injury and that soluble guanylate-cyclase, PKG, and KATP channels play a role in the downstream pathway of SNAP-induced cytoprotection. The present mESC-derived cardiomyocyte based screening platform is a useful tool for discovery of cytoprotective molecules.

Excess nitric oxide impairs liver X receptor α-ATP-binding cassette transporter A1-dependent cholesterol efflux in macrophage foam cells

Excess nitric oxide (NO) promotes the progression of atherosclerosis by increasing the oxidation of low-density lipoprotein (LDL) and inflammatory responses. However, little is known about the impact of NO and its underlying molecular mechanism on lipid metabolism of macrophage foam cells. In this study, Oil-red O staining, cholesterol and triglyceride assay, Dil-oxidized LDL (oxLDL) binding assay, cholesterol efflux assay, real-time RT-PCR and Western blot analysis were used for in vitro experiments. Apolipoprotein E-deficient (apoE(-/-) ) and apoE and inducible nitric oxide synthase-deficient (apoE(-/-) iNOS(-/-) ) mice were as our in vivo models. Treatment with S-nitroso-N-acetyl-D,L-penicillamine (SNAP), an NO donor, exacerbated oxLDL-induced cholesterol accumulation in macrophages, because of reduced efficacy of cholesterol efflux. In addition, SNAP decreased the protein level of ATP-binding cassette transporter A1 (ABCA1) without affecting scavenger receptor type A (SR-A), CD36, ABCG1, or SR-B1 levels. This SNAP-mediated downregulation of ABCA1 was mainly through the effect of NO but not peroxynitrite. Furthermore, the SNAP-downregulated ABCA1 was due to the decrease in the liver X receptor α (LXRα)-dependent transcriptional regulation. Moreover, genetic deletion of iNOS increased the serum capacity of reverse cholesterol efflux and protein expression of LXRα, ABCA1, and SR-BI in aortas and retarded atherosclerosis in apoE(-/-) mice. Our findings provide new insights in the pro-atherogenic effect of excess NO on cholesterol metabolism in macrophages.

Vascular extracellular signal‐regulated kinase mediates migraine‐related sensitization of meningeal nociceptors

To examine changes in the response properties of meningeal nociceptors that might lead to migraine pain and examine endogenous processes that could play a role in mediating them using a clinically relevant model of migraine triggering, namely infusion of the nitric oxide (NO) donor nitroglycerin (NTG). Single-unit recordings made in the trigeminal ganglion of rats were used to test changes in the activity and mechanosensitivity of meningeal nociceptors in response to administration of the migraine trigger NTG or another NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) at doses relevant to the human model of migraine headache. Immunohistochemistry and pharmacological manipulations were used to investigate the possible role of meningeal vascular signaling in mediating the responses of meningeal nociceptors to NO. Infusion of NTG promoted a delayed and robust increase in the mechanosensitivity of meningeal nociceptors, with a time course resembling the development of the delayed migraine headache. A similar sensitization was elicited by dural application of NTG and SNAP. NTG-evoked delayed meningeal nociceptor sensitization was associated with a robust extracellular signal-regulated kinase (ERK) phosphorylation in meningeal arteries. Pharmacological blockade of meningeal ERK phosphorylation inhibited the development of NTG-evoked delayed meningeal nociceptor sensitization. The development of delayed mechanical sensitization evoked by the migraine trigger NTG is potentially of great importance as the first finding of a neurophysiological correlate of migraine headache in meningeal nociceptors. The arterial ERK phosphorylation and its involvement in mediating the NTG-evoked delayed sensitization points to an important, yet unappreciated, role of the meningeal vasculature in the genesis of migraine pain.

The role of nitric oxide in the regulation of neurotransmitter release and processes of exo- and endocytosis of synaptic vesicles in mouse motor nerve endings

In experiments with mouse diaphragm muscle, the effects of a nitric oxide (NO) donor, S-nitroso- N-acetyl-DL-penicillamine (SNAP), and an NO-synthase blocker, NG-nitro-L-arginine methyl ester (LNAME), on transmitter release and processes of exo- and endocytosis of synaptic vesicles in the motornerve ending were studied using electrophysiological and fluorescence techniques. During single stimulation of the motor nerve, SNAP reduced and LNAME did not change the amplitude of the endplate currents and both of the drugs did not affect spontaneous transmitter release. During high-frequency stimulation (20 Hz, 3 min) SNAP increased and LNAME slowed the depression of the amplitudes of endplate potentials (EPPs) compared to the dynamics of EPPs in the control. In experiments using the fluorescent dye FM 1–43, it was shown that the NO donor induced a decrease and LNAME induced an increase in the fluorescence intensity of motor-nerve endings loaded with dye during stimulation at a frequency of 20 Hz for 30 s compared to the control. At the same time, the rate of dye unloading from the terminals that were preloaded with FM 1–43 was higher after preliminary application of SNAP and lower after preliminary application of LNAME. It was suggested that exogenous and endogenous NO in the mouse neuromuscular synapse caused the depression of neurotransmitter release as a result of the suppression of synaptic-vesicle recycling due to a decrease in endocytosis or/and mobilization of synaptic vesicles from a recycling pool to the exocytosis sites.

Nitric Oxide Inhibits IP3R‐mediated Activation of TRPM4 in Cerebral Artery Smooth Muscle Cells

The melastatin transient receptor potential channel TRPM4 is essential for pressure‐induced smooth muscle cell contraction and is activated by Ca2+ released from the SR via inositol trisphosphate receptors (IP3R). Endothelium‐derived nitric oxide (NO) activates soluble guanylyl cyclase (sGC) leading to the generation of cyclic guanosine monophosphate (cGMP), activation of protein kinase G (PKG), and smooth muscle relaxation. We hypothesized that NO blocks IP3R‐dependent activation of TRPM4 channels in cerebral artery myocytes. Using perforated whole‐cell patch clamp electrophysiology, we observe a dose‐dependent reduction in TRPM4 channel activity in response to the NO donor S‐Nitroso‐N‐Acetyl‐D,L‐Penicillamine (SNAP). TRPM4 channel activity is also diminished following exposure to the cell‐permeable cGMP analog dibutyryl‐cGMP. TRPM4 currents inhibited by NO or cGMP did not return following administration of the cell‐permeable IP3 analog, Bt‐IP3. In addition, the sGC inhibitor NS‐2028 prevented NO‐mediated inhibition of TRPM4 channel activity. These findings suggest that NO can relax cerebral artery myocytes by blocking IP3R‐mediated activation of TRPM4. RO1HL091905; F31HL094145–01 (AG)

Shear Stress Regulates Endothelial Microparticle Release

Endothelial activation and apoptosis release membrane-shed microparticles (EMP) that emerge as important biological effectors. Because laminar shear stress (SS) is a major physiological regulator of endothelial survival, we tested the hypothesis that SS regulates EMP release. EMP levels were quantified by flow cytometry in medium of endothelial cells subjected to low or high SS (2 and 20 dyne/cm(2)). EMP levels augmented with time in low SS conditions compared with high SS conditions. This effect was sensitive to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and Rho kinases inhibitors but unaffected by caspase inhibitors. Low SS-stimulated EMP release was associated with increased endothelial Rho kinases and ERK1/2 activities and cytoskeletal reorganization. Overexpression of constitutively active RhoA stimulated EMP release under high SS. We also examined the effect of nitric oxide (NO) in mediating SS effects. L-NG-nitroarginine methyl ester (L-NAME), but not D-NG-nitroarginine methyl ester, increased high SS-induced EMP levels by 3-fold, whereas the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) decreased it. L-NAME and SNAP did not affect Rho kinases and ERK1/2 activities. Then, we investigated NO effect on membrane remodeling because microparticle release is abolished in ABCA1-deficient cells. ABCA1 expression, which was greater under low SS than under high SS, was augmented by L-NAME under high SS and decreased by SNAP under low SS conditions. Altogether, these results demonstrate that sustained atheroprone low SS stimulates EMP release through activation of Rho kinases and ERK1/2 pathways, whereas atheroprotective high SS limits EMP release in a NO-dependent regulation of ABCA1 expression and of cytoskeletal reorganization. These findings, therefore, identify endothelial SS as a physiological regulator of microparticle release.

The nitroxyl donor, Angeli's salt, inhibits inflammatory hyperalgesia in rats

Nitric oxide modulates pain development. However, there is no evidence on the effect of nitroxyl (HNO/NO−) in nociception. Therefore, we addressed whether nitroxyl inhibits inflammatory hyperalgesia and its mechanism using the nitroxyl donor Angeli's salt (AS; Na2N2O3). Mechanical hyperalgesia was evaluated using a modified Randall and Selitto method in rats, cytokine production by ELISA and nitroxyl was determined by confocal microscopy in DAF (a cell permeable reagent that is converted into a fluorescent molecule by nitrogen oxides)-treated dorsal root ganglia neurons in culture. Local pre-treatment with AS (17–450 μg/paw, 30 min) inhibited the carrageenin-induced mechanical hyperalgesia in a dose- and time-dependent manner with maximum inhibition of 97%. AS also inhibited carrageenin-induced cytokine production. AS inhibited the hyperalgesia induced by other inflammatory stimuli including lipopolysaccharide, tumor necrosis factor-α, interleukin-1β and prostaglandin E2. Furthermore, the analgesic effect of AS was prevented by treatment with ODQ (a soluble guanylate cyclase inhibitor), KT5823 (a protein kinase G [PKG] inhibitor) or glybenclamide (an ATP-sensitive K+ channel blocker), but not with naloxone (an opioid receptor antagonist). AS induced concentration-dependent increase in fluorescence intensity of DAF-treated neurons in a l-cysteine (nitroxyl scavenger) sensitive manner. l-cysteine did not affect the NO+ donor S-Nitroso-N-acetyl-DL- penicillamine (SNAP)-induced anti-hyperalgesia or fluorescence of DAF-treated neurons. This is the first study to demonstrate that nitroxyl inhibits inflammatory hyperalgesia by reducing cytokine production and activating the cGMP/PKG/ATP-sensitive K+ channel signaling pathway in vivo.

The effect of NO-donors on chloride efflux, intracellular Ca2 + concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells

Since previous studies showed that the endogenous bronchodilator, S-nitrosglutathione (GSNO), caused a marked increase in CFTR-mediated chloride (Cl−) efflux and improved the trafficking of CFTR to the plasma membrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl− efflux, it was investigated whether the NO-donor properties of GSNO were relevant for its effect on Cl− efflux from airway epithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-N-acetyl-dl-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitric oxide adduct (DEA-NONOate) on Cl− efflux from CFBE (∆F508/∆F508-CFTR) airway epithelial cells was tested. Cl− efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide (MQAE)-technique. Possible changes in the intracellular Ca2+ concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like previously with GSNO, after 4h incubation with the NO-donor, an increased Cl− efflux was found (in the order SNAP>DETA-NO>SNP). The effect of DEA-NONOate on Cl− efflux was not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as with GSNO, after a short (5min) incubation, SNP had no significant effect on Cl− efflux. None of the NO-donors that had a significant effect on Cl− efflux caused significant changes in the intracellular Ca2+ concentration. After 4h preincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOate decreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only in α-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on the expression of CFTR, nor on that of the ENaC subunits in the presence and absence of l-cysteine. It can be concluded that the effect of GSNO on Cl− efflux is, at least in part, due to its properties as an NO-donor, and the effect is likely to be mediated by CFTR, not by Ca2+-activated Cl− channels.

Inducible nitric oxide releasing poly-(ethylene glycol)-fibrinogen adhesive hydrogels for tissue regeneration

ABSTRACTNitric oxide (NO) release can promote healthy tissue regeneration. A PEG-fibrinogen adhesive hydrogel that would allow for inducible NO release was created with mechanical properties that could be tailored to specific applications and tissue types. PEG (4-arm)-fibrinogen hydrogels of varying ratios were derivatized with S-nitroso-N-acetyl-D, L-penicillamine (SNAP)-thiolactone to create an active NO donor material. Controlled release from gels was established using light as the activating source, although temperature, pH, and external mechanical loading are also means to induce active NO release. Gels with varying ratios of fibrinogen to PEG were made, derivatized, and tested. Gels below a ratio of 1.5:1 (fibrinogen:PEG) did not gel, while at ratio of 1.5:1 gelation occurs and NO release can be induced. Interestingly, the release from 1.5:1 gels was significantly lower compared to 2:1 and 3:1 gel formulations. Rheometric data show that lower ratio gels are more elastic than viscous. Derivatized gels exhibited linear elastic moduli, behaving more like other more synthetic hydrogels. Swelling data indicates that as the ratio of fibrinogen to PEG increases the swelling ratio decreases, likely due to the hydrophobic nature of the NO donor. Cells remain viable on both derivatized and non-derivatized gels.

M-Type K+ Channel as Plasma Membrane Nitric Oxide and Reactive Oxygen Species Sensor

M-type K+ channels control excitability of many neuron types. M channel activity is potently enhanced by oxidative modification of triple cysteine pocket within the cytosolic linker between transmembrane domains 2 and 3. Thus, in sensory neurons from dorsal root (DRG) and trigeminal (TG) ganglia M channel activity is increased by the mitochondrial release of reactive oxygen species (ROS) induced by neuromodulator substance P. Here we identified a new signalling role for nitric oxide (NO) in TG sensory neurons. We show that in rat TG neurons, the NO donor, S-Nitroso-N-acetyl-DL-penicillamine (SNAP), inhibited M-current by 53±12% (n=12). This inhibitory effect was blocked by scavenging of NO and inhibition of NO synthases increased M-current, suggesting that tonic NO levels inhibit M-current in TG neurons. NO increased release of calcitonin gene-related peptide (CGRP) from TG neurons (130±10% of control, p≤0.05), consistent with an increase in neuronal excitability. Importantly, incubation with the M-channel opener retigabine completely abolished increases in CGRP release. We further investigated the mechanism of the effects of NO on M channels and identified a site of action of NO to be the same triplet of cysteines, which is also a site of oxidative modification of M channels by ROS. We now show that the same triplet of cysteines can be S-nitrosylated in the presence of SNAP. We show that NO and oxidative modifications have opposing effects on M-current, suggesting that a tightly controlled local redox and NO environment will exert control over M-channel activity and thus neuronal excitability. Together our data have identified a dynamic redox sensor within neuronal M-channels which mediates reciprocal regulation of channel activity by S-nitrosylation and oxidative modification. This sensor may play an important role in controlling neuronal excitability by redox and NO-related mechanisms.

Nitric oxide inhibits succinate dehydrogenase-driven oxygen consumption in potato tuber mitochondria in an oxygen tension-independent manner

NO (nitric oxide) is described as an inhibitor of plant and mammalian respiratory chains owing to its high affinity for COX (cytochrome c oxidase), which hinders the reduction of oxygen to water. In the present study we show that in plant mitochondria NO may interfere with other respiratory complexes as well. We analysed oxygen consumption supported by complex I and/or complex II and/or external NADH dehydrogenase in Percoll-isolated potato tuber (Solanum tuberosum) mitochondria. When mitochondrial respiration was stimulated by succinate, adding the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) or DETA-NONOate caused a 70% reduction in oxygen consumption rate in state 3 (stimulated with 1mM of ADP). This inhibition was followed by a significant increase in the Km value of SDH (succinate dehydrogenase) for succinate (Km of 0.77±0.19 to 34.3±5.9mM, in the presence of NO). When mitochondrial respiration was stimulated by external NADH dehydrogenase or complex I, NO had no effect on respiration. NO itself and DETA-NONOate had similar effects to SNAP. No significant inhibition of respiration was observed in the absence of ADP. More importantly, SNAP inhibited PTM (potato tuber mitochondria) respiration independently of oxygen tensions, indicating a different kinetic mechanism from that observed in mammalian mitochondria. We also observed, in an FAD reduction assay, that SNAP blocked the intrinsic SDH electron flow in much the same way as TTFA (thenoyltrifluoroacetone), a non-competitive SDH inhibitor. We suggest that NO inhibits SDH in its ubiquinone site or its Fe-S centres. These data indicate that SDH has an alternative site of NO action in plant mitochondria.

In vivo regulation of renal expression of (pro)renin receptor by a low-sodium diet

Effects of low salt (LS) on (pro)renin receptor (PRR) expression are not well established. We hypothesized that LS enhances renal PRR expression via the cGMP-protein kinase G (PKG) signaling pathway. Sprague-Dawley rats were fed a normal-salt (NS) or LS diet associated with intrarenal cortical administration of vehicle (V), the nitric oxide (NO) synthase inhibitor nitro-l-arginine methyl ester (l-NAME), the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP), the cGMP analog 8-bromoguanosine (8-Br)-cGMP, the guanylyl cyclase inhibitor 1H-[1, 2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or a PKG inhibitor (PKGi) for 6 days via osmotic minipump. We evaluated the effects of each treatment on renal interstitial fluid (RIF) levels of nitrate/nitrite and cGMP and renal PRR expression. There were no significant changes in blood pressure with any of the treatments. Urinary sodium excretion was significantly lower in rats given a LS diet. Compared with NS + V, RIF nitrate/nitrite and cGMP levels increased in LS + V rats. In NS groups, RIF nitrate/nitrite and cGMP levels did not change with l-NAME, ODQ, or PKGi and increased in response to SNAP. 8-Br-cGMP increased RIF cGMP but not RIF nitrate/nitrite. In LS groups, RIF nitrate/nitrite decreased with l-NAME and did not change with ODQ or PKGi whereas RIF cGMP decreased with l-NAME, ODQ, and PKGi. PRR mRNA and protein increased in LS + V. In NS rats, PRR mRNA and protein increased in response to 8-Br-GMP and were not affected by any of other treatments. In LS rats, PRR mRNA and protein decreased significantly in response to l-NAME, ODQ, and PKGi. We conclude that LS intake enhances renal expression of PRR via cGMP-PKG signaling pathway.

Calcium-permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors Trigger Neuronal Nitric-oxide Synthase Activation to Promote Nerve Cell Death in an Src Kinase-dependent Fashion

In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.

NITRIC OXIDE-INDUCED ENHANCEMENT OF BANANA FRUIT ATTRIBUTES AND KEEPING QUALITY

The effect of nitric oxide (NO) on quality attributes such as texture, colour, respiration, total soluble proteins, sugars, and activities of phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) were studied in ripening banana fruit. Freshly harvested banana fingers, ‘Cavendish’, were soaked in water containing different concentrations (0.1, 0.2, 0.5, 1, 2, 3, 4 and 5 mM) of NO donors (sodium nitroprusside (SNP), nitroso-1-naphthol, nitroso-R-salt, S-nitrosoglutathione (SNG), S-nitroso-N-acetyl D-L-penicillamine (SNAP) and spermine-NO complex) for 3, 4 and 5 h separately. Among treatments, SNP, SNG and SNAP were relatively slowed ripening with colour deviation of 44, 52 and 47%; firmness loss of 49, 48 and 50% and storage period of 27, 29 and 28 days respectively, whilst, control recorded higher colour deviation (62%) and firmness loss (69%) with storage period of only 17 days. A structural analogue of NO donor, potassium ferricyanide having no NO donor effect did not show similar positive effects imparted by NO donor indicating the factual effects of NO. Among the NO-donors, treatment with SNP at 1 mM showed delayed respiratory peak coinciding with delay in the accumulation of total soluble as well as reducing sugars and significantly lower activities of PAL and PPO during ripening. Early and high expression of PAL may serve as a biochemical marker for studying climacteric stage of ‘Cavendish’ banana fruit.

Intravital microscopy reveals endothelial dysfunction in resistance arterioles in Angiotensin II-induced hypertension

It is known that hypertension is associated with endothelial dysfunction and that Angiotensin II (Ang II) is a key player in the pathogenesis of hypertension. We aimed to elucidate whether endothelial dysfunction is a specific feature of Ang II-mediated hypertension or a common finding of hypertension, independently of underlying etiology. We studied endothelial-dependent vasorelaxation in precapillary resistance arterioles and in various large-caliber conductance arteries in wild-type mice with Ang II-dependent hypertension (2-kidney 1-clip (2K1C) model) or Ang II-independent (volume overload) hypertension (1-kidney 1-clip model (1K1C)). Normotensive sham mice were used as controls. Aortic mechanical properties were also evaluated. Intravital microscopy of precapillary arterioles revealed a significantly impaired endothelium-dependent vasorelaxation in 2K1C mice compared with sham mice, as quantified by the ratio of acetylcholine (ACh)-induced over S-nitroso-N-acetyl-D,L-penicillamine (SNAP)-induced vasorelaxation (2K1C: 0.49±0.12 vs. sham: 0.87±0.11, P=0.018). In contrast, the ACh/SNAP ratio in volume-overload hypertension 1K1C mice was not significantly different from sham mice, indicating no specific endothelial dysfunction (1K1C: 0.77±0.27 vs. sham: 0.87±0.11, P=0.138). Mechanical aortic wall properties and endothelium-dependent vasorelaxation, assessed ex vivo in rings of large-caliber conductance (abdominal and thoracic aorta, carotid and femoral arteries), were not different between 2K1C, 1K1C and sham mice. Endothelial dysfunction is an early feature of Ang II- but not volume-overload-mediated hypertension. This occurs exclusively at the level of precapillary arterioles and not in conduit arteries. Our findings, if confirmed in clinical studies, will provide a better understanding of the pathophysiological mechanisms of hypertension.