A Paradoxical Vasodilatory Nutraceutical Intervention for Prevention and Attenuation of Migraine-A Hypothetical Review.

Studies suggest that migraine pain has a vascular component. The prevailing dogma is that peripheral vasoconstriction activates baroreceptors in central, large arteries. Dilatation of central vessels stimulates nociceptors and induces cortical spreading depression. Studies investigating nitric oxide (NO) donors support the indicated hypothesis that pain is amplified when acutely administered. In this review, we provide an alternate hypothesis which, if substantiated, may provide therapeutic opportunities for attenuating migraine frequency and severity. We suggest that in migraines, heightened sympathetic tone results in progressive central microvascular constriction. Suboptimal parenchymal blood flow, we suggest, activates nociceptors and triggers headache pain onset. Administration of NO donors could paradoxically promote constriction of the microvasculature as a consequence of larger upstream central artery vasodilatation. Inhibitors of NO production are reported to alleviate migraine pain. We describe how constriction of larger upstream arteries, induced by NO synthesis inhibitors, may result in a compensatory dilatory response of the microvasculature. The restoration of central capillary blood flow may be the primary mechanism for pain relief. Attenuating the propensity for central capillary constriction and promoting a more dilatory phenotype may reduce frequency and severity of migraines. Here, we propose consideration of two dietary nutraceuticals for reducing migraine risk: L-arginine and aged garlic extracts.

Genipin Derivatives Protect RGC-5 from Sodium Nitroprusside-Induced Nitrosative Stress.

CHR20 and CHR21 are a pair of stable diastereoisomers derived from genipin. These stereoisomers are activators of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). In the rat retinal ganglion (RGC-5) cell model these compounds are non-toxic. Treatment of RGC-5 with 750 µM of sodium nitroprusside (SNP) produces nitrosative stress. Both genipin derivatives, however, protect these cells against SNP-induced apoptic cell death, although CHR21 is significantly more potent than CHR20 in this regard. With Western blotting we showed that the observed neuroprotection is primarily due to the activation of protein kinase B (Akt)/eNOS and extracellular signal-regulated kinase (ERK1/2) signaling pathways. Therefore, LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or PD98059 (a MAPK-activating enzyme inhibitor) abrogated the protective effects of CHR20 and CHR21. Altogether, our results show that in our experimental setup neuroprotection by the diasteromeric pair is mediated through the PI3K/Akt/eNOS and ERK1/2 signaling pathways. Further studies are needed to establish the potential of these compounds to prevent ntric oxide (NO)-induced toxicity commonly seen in many neurodegenerative diseases.

Acute hemolytic vascular inflammatory processes are prevented by nitric oxide replacement or a single dose of hydroxyurea.

Hemolysis and consequent release of cell-free hemoglobin (CFHb) impair vascular nitric oxide (NO) bioavailability and cause oxidative and inflammatory processes. Hydroxyurea (HU), a common therapy for sickle cell disease (SCD), induces fetal Hb production and can act as an NO donor. We evaluated the acute inflammatory effects of intravenous water-induced hemolysis in C57BL/6 mice and determined the abilities of an NO donor, diethylamine NONOate (DEANO), and a single dose of HU to modulate this inflammation. Intravenous water induced acute hemolysis in C57BL/6 mice, attaining plasma Hb levels comparable to those observed in chimeric SCD mice. This hemolysis resulted in significant and rapid systemic inflammation and vascular leukocyte recruitment within 15 minutes, accompanied by NO metabolite generation. Administration of another potent NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) to C57BL/6 mice induced similar alterations in leukocyte recruitment, whereas hemin-induced inflammation occurred over a longer time frame. Importantly, the acute inflammatory effects of water-induced hemolysis were abolished by the simultaneous administration of DEANO or HU, without altering CFHb, in an NO pathway-mediated manner. In vitro, HU partially reversed the Hb-mediated induction of endothelial proinflammatory cytokine secretion and adhesion molecule expression. In summary, pathophysiological levels of hemolysis trigger an immediate inflammatory response, possibly mediated by vascular NO consumption. HU presents beneficial anti-inflammatory effects by inhibiting rapid-onset hemolytic inflammation via an NO-dependent mechanism, independently of fetal Hb elevation. Data provide novel insights into mechanisms of hemolytic inflammation and further support perspectives for the use of HU as an acute treatment for SCD and other hemolytic disorders.

[Effects of Vam3 on sodium nitroprusside-induced apoptosis and SIRT1 and p53 expression in rat articular chondrocytes].

This study is to investigate the effect of Vam3, a dimeric derivative of resveratrol, on SNP-induced apoptosis and its potential mechanism in rat articular chondrocytes. Isolated rat articular chondrocytes were treated with sodium nitroprusside (SNP), a NO donor, to induce apoptosis. Apoptosis percentage was evaluated by Annexin V-PI and nucleus fracture was examined by DAPI staining. Level of intracellular reactive oxygen species (ROS) was detected using 2, 7'-dichlorofluorescin diacetate (DCFH-DA) as a fluorescence probe by fluorescence microplate reader. The change in mitochondrial membrane potential was detected by TMRE staining. Expressions of SIRT1, acetylated p53 (ac-p53), cleaved caspase 9 and cleaved caspase 3 were determined by Western blotting. It showed that Vam3 up to 10 micromol x L(-1) could significantly reduce SNP-induced rat articular chondrocytes apoptosis (P < 0.01) and nucleus fracture, inhibit the increase of intracellular ROS level (P < 0.01) and reverse the decrease in mitochondrial membrane potential (P < 0.01). Simultaneously, Vam3 could upregulate the expression of SIRT1, deacetylate p53, and inhibit the cleavage of caspase 9 and caspase 3 (P < 0.01) of rat articular chondrocytes exposed to SNP. This study indicates Vam3 could protect rat articular chondrocytes against SNP-induced apoptosis, perhaps through the upregulation of SIRT1 and deacetylation of p53.

Peroxynitrite has potent pulmonary vasodilator activity in the rat.

Peroxynitrite (PN) worsens pathological conditions associated with oxidative stress. However, beneficial effects have also been reported. PN has been shown to demonstrate vasodilator as well as vasoconstrictor properties that are dependent upon the experimental conditions and the vascular bed studied. PN-induced vascular smooth muscle relaxation may involve the formation of nitric oxide (NO) donors. The present results show that PN has significant vasodilator activity in the pulmonary and systemic vascular beds, and that responses to PN were not attenuated by L-penicillamine (L-PEN), a PN scavenger, whereas responses to sodium nitroprusside (SNP) were decreased. PN had a small inhibitory effect on decreases in arterial pressure in response to the NO donors diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and S-nitrosoglutathione (GSNO). PN partially reversed hypoxic pulmonary vasoconstriction. PN responses were attenuated by the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and responses to PN and the PN precursor, 3-morpholinosydnonimine (SIN-1), were different. These data show that PN has potent pulmonary vasodilator activity in the rat, and provide evidence that a PN interaction with S-nitrosothiols is not the major mechanism mediating the response. These data suggest that responses to PN are mediated by the activation of sGC, and that PN has a small inhibitory effect on NO responses.

No effect of pure oxygen inhalation on headache induced by glyceryl trinitrate.

Inhalation of hyperbaric oxygen has been used as an experimental treatment for migraine and pure oxygen is an established treatment for cluster headache. Intravenous glyceryl trinitrate (GTN) is an established headache model. In the present study the possibility of decreasing the headache by inhalation of pure oxygen was explored in a double-blind crossover design in 18 healthy subjects. Inhalation of air served as placebo. The subjects received intravenous GTN (0.25 microg/kg/min) for 20 min. Headache was scored for 85 min. Sixteen of 18 (89%) subjects experienced GTN-induced headache after O(2)-inhalation and 17/18 (94%) experienced GTN-induced headache after air. The mean peak headache scores were 1.9 and 2.4, respectively, on a numerical scale of 0-10. Oxygen inhalation did not have effect on GTN-induced headache, most likely because the theoretical decrease in NO levels, due to faster metabolism of NO, is too small to be detected in the GTN headache model.

Urokinase plasminogen activator impairs SNP and PGE2 cerebrovasodilation after brain injury through activation of LRP and ERK MAPK.

Pial artery dilation in response to prostaglandin (PG)E(2) and the nitric oxide (NO) releaser sodium nitroprusside (SNP) are blunted after fluid percussion brain injury (FPI), whereas responses to papaverine are unchanged. Urokinase plasminogen activator (uPA) and ERK mitogen-activated protein kinase (MAPK) are upregulated and contribute to the impairment of cerebrohemodynamics seen after FPI. PA vascular activity is mediated through the low-density lipoprotein receptor (LRP). Therefore, we investigated the role of uPA, LRP, and ERK MAPK in the impaired cerebrovasodilation response to PGE(2) and SNP after FPI. Lateral FPI (2 atm) was induced in anesthetized piglets equipped with a closed cranial window. Cerebrospinal fluid (CSF) ERK MAPK was quantified by enzyme-linked immunosorbent assay (ELISA). Pretreatment with soluble uPA receptor (suPAR), which antagonizes the vascular action of uPA, blunted the impairment of SNP and PGE(2)-mediated dilation seen after FPI. Pretreatment with the LRP antagonist RAP, a monoclonal antibody against LRP (Mab ag LRP) and the ERK MAPK antagonist, U 0126, all provided similar protection, whereas control immunoglobulin G (IgG) had no effect. Responses to papaverine were unchanged after FPI. Upregulation of ERK MAPK phosphorylation in CSF after FPI was blunted in animals pretreated with suPAR, RAP, MAb ag LRP, or U 0126, whereas control IgG had no effect. These data indicate that uPA contributes to the impairment of SNP and PGE(2)-mediated cerebrovasodilation seen after brain injury through activation of LRP and ERK MAPK.

Permeability changes in response to NONOate and NONOate prodrug derived nitric oxide in a blood-brain barrier model formed by primary porcine endothelial cells.

The influence of nitric oxide (NO) and NO-donors on the permeability of the blood-brain barrier (BBB) is still not well understood and the literature about this is quite controversial. Some studies suggest increasing, others decreasing or even no effects of NO-donors on the BBB permeability. In this work we report about the influence of three diazeniumdiolates, which release NO spontaneously and three different diazeniumdiolate prodrugs, which have to be cleaved chemically or enzymatically before releasing NO, on the permeability of an in vitro BBB-model formed by primary porcine endothelial cells. By measuring the flux of a small polar molecule (carboxyfluorescein: CF) we could show, that the NO-releasers PHEPIPERAZI/NO (sodium 1-(phenylpiperazin-1-yl)diazen-1-ium-1,2-diolate), DBA/NO (sodium 1-(N,N-dibutylamino)diazen-1-ium-1,2-diolate) and DETA/NO (1-N,N-di-(2-aminoethyl)amino)diazen-1-ium-1,2-diolate) reduced the BBB-model permeability. In contrast, the NO-prodrugs Et-PHEPIPERAZI/NO (O(2)-Ethyl-1-(phenylpiperazin-1-yl)diazen-1-ium-1,2-diolate) and TOSYL-PYRRO/NO (O(2)-(p-Methylbenzen-sulfonyl)-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate) increased the permeability in all investigated concentrations, whereas the prodrug Et-BUPIPERAZI/NO (O(2)-Ethyl-1-(butylpiperazin-1-yl)diazen-1-ium-1,2-diolate) reduced it at the lowest investigated concentration of 100 microM, at the higher concentrations it increased the permeability. Blocking the effect of the BBB-model permeability reducing compounds could be done by methylene blue, whereas permeability increasing effects could not be blocked.

Glycyrrhizin protection against 3-morpholinosydnonime-induced mitochondrial dysfunction and cell death in lung epithelial cells.

The present study was designed to assess the preventive effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against mitochondrial damage and cell death in lung epithelial cells exposed to 3-morpholinosydnonime, a donor of nitric oxide and superoxide. Treatment of lung epithelial cells with 3-morpholinosydnonime resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. Treatment of glycyrrhizin and 18beta-glycyrrhetinic acid attenuated the 3-morpholinosydnonime-induced mitochondrial damage, formation of reactive oxygen species and GSH depletion and revealed a maximal inhibitory effect at 10 and 1 muM, respectively; beyond these concentrations the inhibitory effect declined. Melatonin, carboxy-PTIO, rutin and uric acid reduced the 3-morpholinosydnonime-induced cell death. The results show that glycyrrhizin and 18beta-glycyrrhetinic acid seem to prevent the toxic effect of 3-morpholinosydnonime against lung epithelial cells by suppressing the mitochondrial permeability transition that leads to the release of cytochrome c and activation of caspase-3. The preventive effect may be ascribed to the inhibitory action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that licorice compounds seem to prevent the nitrogen species-mediated lung cell damage.

Trophic factors attenuate nitric oxide mediated neuronal and axonal injury in vitro: roles and interactions of mitogen-activated protein kinase signalling pathways.

Inflammation in the central nervous system occurs in diseases such as multiple sclerosis and leads to axon dysfunction and destruction. Both in vitro and in vivo observations have suggested an important role for nitric oxide (NO) in mediating inflammatory axonopathy. The purposes of this study were to model inflammatory axonopathy in vitro and identify modulators of the process. Rat cortical neurones were cultured and exposed to an NO-donor plus potential protective factors. Cultures were then assessed for neuronal survival, axon survival and markers of intracellular signalling pathways. The NO-donor produced dose-dependent neuronal loss and a large degree of axon destruction. Oligodendrocyte conditioned medium (OCM) and insulin-like growth factor type-1 (IGF-1), but not glial cell line-derived neurotrophic factor (GDNF), improved survival of neurones exposed to NO donors. In addition p38 MAP kinase was activated by NO exposure and inhibition of p38 signalling led to neuronal and axonal survival effects. OCM and IGF-1 (but not GDNF) reduced p38 activation in NO-exposed cortical neurones. OCM, IGF-1 and GDNF improved axon survival in cultures exposed to NO, a process dependent on mitogen-activated protein kinase/extracellular signal-related kinase signalling. This study emphasizes that different mechanisms may underlie neuronal/axonal destructive processes, and suggests that trophic factors may modulate NO-mediated neurone/axon destruction via specific pathways.

Differential mechanisms of nitric oxide- and peroxynitrite-induced cell death.

Nitric oxide (NO) contributes to cellular degeneration in various disorders, particularly in the nervous system. NO targets cell proteins such as soluble guanylyl cyclase, but its detrimental effects are generally attributed to its reaction product with superoxide, peroxynitrite. To understand the mechanisms of NO-induced cell stress, we studied the effects of the NO donors diethylenetriamine and spermine NONOate and the peroxynitrite donor 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1) in SH-SY5Y and NG108-15 neuroblastoma cells. All three compounds induced a dose- and time-dependent decrease in viable cells, which was not blocked by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The two NONOates were approximately 15-fold more potent in SH-SY5Y than in NG108-15 cells, whereas the EC50 values of SIN-1 in SH-SY5Y and NG108-15 cells were in the same order. This led us to conclude that the mechanisms of NO and peroxynitrite did not converge. This was supported by our other findings. NONOates induced DNA fragmentation and an increase in cellular caspase-3 activity that preceded the gradual decline in cell viability. In contrast, SIN-1 induced a transient decline in ATP levels and a delayed loss of cell viability with no significant increase in caspase-3 activity or DNA laddering. Moreover, post-treatment with insulin inhibited caspase-3 activation and loss of cell viability in NONOate- but not in SIN-1-exposed cells. These findings suggest that NO is a potent toxin independent of peroxynitrite formation.

Nitric oxide stimulates Nrf2 nuclear translocation in vascular endothelium.

Vascular endothelial cells respond to nitric oxide by activating MAPK pathways and upregulating stress-activated proteins such as gamma-glutamylcysteine synthetase (gamma-GCS) and heme oxygenase-1 (HO-1). Since consensus sequences for the antioxidant response element (ARE) are found in the promoters of the gamma-GCS and HO-1 genes, we examined nuclear translocation of Nrf2, a CNC-bZIP protein which binds to and activates the ARE. We found a dramatic increase in Nrf2 nuclear translocation 1-8h following the nitric oxide donor spermine NONOate. Translocation was inhibited by pretreatment of cells with N-acetylcysteine suggesting involvement of an oxidative mechanism in this response. Translocation was also blocked by PD 98059 and SB 203580, inhibitors of ERK and p38 pathways, respectively. In addition to effects on Nrf2 subcellular localization, spermine NONOate increased Nrf2 protein levels by a mechanism which was inhibited by PD 98059. Pretreatment with N-acetylcysteine, PD 98059, and SB 203580 decreased HO-1 upregulation in spermine NONOate-treated cells. These results suggest that ERK and p38 pathways may regulate nitric oxide-mediated adaptive responses in vascular endothelium via translocation of Nrf2 and activation of the ARE.

Reversible inhibition of the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 by S-nitrosothiols.

Human arylamine N-acetyltransferase 1 (NAT1) is a polymorphic phase II xenobiotic-metabolizing enzyme which catalyzes the biotransformation of primary aromatic amines, hydrazine drugs, and carcinogens. Structural and functional studies have shown that the NAT1 and factor XIII transglutaminase catalytic pockets are structurally related with the existence of a conserved catalytic triad (Cys-His-Asp). In addition, it has been reported that factor XIII transglutaminase activity could be regulated by nitric oxide (NO), in particular S-nitrosothiols (RSNO). We thus tested whether NAT1 could be a target of S-nitrosothiols. We show here that human NAT1 is reversibly inactivated by S-nitrosothiols such as SNAP (S-nitroso-N-acetyl-DL-penicillamine). A second-order rate constant for the inactivation of NAT1 by SNAP was determined (k(inact)=270M(-1)min(-1)) and shown to be in the same range of values reported for other enzymes. The inhibition of NAT1 by S-nitrosothiols was reversed by dithiothreitol and reduced glutathione, but not by ascorbate. As reported for some reactive cysteine-containing enzymes, our results suggest that inactivation of NAT1 by S-nitrosothiols is due to direct attack of the highly reactive cysteine residue in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between these NO-derived oxidants and NAT1. Finally, our findings suggest that, in addition to the polymorphic-dependent variation of NAT1 activity, NO-derived oxidants, in particular S-nitrosothiols, could also regulate NAT1 activity.

Mesencephalic trigeminal neurons are innervated by nitric oxide synthase-containing fibers and respond to nitric oxide.

In the present study we found that mesencephalic trigeminal (Mes-V) neurons of the rat are innervated by nitrergic fibers and that nitric oxide (NO) modifies the electrophysiological properties of these cells. Mes-V neurons were surrounded by a network of fibers that contained neuronal nitric oxide synthase (nNOS); these fibers gave rise to terminal-, bouton-like structures which ended in Mes-V cells bodies. These cells, which did not display nNOS-like immunoreactivity were immunoreactive to a cGMP antibody. By performing intracellular recordings in the adult rat brain slice preparation, the effects of diethylenetriamine/NO adduct (DETA/NO) applications were examined. DETA/NO induced a depolarization that averaged 2.2 mV (range: 1-6 mV) in nine of 22 neurons. In 15 of 22 neurons (68% of the cells), there was a decrease in current threshold from 0.74 to 0.60 nA (19%; P<0.001). The excitatory effects of DETA/NO were abolished by ODQ, a blocker of soluble guanylate cyclase. Input resistance (R(in)) decreased in 80% of the cells from a mean of 24.8 to 20.6 Momega (17%; P<0.001) and the membrane time constant (tau(m)) decreased from 7.5 to 5.6 ms (25%; P<0.05). The 'sag' seen in the membrane response of these cells to current pulses was augmented during DETA/NO application. These findings indicate that there is a nitrergic innervation of Mes-V neurons and that these sensory cells are target for NO that may act on them as an excitatory neuromodulator promoting the synthesis of intracellular cGMP.

Effect of L-arginine on memory in rats.

Effect of intracerebroventricularly (icv) or subcutaneously (sc) injected L-arginine (L-Arg) on memory was determined using the procedure of passive avoidance test. Moreover, locomotor and exploratory activity was determined in rats in an open field test. We found that either the peripheral (sc) or icv administration of L-Arg significantly prolonged latency time in the passive avoidance test. This effect appeared at 20-100-fold higher doses in comparison to such effect of arginine vasopressin (AVP) observed in our previous study. This memory improving effect was not correlated with the inhibition of locomotor and exploratory activity. The effect of the lower icv dose (10 nmoles) of L-Arg was blocked by L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor. Moreover, the effect of both used doses (10 and 100 nmoles) of L-Arg was also blocked by S-methylisothiourea (Mtu), a selective inhibitor of inducible isoform of NOS. On the other hand, the effect of higher icv dose of L-Arg (100 nmoles) was prevented by 7-nitroindazole (7-NI), an inhibitor of neuronal NOS. We conclude that a uniform effect of L-Arg on memory is mediated by different isoforms of NOS, mainly by neuronal and inducible NOS.

Ginkgolide A, B, and huperzine A inhibit nitric oxide-induced neurotoxicity.

Nitric oxide (NO) is believed to play important roles in neuronal degeneration. In this study, the effects of NO on cell growth and apoptosis have been examined in human neuroblastoma cell line SK-N-SH. Sodium nitroprusside (SNP), a NO donor, was found to significantly inhibit cell growth and to induce apoptosis. The inhibitory and apoptotic activities of SNP followed a dose- and time-dependent manner. Ginkgolide A, B (GA, B), and huperzine A (Hup A), the three compounds isolated from Chinese herbs, blocked the inhibition of cell growth and apoptosis induced by SNP. The results suggest that inhibition of NO-induced neurotoxicity may be one mechanism of the above three therapeutic agents in neurodegenerative diseases.

Endothelium-dependent relaxation of rat mesenteric arterial rings by a Phoneutria nigriventer venom fraction.

Phoneutria nigriventer spider venom has been described as acting on several cardiovascular sites. In the present paper, a semi-purified fraction of this spider venom was studied to observe any contractile or relaxing effect in rat mesenteric arterial rings (MAR). Spider venom was first fractionated by gel filtration and subsequently by gradual isocratic steps in 0.1% trifluoroacetic acid. The first fraction of this last fractionation step is studied in the present paper and due to its main effect, it was named NORF (nitric oxide releasing fraction). No direct contractile effect was induced by NORF in relaxed MAR, suggesting no NORF-induced neurotransmitter release in this preparation. No significant influence of NORF was observed on concentration-response curves to phenylephrine on endothelium-denuded MAR, but a significant inhibitory shift of concentration-respense curves was observed on endothelium-preserved MAR (EC50 = 0.39 +/- 0.07 microM for control and EC50 = 0.68 +/- 0.14 microM with NORF). NORF induced concentration-dependent relaxation in endothelium-preserved phenylephrine pre-contracted MAR but not in endothelium-denuded MAR. NORF-induced relaxation was inhibited by the nitric oxide synthase inhibitor L-NAME (N(omega)-nitro-arginine methyl ester). Indomethacin or HOE-140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin) had no significant effect on NORF-induced relaxation. Acetylcholine- and NORF-induced relaxation of pre-contracted MAR were differently inhibited by atropine. The pA2 value for atropine-acetylcholine was 9.78 +/- 0.06 and that for atropine-NORF was 8.53 +/- 0.30 (P<0.01). These observations suggest that NORF induces concentration-dependent liberation of nitric oxide from MAR endothelium and that a non-muscarinic mechanism might be involved in this effect. Our data suggest no involvement of prostanoids or bradykinin in the relaxing mechanism.

SIN-1-induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay).

Human lymphocytes were exposed to increasing concentrations of SIN-1, which generates superoxide and nitric oxide, and the formation of single-strand breaks (SSB) in individual cells was determined by the single-cell gel electrophoresis assay (comet assay). A dose- and time-dependent increase in SSB formation was observed rapidly after the addition of SIN-1 (0.1-15 mM). Exposure of the cells to SIN-1 (5 mM) in the presence of excess of superoxide dismutase (0.375 mM) increased the formation of SSB significantly, whereas 1000 U/ml catalase significantly decreased the quantity of SSB. The simultaneous presence of both superoxide dismutase and catalase before the addition of SIN-1 brought the level of SSB to that of the untreated cells. Moreover, pretreatment of the cells with the intracellular Ca(2+)-chelator BAPTA/AM inhibited SIN-1-induced DNA damage, indicating the involvement of intracellular Ca(2+) changes in this process. On the other hand, pretreatment of the same cells with ascorbate or dehydroascorbate did not offer any significant protection in this system. The data suggest that H2O2-induced changes in Ca(2+) homeostasis are the predominant pathway for the induction of SSB in human lymphocytes exposed to oxidants.

Mechanisms that produce nitric oxide-mediated relaxation of cerebral arteries during atherosclerosis.

BACKGROUND AND PURPOSE: The first goal of the present study was to examine the hypothesis that relaxation of cerebral arteries to nitric oxide in primates is dependent on activation of soluble guanylate cyclase (sGC). The second goal was to determine whether the role of sGC in mediating responses to nitric oxide is altered in atherosclerosis. METHODS: Basilar arteries from normal and atherosclerotic monkeys were studied in vitro. After precontraction with prostaglandin F(2alpha) (0.1 to 1 micromol/L), concentration-response curves to authentic nitric oxide (1 nmol/L to 1 micromol/L), sodium nitroprusside (10 nmol/L to 10 micromol/L; a nitric oxide donor), and papaverine (10 nmol/L to 10 micromol/L; a non-nitric oxide, non-sGC-dependent stimulus) were generated in the presence and absence of 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 and 10 micromol/L; an inhibitor of sGC). The effect of ODQ on basal tone of basilar arteries from normal and atherosclerotic monkeys was also examined. RESULTS: Nitric oxide, sodium nitroprusside, and papaverine produced relaxation that was similar (P:>0.05) in normal and atherosclerotic monkeys. ODQ produced marked inhibition (P:<0.05) of vasorelaxation in response to nitric oxide and nitroprusside but not papaverine. For example, relaxation of the basilar artery in response to nitric oxide (0.1 micromol/L) was inhibited by approximately 85% and 73% by ODQ (1 micromol/L) in normal and atherosclerotic monkeys, respectively. ODQ produced contraction of the basilar arteries, and the increase in tension to ODQ was greater in normal (2.7+/-0.3 g; mean+/-SE) than in atherosclerotic monkeys (1.4+/-0.4 g; P:<0.05). In contrast, contraction to prostaglandin F(2alpha) was similar in the basilar artery from normal and atherosclerotic monkeys. CONCLUSIONS: These findings suggest that (1) relaxation of cerebral arteries in primates in response to nitric oxide is normally dependent, in large part, on activation of sGC and (2) the influence of sGC (via reduced production and/or activity of basal nitric oxide) on cerebral vascular tone is reduced in atherosclerosis.