Dermal application of nitric oxide in vivo: kinetics, biological responses, and therapeutic potential in humans.

Many local hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). Previous findings point to a therapeutic potential of dermal NO application in the treatment of hemodynamic disorders, but no reliable data are available on the mechanisms, kinetics, or biological responses relating to cutaneous exposure to NO in humans in vivo. Here we show that, owing to its excellent diffusion capacity, cutaneously applied NO rapidly penetrates the epidermal barrier in significant amounts, strongly enriching skin tissue and blood plasma with its vasoactive derivates. In parallel, it significantly increased vasodilatation and blood flow and reduced thrombocyte aggregation capacity. Data presented here for the first time show that, in humans, dermal application of NO has strong potential for use in the therapy of local hemodynamic disorders arising from insufficient availability of NO or its bioactive derivates.

Nitric oxide synthase reduces nitrite to NO under anoxia.

Cultured bEND.3 endothelial cells show a marked increase in NO production when subjected to anoxia, even though the normal arginine pathway of NO formation is blocked due to absence of oxygen. The rate of anoxic NO production exceeds basal unstimulated NO synthesis in normoxic cells. The anoxic release of NO is mediated by endothelial nitric oxide synthase (eNOS), can be abolished by inhibitors of NOS and is accompanied by consumption of intracellular nitrite. The anoxic NO release is unaffected by the xanthine oxidase inhibitor oxypurinol. The phenomenon is attributed to anoxic reduction of intracellular nitrite by eNOS, and its magnitude and duration suggests that the nitrite reductase activity of eNOS is relevant for fast NO delivery in hypoxic vascular tissues.

NO trapping in biological systems with a functionalized zeolite network.

Zeolite-Y powder has been functionalized with ferric iron-diethyldithiocarbamate complexes and applied to trap nitric oxide radicals in liquids and biological systems. The complexes have been assembled in situ in the pores of zeolite-Y and act as traps for nitric oxide radicals. The resulting mononitrosyl-iron complexes form a mixture of diamagnetic ferric and paramagnetic ferrous complexes. The yield of trapped NO may be determined ex situ using electron paramagnetic resonance. The material may be anchored on solid surfaces, mixed into a composite or compressed into small pellets. The material was used to detect endogenous NO in endothelial cell cultures and spinach leaves. The sensitivity of the functionalized zeolite is significantly better than that achieved in conventional trapping of NO with iron-diethyldithiocarbamate complexes.

Ferric saccharate induces oxygen radical stress and endothelial dysfunction in vivo.

BACKGROUND: Intravenous iron supplementation is used widely in haemodialysis patients. However, nontransferrin-bound iron (NTBI), which increases after intravenous supplementation of ferric saccharate, has been suggested to act as a catalytic agent in oxygen radical formation in vitro and may thus contribute to endothelial impairment in vivo. MATERIALS AND METHODS: In 20 healthy volunteers the effect of 100 mg ferric saccharate infusion was investigated. Vascular ultrasound was used to assess endothelium-dependent vasodilatation at baseline, and 10 and 240 min after ferric saccharate infusion. Whole blood was collected to measure NTBI and in vivo radical formation was assessed by electron spin resonance. A time-control study was performed using saline infusion. RESULTS: Infusion of ferric saccharate induces a greater than fourfold increase in NTBI, as well as a transient, significant (P < 0.01) reduction of flow-mediated dilatation 10 min after infusion of ferric saccharate, when compared with saline. The generation of superoxide in whole blood increased significantly 10 and 240 min after infusion of ferric saccharate by, respectively, 70 and 53%. CONCLUSIONS: Iron infusion at a currently used therapeutic dose for intravenous iron supplementation leads to increased oxygen radical stress and acute endothelial dysfunction.

Placental superoxide is increased in pre-eclampsia.

One of the current hypotheses on the pathophysiology of pre-eclampsia (PE) states that the placenta secretes one or more cytotoxic factors resulting in maternal endothelial dysfunction. Among the candidate factors are the products of increased oxidative stress. Although there is circumstantial evidence of such an increase, direct evidence is still lacking. Electron paramagnetic spin trap resonance (EPR), the most direct method to detect free radicals in tissues, was used to measure superoxide levels in placentae from normal pregnancies (n=13) and pregnancies complicated by PE (n=10). The superoxide level was significantly increased in the placental tissue of pre-eclamptic women. Moreover, upon inhibition of Cu-Zn superoxide dismutase (SOD) activity the relative increase of the superoxide levels was significantly smaller in the placentae from the PE patients, implying decreased basal Cu-Zn SOD activity. These findings lend direct support to the hypothesis that oxidative stress in placental tissue is increased in PE.

Antioxidant capacity of mononitrosyl-iron-dithiocarbamate complexes: implications for NO trapping.

Using EPR spectroscopy, we show that the water-soluble mononitrosyl iron complexes with N-methyl-D-glucamine dithiocarbamate (MNIC-MGD) ligands can easily react with superoxide and with peroxynitrite. The reaction with superoxide transforms the paramagnetic MNIC-MGD complex into an EPR silent complex with a reaction rate of 3 x 10(7) (M.s)(-1). Suppletion of ascorbate partially restores the complexes to their original paramagnetic state. We propose that the reaction of MNIC-MGD with either superoxide or peroxynitrite leads to identical EPR silent complexes. Our results have important implications for the technique of NO trapping in biosystems with Fe-dithiocarbamate complexes, where mononitrosyl-iron complexes (hydrophilic as well as hydrophobic) are formed as adducts in the trapping reaction. This principle is illustrated by NO trapping experiments on viable cultured endothelial cells. We find that MNIC-MGD acts as a very potent and water-soluble antioxidant with an efficiency exceeding most SOD mimics. Moreover, by accounting for the EPR silent fraction of iron complexes, the sensitivity of NO trapping can be enhanced considerably. The method was demonstrated for hydrophobic iron-dithiocarbamate complexes in endothelial cell cultures, where sensitivity for NO detection was enhanced by a factor of 5.

Folic acid reverts dysfunction of endothelial nitric oxide synthase.

5-methyltetrahydrofolate (MTHF), the active form of folic acid, has been reported to restore NO status in hypercholesterolemic patients. The mechanism of this effect remains to be established. We assessed the effects of L- and D-MTHF on tetrahydrobiopterin (BH(4))-free and partially BH(4)-repleted endothelial NO synthase (eNOS). Superoxide production of eNOS and the rate constants for trapping of superoxide by MTHF were determined with electron paramagnetic resonance using 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) as spin trap for superoxide. NO production was measured with [(3)H]arginine-citrulline conversion or nitrite assay. The rate constants for scavenging of superoxide by L- and D-MTHF were similar, 1.4 x 10(4) ms(-1). In BH(4)-free eNOS, L- and D-MTHF have no effect on enzymatic activity. In contrast, in partially BH(4)-repleted eNOS, we observe a 2-fold effect of MTHF on the enzymatic activity. First, superoxide production is reduced. Second, NO production is enhanced. In cultured endothelial cells, a similar enhancement of NO production is induced by MTHF. In the present study, we show direct effects of MTHF on the enzymatic activity of NO synthase both in recombinant eNOS as well as in cultured endothelial cells, which provides a plausible explanation for the previously reported positive effects of MTHF on NO status in vivo.

Dinitrosyl iron complexes with thiol-containing ligands and S-nitroso-D,L-penicillamine as inductors of heat shock protein synthesis in H35 hepatoma cells.

The concentration-dependent effect of various nitric oxide donors on synthesis of different heat shock proteins was evaluated in Reuber H35 hepatoma cells and their heat shock protein-inducing ability was compared with the effect of a heat shock. A 6 h incubation of H35 cells with the dimeric (diamagnetic) form of dinitrosyl iron complex with glutathione or N-acetyl-L-cysteine activated synthesis of various heat shock proteins, heat shock protein 28, 32, 60, 70, 90 and 100. Synthesis of these proteins was evaluated by [35S]methionine and [35S]cysteine labelling with subsequent separation of proteins by polyacrylamide gel electrophoresis. The dinitrosyl iron complex with glutathione appeared to be the most efficient inductor of heat shock protein synthesis and initiated the synthesis of heat shock protein 28 even more efficiently than a 30 min heating of cells. In the same experiments, S-nitroso-D,L-penicillamine exerted a considerably lesser effect on the synthesis of heat shock proteins. It was suggested that the active moiety of dinitrosyl iron complexes as inductors of heat shock protein synthesis is represented by their Fe+(NO+)2 groups which move to thiol groups of the proteins participating in the initiation of heat shock protein synthesis.

Origin of superoxide production by endothelial nitric oxide synthase.

Using fluorescence optical and electron spin resonance spectroscopy, we have investigated the production of superoxide by bovine endothelial nitric oxide synthase (NOS). In contrast to neuronal NOS, the heme moiety is identified as the exclusive source of superoxide production by endothelial NOS. Thus, calmodulin-mediated enzyme regulation affects production of nitric oxide and superoxide simultaneously and inseparably. The balance between the nitric oxide/superoxide reaction pathways may be shifted by addition of exogenous heme-specific agents, such as tetrahydrobiopterin. Our results have direct relevance for the pathophysiology of atherosclerosis.

Oxygen radical stress in vascular disease: the role of endothelial nitric oxide synthase.

Impaired nitric oxide (NO) activity in proatherosclerotic states has been suggested to be caused mainly by increased degradation of NO by oxygen radicals. In recent years, endothelial NO synthase has been identified as a system that contributes to oxygen radical stress under pathophysiologic conditions. We discuss the origin of NO synthase-derived superoxide production, as well as possibilities to modulate (pathologic) shifts in NO/superoxide production by endothelial NO synthase.

Rotational motion of Ca-ATPase monitored by electron spin echoes.

Electron spin echoes are used to study the dynamics of different aggregational forms of spin-labeled Ca-ATPase in the sarcoplasmic reticulum membrane. The 2D-ESE measurements are sensitive to motions on the microsecond time scale. The motional information is extracted from the variation of the echo decays across the CW-ESR absorption spectrum. The motional contribution to the decays is described by assuming that the Ca-ATPase molecule is perfectly oriented along the normal to the membrane surface and only undergoes rotational motion about its long axis. The echo-amplitude decays have been evaluated in the time domain by solving the Bloch equations for the stochastic spin Hamiltonian on making use of stochastic trajectories for the orientational behavior of the spin-labeled protein. This approach provides a useful insight into the information provided by the 2D-ESE measurements and affords a direct comparison of the results obtained with different experimental techniques. It is shown that the 2D-ESE technique monitors the orientational motions of dimers or larger aggregates of Ca-ATPase molecules whose rotational correlation times vary between 200 microseconds and 1 ms for the temperature range between 37 and 4 degrees C.

Effect of lipid molecular structure and gramicidin A on the core of lipid vesicle bilayers. A time-resolved fluorescence depolarization study.

We have investigated the molecular orientational order and reorientational dynamics of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in the core of the membrane bilayer. Vesicles of lipids of varying unsaturation and headgroup (POPC, DOPC, DLPC, DLLPC, EGGPG, DOPG, DGDG, and SQDG) were studied using the time-resolved fluorescence anisotropy of DPH. Generally, values of the second order parameter for DPH are found to be very small. However, this should not be interpreted as DPH having low orientational order as witnessed by large values of the next relevant order parameter . This implies considerable transverse populations of DPH molecules within the bilayer. In phosphatidylcholines with an acyl chain of 18 carbon atoms, the value of for DPH decreases with increasing lipid unsaturation and even attains negative values. No effect of the lipid headgroup on the order and dynamics of DPH is detected. Furthermore, we study the peptide-lipid interaction of the hydrophobic antibiotic gramicidin A (gA) in DOPC vesicles using DPH. The nonchannel conformation has an ordering effect on DPH in the bilayer core, which the channel confirmation lacks. This can be understood in terms of the geometrical shape of the gA dimer, as shown previously with the probes TMA-DPH and DPHPC [Muller, J. M., et al. (1995) Biochemistry 34, 3092]. We find that for DPH data the conventional Brownian rotational diffusion (BRD) model and the compound motion model (CMM) give equivalent fits. In this respect, DPH differs from TMA-DPH and DPHPC, for which probes only the CMM allowed a consistent interpretation of the molecular orientation.

Effect of gramicidin A on structure and dynamics of lipid vesicle bilayers. A time-resolved fluorescence depolarization study.

We investigated the effects of the hydrophobic small peptide antibiotic gramicidin A (gA) on the properties of vesicle bilayers in the liquid crystalline state. Time-resolved fluorescence anisotropy experiments were performed with unilamellar vesicles of the lipids DMPC, POPC, DOPC, EGGPC, DLPC, DOPG, and SQDG containing various concentrations of gA in two different conformations using TMA-DPH and DPHPC as fluorescent probes. These analogues of DPH were taken to study the gA induced change in the structural and dynamical properties of the lipid bilayer in different portions of the hydrophobic region. The time-resolved anisotropy data were analyzed using the recently introduced compound motion model [van der Sijs, D. A., et al. (1993) Chem. Phys. Lett. 216, 559; Muller, J. M., et al. (1994) Chem. Phys. 185, 393]. In general, gA raises the order and reduces the rotational diffusion coefficient for the probes in the bilayer. In DOPC vesicles this ordering effect of gA on the bilayer is found to depend on both the conformation of the peptide and the depth in the bilayer at which the order is probed. This significant effect of gA conformation on the lipid order parameter profile suggests that the shape of the gA dimer in the bilayer, which is determined by its conformation, affects the order of the adjacent DOPC lipid acyl chains.

Copper, zinc-superoxide dismutase and hydrogen peroxide: a hydroxyl radical generating system.

Because superoxide (O2-.) is a mediator of inflammation, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) has been employed as an anti-inflammatory compound. However, Cu,Zn-SOD can increase intra- and extracellular H2O2. This may react with the Cu atom of SOD in a Fenton-type reaction producing the hydroxyl radical (.OH). With a non-physiological concentration of H2O2 (0.8 mmol/l) to stimulate chemiluminescence (CL) at a level < 2 mV, it was observed that the addition of Cu,Zn-SOD (100 micrograms/ml) yielded an increase of 204.7 +/- 78.2 mV (P < 0.05). This increase in CL depended on the concentrations of H2O2 and Cu,Zn-SOD and was only seen with luminol (reacts with O2-. and .OH) but not with lucigenin (reacts with O2-.). No CL was observed when Cu,Zn-SOD was heat inactivated, or when Mn-SOD was used. Dissipators of H2O2, copper chelators and .OH scavengers attenuated this CL. In electron paramagnetic resonance, with the use of the spin-trap dimethylpyrroline-N-oxide (DMPO), it was demonstrated that, in the reaction between H2O2 and Cu,Zn-SOD, .OH was generated. The oxidation of keto-methylthiobutyric acid (KMB) to ethylene, assessed by gas chromatography, demonstrated that H2O2/Cu,Zn-SOD-generated .OH can react with KMB and not only with the SOD molecule itself. We conclude that H2O2 reduces SOD-bound Cu2+ to Cu1+ which, in reaction with H2O2 catalyses its reduction to OH. Whether this 'pro-inflammatory' reaction occurs in vivo remains to be established.

The interpretation of the time-resolved fluorescence anisotropy of diphenylhexatriene-phosphatidylcholine using the compound motion model.

Time-resolved fluorescence anisotropy experiments on lipid membranes can provide estimates of the molecular order and motion on microscopic scales. For the analysis of anisotropy data the so-called compound motion model was recently introduced to overcome problems with conventional models. We show that this novel model gives good fits for the time-resolved anisotropy of the fluorescent probe diphenylhexatriene-phosphatidylcholine (DPHPC) and can be successfully used to interpret experiments with DPHPC embedded in small unilamellar vesicles of the lipids DMPC, POPC, DOPC, DLPC, DERPC, DOPE, POPE, EGGPG and SQDG. The lifetime and order parameters are found to be intermediate between those found for the related DPH and TMA-DPH fluorescent probes, while the rotational diffusion of DPHPC is much slower. These findings can be rationalised in terms of the position of the DPH-fluorophore of DPHPC in the bilayer.

An electron paramagnetic resonance study of the antioxidant properties of the nitroxide free radical TEMPO.

Previous reports of superoxide scavenging and ferroxidase-like activity of nitroxide free radicals have greatly increased interest in the ability of these compounds to protect cells against oxidative cellular damage. In the present study we investigated the antioxidant properties of the six membered nitroxide 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in various assays. TEMPO (5 mM) inhibited the hydroxyl radical mediated generation of ethylene from 2-keto-4-methylthiobutyric acid to 31.2 +/- 4.0% of control values. Furthermore, we noted that TEMPO had the ability to maintain iron in its ferric form, a finding with strong implications for the interpretation of the ferricytochrome c reduction assay. TEMPO may be reduced to an electron paramagnetic resonance silent hydroxylamine by a number of pathways. TEMPO absorption intensity decay (TAID) was monitored to investigate the effects of hydrogen peroxide on Cu,Zn-SOD. TEMPO was found to effectively scavenge or suppress formation of hydroxyl radicals inside Cu,Zn-SOD. The generation of hydroxyl radical was confirmed by employing the conventional spin trapping agent DMPO. Using radical scavengers unable to penetrate the Cu,Zn-SOD enzyme (e.g., mannitol, ethanol, albumin) or compounds with access to copper within the Cu,Zn-SOD enzyme (azide and cyanide), we could not detect hydroxyl radicals outside the enzyme. Finally, since the electron paramagnetic resonance absorption intensity is directly proportional to the concentration of TEMPO spins, loss of absorption intensity provided information concerning radical-mediated processes. Therefore, the decay kinetics of TEMPO may be used as a very sensitive alternative to conventional spin traps.

Doxorubicin-mediated free radical generation in intact human tumor cells enhances nitroxide electron paramagnetic resonance absorption intensity decay.

The decay of nitroxide spin label electron paramagnetic resonance (EPR) absorption intensity was used to investigate the doxorubicin-mediated intracellular generation of free radicals. The effects of 50-500 micrograms/ml doxorubicin on human tumor cells (MCF-7, breast cancer cells, and HL-60, promyelocytic leukemia, cells) were studied by measuring 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) absorption intensity decay (TAID) at a TEMPO concentration of 10 microM. Doxorubicin accelerated the TAID in both cell lines with a detection limit of 50 micrograms/ml for MCF-7 cells and 500 micrograms/ml doxorubicin for HL-60 cells. Preincubation of cells with the iron chelating agent, deferoxamine (5 mM), partially prevented the effects of doxorubicin on the TAID. Catalase and copper, zinc-superoxide dismutase (Cu,Zn-SOD) had no influence on the effects of doxorubicin on the TAID in intact cells. However, Cu,Zn-SOD completely abolished the effects of doxorubicin on the TAID in a MCF-7 cell-free system. Our findings suggest that doxorubicin mediates the intracellular generation of O2.- and that iron is involved in this process.

On the interpretation of fluorescence anisotropy decays from probe molecules in lipid vesicle systems.

Measurements of fluorescence depolarization decays are widely used to obtain information about the molecular order and rotational dynamics of fluorescent probe molecules in membrane systems. This information is obtained by least-squares fits of the experimental data to the predictions of physical models for motion. Here we present a critical review of the ways and means of the data analysis and address the question how and why totally different models such as Brownian rotational diffusion and "wobble-in-cone" provide such convincing fits to the fluorescence anistropy decay curves. We show that while these models are useful for investigating the general trends in the behavior of the probe molecules, they fail to describe the underlying motional processes. We propose to remedy this situation with a model in which the probe molecules undergo fast, though restricted local motions within a slowly rotating cage in the lipid bilayer structure. The cage may be envisaged as a free volume cavity between the lipid molecules, so that its position and orientation change with the internal conformational motions of the lipid chains. This approach may be considered to be a synthesis of the wobble-in-cone and Brownian rotational diffusion models. Importantly, this compound motion model appears to provide a consistent picture of fluorescent probe behavior in both oriented lipid bilayers and lipid vesicle systems.