Effect of thiol reagents and ionizing radiation on the permeability of erythrocyte membrane for spin-labeled non-electrolytes

Four different thiol reagents: p-chloromercuribenzoic acid (pCMB), mercuric chloride (HgCl2), N-ethylmaleimide (NEM), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were employed as agents modifying the transport of a hydrophilic and hydrophobic non-electrolyte spin labels: 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) into bovine erythrocytes. Gamma-irradiation of erythrocytes amplified the effects of pCMB, HgCl2 and NEM of inhibition of TEMPOL transport and attenuated them in the case of TEMPO transport. These results suggest that the transport of TEMPOL across the erythrocyte membrane is controlled by both superficially and more deeply located membrane -SH groups while only superficial -SH groups control the transport of TEMPO. The lower extent of inhibition of TEMPO transport indicates a higher contribution of diffusion through the lipid phase to the transport of TEMPO across the erythrocyte membrane as compared with TEMPOL.     

Effect of gamma radiation on the transport of spin-labeled compounds across the erythrocyte membrane

Summary The effect of ionizing radiation on the non-electrolyte, anion and cation permeability of the erythrocyte membrane was studied by measurement of the reduction rate of appropriate nitroxyl derivatives. Irradiation of bovine erythrocytes in the dose-range of 2–50 krad resulted in a regular dose-dependent increase in the reduction rates of a cation (TEMPO-choline) and a hydrophobic non-electrolyte (TEMPO), and non-regular changes in the reduction rate of a hydrophilic non-electrolyte (TEMPOL). The permeation constant for TEMPO-choline also showed a non-regular response to radiation, similar to the response pattern of other red blood cell parameters. These results also demonstrate that the effects of radiation on the transport of various solutes can be used as a means of distinguishing between different channels of membrane transport.     

Effect of hyperthermia on lymphocyte membrane. 2. Spin-labelled non-electrolyte permeability

The effect of elevated temperatures on the permeability of non-electrolyte spin labels, hydrophilic TEMPOL and more hydrophobic TEMPO across the porcine lymphocyte membrane was investigated. In the range of 41-44 degrees C, temperature-induced changes in the permeation constant were lower for TEMPO than TEMPOL. The data obtained may suggest that the permeability of spin labels across the membrane is sensitive to changes of temperature especially above 43 degrees C.     

Effect of cupric ions on the permeability of erythrocyte membrane to non-electrolyte spin labels

The addition of cupric ions caused decreased permeability to hydrophilic molecules and increased permeability to hydrophobic molecules. These results suggest that TEMPOL penetrates the erythrocyte in a different way than TEMPO. Penetration of TEMPOL is controlled by-SH groups, while TEMPO probably diffuses through the lipid bilayer. Cupric ions increase the permeability of erythrocyte membranes to both non-electrolytes in vivo.     

Interaction of dipyridamole with micelles of lysophosphatidylcholine and with bovine serum albumin: fluorescence studies.

The interaction of the coronary vasodilator dipyridamole with biological systems, protein and membranes has been studied through optical absorption and fluorescence spectroscopies. Using the analysis of the spectra and fluorescence intensity of dipyridamole (DIP) in solution, the interaction of this compound with the transport protein albumin (BSA) and with a model of cell membranes, namely micelles of lysophosphatidylcholine (L-PC), was investigated. Measurements were performed at pH 5.0 and pH 7.0 where the molecule of DIP is fully protonated and partially protonated, respectively. The quenching of fluorescence with nitroxide-stable radicals 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) as well as with acrylamide and iodide allowed the localization of the drug in the polar interface of micelles. Quenching by acrylamide and iodide in L-PC micelles demonstrated the effect of micelle protonation which increased the accessibility of iodide to the chromophore. An effective association constant was obtained both at pH 7.0 (7.5 x 10(3) M-1) and pH 5.0 (2.5 x 10(3) M-1) and a very good agreement with the proposed binding model was observed. The quantum yields of fluorescence data agree very well with the fluorescence lifetimes. The measurement of lifetimes was important to understand the kinetic data obtained from Stern-Volmer plots both of radical, acrylamide and iodide quenching of fluorescence. It was observed that, in the presence of micelles, the kq value increased for TEMPO while decreased for TEMPOL. This result, together with the vanishing solubility of DIP in saturated hydrocarbons and the preferential partition of TEMPO in micelles, suggested the localization of DIP in the polar micellar interface. This is also supported by the enhanced iodide quenching at pH 5.0, constancy of acrylamide quenching in the range of pH 7.0-5.0 and the partition of TEMPO and TEMPOL in SDS micelles. The association constant of DIP to BSA was also estimated both at pH 7.0 (2 x 10(4) M-1) and pH 5.0 (4 x 10(3) M-1). Quenching studies with nitroxide radicals, acrylamide and iodide also suggested the binding of the drug to a hydrophobic region of the protein. At pH 5.0, the protein undergo a conformational change which leads to a loosening of the overall structure so that the accessibility of the nitroxide radicals for DIP is increased at this pH. The differences in kq values at pH 7.0 and pH 5.0 suggested that at pH 7.0 the chromophore is protected in the protein site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxidant stress in malaria as probed by stable nitroxide radicals in erythrocytes infected with Plasmodium berghei. The effects of primaquine and chloroquine

Erythrocytes from normal mice and mice infected with the malarial parasite Plasmodium berghei reduce the water-soluble spin probes 2,2,6,6-tetramethylpiperidine-4-hydroxy-N-oxyl (TEMPOL), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and 2,2,6,6-tetramethylpiperidine-4-keto-N-oxyl (TEMPONE) at similar rates under both air and N2 atmospheres. The ESR signal of the lipid-soluble spin probe 5-doxyl-stearate is stable on incorporation into erythrocytes from normal mice. In contrast, parasitized red cells reduce this nitroxide probe, at a rate which increases with the level of parasitemia. Inhibitors of electron transport such as KCN and NaN3, increase the rate of reduction. We propose that nitroxide reduction occurs via the electron transport chain in the parasite. The antimalarial drug primaquine causes reduction of both water-soluble and lipid-soluble spin probes. This action of primaquine is independent of its ability to release H2O2 from oxyhemoglobin, and is ascribed to the ability of primaquine to accelerate flux through the hexose monophosphate shunt. The increased production of NADPH results in increased rates of reduction of the nitroxide radicals. Methylene blue, which also increases flux through the shunt, is even more effective than primaquine at reducing the nitroxides. Chloroquine has no such effect. Parasitized mice treated with chloroquine six hours prior to ESR measurements show less nitroxide reducing capacity than do untreated mice. Chloroquine is known to decrease flux through the hexose monophosphate shunt. The metabolic influences of the two antimalarial drugs are, thus, quite different.     

A mitochondria-targeted nitroxide is reduced to its hydroxylamine by ubiquinol in mitochondria

Piperidine nitroxides such as TEMPOL act as antioxidants in vivo due to their interconversion among nitroxide, hydroxylamine, and oxoammonium derivatives, but the mechanistic details of these reactions are unclear. As mitochondria are a significant site of piperidine nitroxide metabolism and action, we synthesized a mitochondria-targeted nitroxide, MitoTEMPOL, by conjugating TEMPOL to the lipophilic triphenylphosphonium cation. MitoTEMPOL was accumulated several hundred-fold into energized mitochondria where it was reduced to the hydroxylamine by direct reaction with ubiquinol. This reaction occurred by transfer of H() from ubiquinol to the nitroxide, with the ubisemiquinone radical product predominantly dismutating to ubiquinone and ubiquinol, together with a small amount reacting with oxygen to form superoxide. The piperidine nitroxides TEMPOL, TEMPO, and butylTEMPOL reacted similarly with ubiquinol in organic solvents but in mitochondrial membranes the rates varied in the order: MitoTEMPOL > butylTEMPOL > TEMPO > TEMPOL, which correlated with the extent of access of the nitroxide moiety to ubiquinol within the membrane. These findings suggest ways of using mitochondria-targeted compounds to modulate the coenzyme Q pool within mitochondria in vivo, and indicate that the antioxidant effects of mitochondria-targeted piperidine nitroxides can be ascribed to their corresponding hydroxylamines.     

Prooxidative effects of TEMPO on human erythrocytes

The prooxidative effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were observed in human erythrocytes. Incubation of red blood cells with the membrane-permeable TEMPO leads to a decrease in the concentration of intracellular reduced glutathione, accompanied by the reduction of TEMPO. Extracellular ferricyanide inhibited the loss of glutathione and reduction of TEMPO. TEMPO induced glutathione release from the cells and oxidation of hemoglobin to methemoglobin; ferricyanide prevented these effects. These results indicate that TEMPO may act as an oxidant to erythrocytes, whilst extracellular ferricyanide protects against its effects.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Immunomodulating effect of autohaemotherapy (a literature review)

An analysis is presented of experimental and clinical data from different authors on the stimulating effect of autohaemotherapy with regard to the immunological reactivity of humans and animals as well as in vitro experiments with lymphocytes. Erythrolysate has been found to exert a more powerful effect than intact erythrocytes. The stimulating effect of autohaemotherapy on both irradiated and non-irradiated animals manifests itself in an increase in resistance to infection (increased LD50 in experimental infection), enhanced production of antibodies to microbial and tissue antigens and activated functioning of cell-mediated immune defence mechanisms. The favourable influences on radioresistance and the antitumour effect of authohaemotherapy are described. Induced desensitization plays an important part in the mechanism of action of autohaemotherapy. The administration of large doses of erythrocytes or of erythrolysate results in immunosuppression. Autohaemotherapy does not cause side effects and is feasible both on an in-and out-patient basis.     

Electron paramagnetic resonance of electron transport in photosynthetic systems. XI. Effects of photosynthetic control: dependence of the rate of electron transport on the energization of bean chloroplast thylakoid membrane

The kinetics of light-induced P700 redox transients in bean chloroplast was studied. It has been shown that the rate of electron transport decreased during few seconds of illumination of coupled chloroplasts without addition of ADP and inorganic phosphate. The evidence were obtained that there is a feedback inhibition of electron transport governed by the internal pH of thylakoid. This results in the overshoot in the kinetics of P700 redox transients induced by continuous actinic light. Under the phosphorylation condition (addition of Mg-ADP and inorganic phosphate) the effect of decreasing of the rate of electron transport between two photosystems was not observed. Addition of uncouplers (FCCP or gramicidine) also increased the steady-state rate of noncyclic electron transport. After adding only Mg-ADP (without phosphate) or Mg-ATP to coupled chloroplasts the effect of the light-driven inhibition of electron transport was observed as in the case of chloroplasts without any additions. We showed that the regulation for the electron transport rate was realized at the step of the plastoquinol oxidation by photosystem 1. Light-driven energization of the thylakoid membrane also leads to the the slowing of the reduction of spin label TEMPO. Evidences were obtained that TEMPO interacts with the semiquinone localized in the acceptor side of photosystem 2. From the comparative study of P700+ and TEMPO reduction by photosystem 2 we have concluded that there are two points of inhibitory action of DCMU localized at the acceptor and donor sides of photosystem 2. The mechanisms of photosynthetic control and the role of transmembrane proton gradient for energy transmission in chloroplasts are discussed.     

Action of calcium channel and beta-adrenergic blocking agents in bilayer lipid membranes

The action of beta-adrenergic blockers (propranolol, exprenolol, metoprolol, sotalol, atenolol, timolol) and calcium-channel blockers (verapamil, diltiazem) on the electrical properties and fluidity of bilayer lipid membranes (BLM and liposomes) has been investigated. When antibiotic ionophore substances were used as a probe, the electrical measurements showed that many of the drugs inhibited the cation transport across the membrane facilitated by the mobile carrier valinomycin, while having no significant effect on the cation transport through channels formed by gramicidin. The ability of the drugs to decrease the carrier-dependent membrane conductance was correlated to their partition into the lipid bilayer and the magnitude of transmembrane potential induced by them. In the TEMPO ESR spectral measurements, a number of beta-adrenergic and calcium blockers showed the fluidizing effect on liposomes composed of different lipids. The drug concentration required for a detectable change in TEMPO spectra parameter (f) was rather high (0.01 M verapamil), and the variation of pH from 6.5 to 3.0 did not affect the fluidizing effect of the drugs.     

Effect of membrane potential on band 3 conformation in the human erythrocyte membrane detected by triplet state quenching experiments.

The triplet lifetime and absorption anisotropy decay of eosin-labeled band 3 was measured in resealed erythrocyte ghosts. Membrane potentials were generated by the addition of valinomycin in the presence of a K+ gradient. Neither negative nor positive membrane potentials had any detectable effect on the rotational diffusion of band 3 nor on the eosin triplet lifetime. The membrane potential did, however, affect quenching of the eosin triplet state by I- and TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Quenching was enhanced by a negative membrane potential (negative inside) and reduced by a positive membrane potential. In addition, it was found that a negative membrane potential enhanced the efficiency of eosin labeling of band 3 in intact erythrocytes. A positive membrane potential had the opposite effect. These results indicate that the eosin binding site on band 3 becomes more accessible to the extracellular aqueous phase in the presence of a negative membrane potential and less accessible in the presence of a positive membrane potential. Quenching by I- and TEMPO of the triplet state of eosin-labeled band 3 was further investigated as a function of pH. Quenching by TEMPO and its dependence on membrane potential were relatively insensitive to pH. In contrast, the rate of quenching by I- showed a marked decrease over the range pH 5.5-9.5. Moreover, the effect of a negative membrane potential on I- quenching also varied with pH. These results are discussed on the supposition that the eosin probe is located in the anion access channel of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.     

Reactive effect of low intensity He-Ne laser upon damaged ultrastructure of human erythrocyte membrane in Fenton system by atomic force microscopy

To find out the mechanism of modulating the deformability of erythrocytes with low intensity He-Ne laser action, we studied the effect of low intensity He-Ne laser on the ultrastructure of human erythrocyte membrane. Erythrocytes were treated with free radicals from a Fenton reaction system before exposing them to low intensity He-Ne laser. The ultrastructure of damaged erythrocyte membrane was examined by atomic force microscopy. The results showed that the erythrocyte membrane became very rough and the molecules on the surface of the membrane congregated into particles of different magnitudes sizes after treating with free radicals. Comparing the degree of congregation of the molecular particles in the non-irradiated group and the He-Ne laser irradiated （9 mW and 18 mW） group, we found the average size of molecular particles in the laser irradiated group was smaller than that in the non-irradiated group, indicating that the low intensity laser had repairing function to the damage of erythrocyte membrane produced by the free radicals.     

Evidence for different transport systems for oxidized glutathione and S-dinitrophenyl glutathione in human erythrocytes

The effect of oxidized glutathione (GSSG) on the ATP-dependent transport of S-dinitrophenyl glutathione (Dnp-SG) by inside-out vesicles prepared from human erythrocytes and by intact erythrocytes has been studied. It is demonstrated that the transport of Dnp-SG is not inhibited by GSSG in either intact erythrocytes or in inside-out vesicles. These results suggest that Dnp-SG and GSSG are transported out of human erythrocytes by separate systems.     

Effect of erythropoietin on the glucose transport of rat erythrocytes and bone marrow cells

The effect of Ep on radioactive glucose and methyl-alpha-D-glucoside transport by rat erythrocytes and bone marrow cells were studied. There is initial linearity followed by saturation kinetics of [14C]glucose transport by the erythrocytes of starved and starved plus Ep-treated rats at different concentrations of glucose. Starvation caused slight inhibition of glucose transport which increased markedly on Ep administration to starved rats. Normal animals failed to show any significant change in glucose transport after Ep treatment. Methyl-alpha-D-glucoside inhibited the Ep-stimulated glucose transport significantly. Ep also stimulated the transport of radioactive methyl-alpha-D-glucoside which was competitively inhibited in presence of D-glucose. Glucose transport in erythrocytes was found to be sensitive to metabolic inhibitors like azide and DNP. A sulfhydryl reagent and ouabain also inhibited the transport process. Ep stimulated glucose and methyl-alpha-D-glucoside transport in the bone marrow cells of starved rats. The sugar analog competitively inhibited the glucose transport in bone marrow cells and vice versa.     

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N^ω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.     

Identification by an EPR technique of decreased mitochondrial reducing activity in puromycin aminonucleoside-induced nephrosis

The temporal changes in the electron paramagnetic resonance (EPR) signal intensities of a nitroxide radical, 4-hydroxy 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidney in rat puromycin aminonucleoside (PAN) nephrosis were investigated in vivo and in vitro. The rats of the PAN nephrosis group received intraperitoneal injections of PAN at 75 mg/kg body weight while those of control group received saline. The in vivo renal half-lives of TEMPOL were calculated from the decay curve of EPR signal intensities after the intravenous injection of the TEMPOL solution. The mitochondrial half-lives were obtained from the decay curve of the EPR signals after mixing the mitochondrial fraction of the kidney and TEMPOL solution. The in vivo half-lives of TEMPOL of the kidney from 7 to 14 d after PAN administration were significantly longer than those of the controls. The mitochondrial half-lives of TEMPOL on the 9th day after the PAN administration prolonged remarkably compared to the controls (378 +/- 69 vs. 676 +/- 183 s, p <.01). These findings indicate that the in vivo and mitochondrial reducing activity in PAN treated rats decreased markedly, because the half-life of TEMPOL in the kidney reflects the renal reducing activity.