Carbamylation of proteins leads to alterations in the membrane structure of erythrocytes

The effect of the sodium cyanate-induced carbamylation (carbamoylation) of proteins in erythrocytes was studied using spin labelling and spectrophotometric methods. The experiments were conducted in whole blood and in erythrocytes in phosphate buffer using 25 mmol/L of sodium cyanate. Lipid membrane fluidity was determined using three spin-labelled fatty acids: 5-, 12- and 16-doxylstearic acids (5-DS, 12-DS, 16-DS). Internal viscosity was measured with Tempamine, using also EPR spectroscopy. Osmotic fragility was determined spectrophotometrically. Incubation of whole blood with sodium cyanate led to an increase in lipid membrane fluidity in the deeper region of the lipid layer, indicated by 12- and 16-doxylstearic acid, and a decrease near the surface (5-DS). Statistically significant results were obtained for the internal viscosity and osmotic fragility of erythrocytes. An increase in internal viscosity and increase in osmotic fragility were found in erythrocytes after incubation of whole blood, as well as in erythrocytes incubated with sodium cyanate in buffer. Alterations in internal viscosity were stronger in erythrocytes incubated with sodium cyanate in blood than in erythrocytes in the buffer. On the other hand, higher osmotic fragility was observed for erythrocytes in the buffer.     

Distribution of 5-doxylstearic acid in the membranes of mammalian cells

Concentration-dependent spin broadening of ESR spectra of the nitroxide 5-doxylstearic acid has been used to evaluate the distribution of 5-doxylstearic acid in the membranes of intact mouse thymus-bone marrow (TB) and Chinese hamster ovary (CHO) cells. TB cells, CHO cells, erythrocytes, and isolated plasma membranes from CHO cells were labelled with 5-doxylstearic acid and the peak to peak linewidths of the central line of the resulting ESR spectra were measured. The measured line widths were linearly dependent on the amount of 5-doxylstearic acid incorporated into the sample over the range of 0-0.18 mol nitroxide per mol lipid. In erythrocytes, the relationship between linewidths approximated a linear function at lower concentrations of 5-doxylstearic acid, up to 0.07 mol nitroxide per mol lipid. The amount of broadening of the central line for a given amount of 5-doxylstearic acid was far less for intact cells than for either erythrocytes or plasma membrane, indicating that the 5-doxylstearic acid samples a much larger lipid pool in the intact cells. With the broad assumption that the mobility of the 5-doxylstearic acid is similar in different membranes, the size of the lipid pool sampled by 5-doxylstearic acid is approximately equal to the total cellular lipid in intact cells. If a given concentration of 5-doxylstearic acid sampled only the plasma membrane of TB or CHO cells, we would expect to see a linewidth corresponding to a 12-20-fold greater local concentration of 5-doxylstearic acid than was observed, since the plasma membranes of CHO and TB cells represent only 5-8 percent of the total cellular lipid. Therefore, the 5-doxylstearic acid must distribute into most or all cellular membranes of intact cells and is not localized in the plasma membrane alone.     

Studies on the superoxide releasing site in plasma membranes of neutrophils with ESR spin-labels

Superoxide (O2-)-generating membranes of pig blood neutrophils were studied by the ESR spin-label method. Neutrophils were spin-labeled with doxylstearic acids, consisting of nitroxide free radicals bonded to the 5, 7, 12, or 16 position of stearic acid (5-, 7-, 12-, or 16-DS), to detect the reduction of their nitroxide radicals at different positions in the membrane. The spin-labeled cells were then stimulated with phorbol myristate acetate (PMA). Stimulation of the labeled cells resulted in a marked decrease in the spin concentration of 5-DS due to the reduction by O2-, but not in those of the other three DS labels. This reduction of 5-DS was completely inhibited by copper salicylate (CS), a hydrophobic and permeable O2(-)-scavenger, but not by superoxide dismutase (SOD). CS was not inhibitory on the respiratory burst, i.e., O2(-)-generating activity of neutrophils. On the contrary, if the spin-labels were present in the extracellular medium, SOD inhibited the reduction of all four DS labels due to O2- released from PMA-stimulated cells. These results suggest that the O2(-)-releasing site is not located at the outer surface of the plasma membrane but in an inner hydrophobic environment a short distance (around 4-5 A) from its outer surface.     

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

Evaluation of hydralazine and procainamide effects on fibroblast membrane fluidity

In this study the membrane fluidity of fibroblasts under different pharmacological treatment was investigated. Two drugs, hydralazine and procainamide, were used to treat the immortalized mouse NIH 3T3 and hamster B14 fibroblasts. Membrane lipid dynamics was measured by fluorescence spectroscopy and electron spin resonance techniques. Two kinds of fluorescent probes (TMA-DPH and 12-(9-anthroyloxy)-stearic acid (12-AS)) and two spin labels (5-doxylstearic acid (5-DS) and 12-doxylstearic acid (12-DS)) were used to monitor fluidity in the upper polar and in the hydrophobic core regions of the lipid bilayer. The drugs influenced the membrane hydrophobic core, of which hydralazine induced fluidization and procainamide increased the rigidity. The membrane fluidity at the surface of the lipid bilayer was not modified by the drugs which indicates that both drugs intercalated mainly into the inner core of the cell membrane.     

3H]Ouabain binding and 86 rubidium uptake during the dimethyl sulfoxide-induced differentiation of human promyelocytic leukemia HL-60 cells

When HL-60 cells were induced to differentiate into granulocyte-like cells by culture in the presence of 1.3% dimethyl sulfoxide, an increase in the rate of ouabain-sensitive 86Rb transport was observed during the first 6 h followed by a constant decrease which became stable on day 4 at about 40% of control level. By contrast, the number of ouabain binding sites remained constant during the first 24 h then decreased with the same kinetics as that of the 86Rb transport rate. These results suggest that alterations in ionic fluxes, through activation of the sodium pump, are part of the early events resulting in HL-60 cell differentiation.     

Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and noninvasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP was reduced 3 to 11 times slower (depending on the tissue) than Tempol in vivo and that maximum tissue concentration varies substantially between tissues (0.6-7.2mM). For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance spectroscopy, we showed that the nitroxide reduction rate depends only weakly on cellular pO(2) in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO(2) is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo.     

Organophosphorous plant growth regulator Melaphen: Resistance of plant and animal cells to stress factors

The addition of the organophosphorous plant growth regulator Melaphen (4 × 10^?12 M) to the incubation medium increases the maximum rate of oxidation of NAD-dependent substrates in rat liver and sugar beet root mitochondria. In addition, Melaphen stimulates electron transport during oxidation of succinate by rat liver mitochondria, but has no effect on the rate of this substrate oxidation in sugar beet root mitochondria. In storage organs of plants, the rate of oxidation of NAD-dependent substrates by mitochondria is relatively low. By stimulating the activity of NAD-dependent dehydrogenases, Melaphen stimulates energy metabolism in the cells and manifests adaptogenic activity by accelerating the germination of seeds. Melaphen does not influence the fluorescence of lipid peroxidation (LPO) products in mitochondria non-exposed to stress, but decreases 1.5–2 fold the LPO fluorescence in rat liver mitochondria exposed to cold stress and artificially “aged” sugar beet root mitochondria. Besides, Melaphen increases the rate of electron transport in a terminal site of respiratory chains of plant and animal mitochondria and decreases LPO. The data obtained testify to antistress activity of Melaphen.     

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

Abstract

Nitroxides are widely used in biology as antioxidants, spin labels, functional spin probes for pH, oxygen and thiol levels, and tissue redox status imaging using electron paramagnetic resonance (EPR); however, biological applications of nitroxides is hindered by fast bioreduction to EPR-silent hydroxylamines and rapid clearance. In this work, we have studied pyrrolidine nitroxides with acetoxymethoxycarbonyl groups which can undergo hydrolysis by cellular esterases to hydrophilic carboxylate derivatives resistant to bioreduction. Nitroxides containing acetoxymethoxycarbonyl groups were rapidly absorbed by cells from the media, 3,4-bis-(acetoxymethoxycarbonyl)-proxyl (DCP-AM2) and 3-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)acetamido)-proxyl (DCAP-AM2) showing the strongest EPR signal of the cellular fraction. Remarkably, the EPR parameters of 3,4-dicarboxy-proxyl (DCP) and its mono- and di-acetoxymethyl esters are different, and consequent intracellular hydrolysis of acetoxymethoxycarbonyl groups in DCP-AM2 can be followed by EPR. To elucidate intracellular location of the resultant DCP, the mitochondrial fraction has been isolated. EPR measurements showed that mitochondria were the main place where DCP was finally accumulated. TEMPO derivatives showed expectedly much faster decay of EPR signal in the cellular fraction, compared to pyrrolidine nitroxides. It was found that supplementation of endothelial cells with 50?nM of DCP-AM2 completely normalised the mitochondrial superoxide level. Moreover, administration of DCP-AM2 to mice (1.4?mg/kg/day) resulted in substantial nitroxide accumulation in the tissues and significantly reduced hypertension. We found that hydroxylamine derivatives of dicarboxyproxyl nitroxide DCP-AM-H can be used for the detection of superoxide in vivo in angiotensin II model of hypertension. Infusion of DCP-AM-H in mice leads to accumulation of persistent EPR signal of nitroxide in the blood and vascular tissue in angiotensin II-infused wild-type but not in SOD2 overexpressing mice. Our data demonstrate that acetoxymethoxycarbonyl group containing nitroxides accumulate in mitochondria and demonstrate site-specific antioxidant activity.     

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.     

High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25 kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in ‘broken mitochondria’ (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.     

Modulation of potassium transport in cultured retinal pigment epithelium and retinal glial cells by serum and epidermal growth factor.

The ionic environment of retinal photoreceptors is partially controlled by potassium transporters on retinal glial and retinal pigment epithelial cells (RPE). In this study, serum and epidermal growth factor (EGF) were examined as modulators of potassium transport in confluent cultures of human RPE and rabbit retinal glia. EGF is a known mitogen for confluent RPE cultures and was shown here to also stimulate [3H]thymidine incorporation in cultures of retinal glia. For potassium transport studies 86Rb was used as a tracer during a 17-min incubation. For both retinal cell types the mean total 86Rb uptake in 10% serum was approximately 60% above basal, serum-free controls. For EGF, tested in several experiments in a concentration range from 1 to 100 ng/ml, maximal total uptake was 33 and 24% above controls for RPE and glia, respectively. Inhibitor studies suggested that basal and serum-stimulated uptake for both cell types occurred by the ouabain-sensitive Na-K ATPase pump and by the furosemide- or bumetanide-sensitive Na-K-Cl cotransporter. EGF-stimulated uptake appeared to be due predominantly to the cotransporter. The data suggest that serum components and EGF, which may be available to retina-derived cells under pathologic conditions, may not only stimulate proliferation but may also act as short-term modulators of potassium ion movement and thus affect physiologic processes that are sensitive to ion homeostasis.     

Characterization of spin-labelled fatty acids and hematoporphyrin binding sites interactions in serum albumin

Electron paramagnetic resonance (EPR) was used to investigate the spin-labelled fatty acid (SLFA) binding equilibrium to human (HSA) and bovine (BSA) serum albumin. The number of 5-doxyl stearate (5-DS) and 16-doxyl stearate (16-DS) binding sites on HSA and BSA were found to be equal, while the association constants, KA values (especially those of the primary binding site) were different. The applied EPR spectra analysis permitting a quantitative distinguishing between slow macromolecular rotation (pi c) and fast anisotropic motion (steric restriction, S) of bound SLFA, allowed SLFA oxazolidinyl ring mobility to be estimated. The 5-DS nitroxide radical is completely immobilized within the HSA protein matrix (S approximately 1.0, pi c approximately 56 +/- 1 ns). The 5-DS when bound to BSA exhibited the presence of more extensive fluctuations (lower S and pi c values) and its immersion depth with respect to BSA surface was calculated to be 4 +/- 2 A. The 16-DS oxazolidinyl radical bound to HSA was found to undergo moderated fluctuations (both S and pi c are smaller with respect to 5-DS) and it is buried deeper within the protein core (rimm = 10 +/- 2 A with respect to BSA surface). The tetrapyrrole ligands hematoporphyrin (Hp) and hematoporphyrin derivative (HpD) were found to induce well detectable changes in the SLFA binding patterns to serum albumin. The action mode was determined to be different for 16-DS (primary) and 5-DS (secondary) serum albumin binding sites: (i) 5-DS is extruded from several binding sites accompanied by an increase in KA in the remaining ones; (ii) simultaneous binding of 16-DS and Hp consists of cooperative and non-cooperative phases (both the number of the independent sites and the parameter of cooperativity, alpha, being dependent on Hp/HSA ratio); (iii) in principal the mobilities of 5-DS and 16-DS bound to HSA are changed, depending on the porphyrin/HSA ratio; and (iv) the effective immersion depth of the paramagnetic centres with respect to the protein surface is increased when Hp is present as a second ligand (rimm = 7 +/- 2 and 16 +/- 2 A for 5-DS and 16-DS, respectively).     

Potassium Transport and the Relationship Between Intracellular Potassium Concentration and Amino Acid Uptake by Cells of a Marine Pseudomonad

Transport of K(+) by K(+)-depleted cells of marine pseudomonad B-16 (ATCC 19855) exhibited saturation kinetics. Rb(+) inhibited both K(+) transport and the K(+)-dependent transport of alpha-aminoisobutyric acid (AIB) into K(+)-depleted cells of the organism in proportion to the concentration of Rb(+) in the suspending medium. Inhibition of the K(+)-dependent uptake of AIB into K(+)-depleted cells by Rb(+) could be overcome by increasing the concentration of K(+) in the medium. When AIB and K(+) were added simultaneously to a suspension of K(+)-depleted cells, the uptake of K(+) occurred immediately and rapidly, whereas the accumulation of AIB occurred only after a lag. The initial uptake rate of AIB was directly proportional to the intracellular K(+) concentration. The intracellular concentration of K(+) and AIB at their steady-state levels increased to a maximum as the Na(+) concentration in the suspending medium was increased. At Na(+) concentrations between 0.2 and 0.3 M, the molar ratio of K(+) to AIB at their intracellular steady-state concentrations was constant at 1.6. At external Na(+) concentrations less than 0.2 M, the cells maintained a relatively higher K(+) intracellular steady-state level than AIB.     

Electron spin resonance investigation of the interaction of the anion and glucose transport inhibitor, p-azidobenzylphlorizin, with the human red cell membrane

The membrane perturbations caused by the interaction of p-azidobenzylphlorizin (p-AzBPhz), a potential photoaffinity labeling agent of the anion and D-glucose transporters in the human erythrocyte, have been studied using electron spin resonance (ESR) spectrometry. Two lipid-specific spin labels have been employed; one of these agents, a hexadecyl-quarternary amine with the nitroxide reporter group covalently attached to the cationic nitrogen, (CAT-16), has been used to monitor changes in the physical state of the membrane's extracellular phospholipid/water interface. The other spin label, 5-doxylstearic acid (5-NS), is designed to examine the order and motion of the lipid bilayer near the cell surface. In separate experiments, intact human red cells labeled with these lipid-specific spin labels were exposed to small amounts of the phlorizin azide. A dose-dependent alteration in CAT-16 motion was observed, but the p-AzBPhz interaction with the membrane had no effect on the spectrum of 5-NS. The half-maximal effect of the phlorizin derivative on the CAT-16 spectrum occurred when about 2 million molecules were bound to each cell. This is also the combined amount of band 3 and band 4.5 present in the red cell membrane and represents the concentration necessary to inhibit both anion and glucose transport. Our results suggest that the first p-AzBPhz molecules binding to the red cell membrane interact with the anion and sugar transporters, and not with the bulk lipid bilayer.     

Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes: a pulse ESR spin labeling study.

Transport and diffusion of molecular oxygen in phosphatidylcholine (PC)-cholesterol membranes and their molecular mechanism were investigated. A special attention was paid to the molecular interaction involving unsaturated alkyl chains and cholesterol. Oxygen transport was evaluated by monitoring the bimolecular collision rate of molecular oxygen and the lipid-type spin labels, tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid. The collision rate was determined by measuring the spin-lattice relaxation times (T1's) in the presence and absence of molecular oxygen with long-pulse saturation-recovery ESR techniques. In the absence of cholesterol, incorporation of either a cis or trans double bond at the C9-C10 position of the alkyl chain decreases oxygen transport at all locations in the membrane. The activation energy for the translational diffusion of molecular oxygen in the absence of cholesterol is 3.7-6.5 kcal/mol, which is comparable to the activation energy theoretically estimated for kink migration or C-C bond rotation of alkyl chains [Tr?uble, H. (1971) J. Membr. Biol. 4, 193-208; Pace, R. J., & Chan, S. I. (1982) J. Chem. Phys. 76, 4241-4247]. Intercalation of cholesterol in saturated PC membranes reduces oxygen transport in the headgroup region and the hydrophobic region near the membrane surface but little affects the transport in the central part of the bilayer. In unsaturated PC membranes, intercalation of cholesterol also reduces oxygen transport in and near the headgroup regions. In contrast, it increases oxygen transport in the middle of the bilayer. On the basis of these observations, a model for the mechanism of oxygen transport in the membrane is proposed in which oxygen molecules reside in vacant pockets created by gauche-trans isomerization of alkyl chains and the structural nonconformability of neighboring lipids, unsaturated PC and cholesterol in particular, and oxygen molecules jump from one pocket to the adjacent one or move along with the movement of the pocket itself. The presence of cholesterol decreases oxygen permeability across the membrane in all membranes used in this work in spite of the increase in oxygen transport in the central part of unsaturated PC-cholesterol membranes because cholesterol decreases oxygen transport in and near the headgroup regions, where the major barriers for oxygen permeability are located. Oxygen gradients across the membranes of the cells and the mitochondria are evaluated. Arguments are advanced that oxygen permeation across the protein-rich mitochondrial membranes can be a rate-limiting step for oxygen consumption under hypoxic conditions in vivo.     

Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H₂O₂) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H₂O₂ alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 μmol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 μmol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.     

Measurement of bioreduction rates of cells with distinct responses to ionizing radiation and cisplatin.

A low frequency electron paramagnetic resonance (EPR) spectrometer has been used to measure the bioreduction rate of an exogenously added nitroxide free radical species. Measurements have been made in a well controlled, in vitro system using an X-ray and cisplatin sensitive Chinese hamster ovary (CHO) cell line, xrs-5, and partial revertants which display wild-type levels of sensitivity to X-rays but retain xrs-5 levels of cisplatin sensitivity. The xrs-5 cells reduce this radical species at a rate which is approx. 50% that of the wild-type CHO cell line, K1. The partial revertants maintain this defect in bioreduction despite their decrease in radiosensitivity. However, the bioreduction rate observed in these cells correlates with their sensitivity to the chemotherapeutic drug cisplatin. Low frequency EPR allows measurements and imaging of living tissue and may be of value as a predictive assay of human tumor response to chemotherapy.     

Electron spin resonance studies on the selective labeling of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide of rat liver mitochondria

N-(1-Oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide (MSL) was incorporated into rat liver mitochondria and the nitroxide radical incorporated was found to decay considerably. The incorporation was blocked by a high concentration of NEM, but not by pCMB. Spin labeled fatty acid derivatives, 2-(3-carboxypropyl)-2-tridecyl-4,4-dimethyl-3-oxazolidinyloxyl (FSL1) and 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (FSL2), were also incorporated and the nitroxide radical decayed. However, incorporation of FSL1 or FSL2 was not blocked by NEM or pCMB. The ESR spectrum of 3-carboxyl-2,2,5,5-tetramethyl-pyrroline-1-oxyl (CSL) did not change on reaction with the mitochondria. The labeled MSL exhibited an ESR spectrum composed of both strongly immobilized and weakly immobilized components. A similar reaction with FSL1 gave an ESR spectrum mainly composed of a strongly immobilized component, the weakly immobilized component was negligibly small, while FSL2 exhibited an ESR spectrum in which free-like signals of the nitroxide radical were predominant. The results suggest that MSL is labeled selectively in the mitochondrial membrane through those SH groups that are not reactive to pCMB, and the labeled nitroxide radical is reduced in situ. The mode of incorporation into the mitochondria differs between MSL and the other spin labeled reagents, and labeling of MSL at the binding site may precede reduction of the nitroxide radical. The incorporation of MSL was dependent on the concentration of MSL used. ADP-acceleration of mitochondrial oxygen uptake with succinate was inhibited by labeling the mitochondria with MSL without loss of the electron transferring activity.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.