Superoxide release

C. Evaluation of Biological Activity In Vitro Superoxide Release from Cultured Cells... 

Figure 7 Differentiation of HL-60 cells by the incubation with dimethylsulfoxide [DMSO] (a), and the assay of superoxide release in the DMSO-differentiated HL-60 cells by cytochrome C method (b).

Figure 9 Effect of low-molecular weight (3,400) po-ly(MA-CDA)s on the superoxide release in the DMSO-differentiated HL-60 cells. The data are the mean of three experimental results. control [J = unmodified...

Since it might be possible that the perturbation of membrane directly stimulated the NADPH-oxidase located on the cell membrane, which is the enzyme for the production of superoxide [24], the possibility was examined by the assay using detergent (Triton X-100) instead of polymers. At 0.001% of Triton X-100, no stimulation of superoxide release from DMSO-differentiated HL-60 cells was observed. At 0.01% of Triton X-100, a... 

C. Privat, O. Stepien, M. David-Dufilho, A. Brunet, F. Bedioui, P. Marche, J. Devynck, and M.-A. Devynck, Superoxide release from interleukin-1 (3-stimulated human vascular cells in situ electrochemical measurement. Free Radicals Bio. Med. 27, 554-559 (1999). 

There are numerous in vitro and in vivo studies, in which the damaging free radical-mediated effects of iron have been demonstrated. Many such examples are cited in the following chapters. However, recent studies [170,171] showed that not only iron excess but also iron deficiency may induce free radical-mediated damage. It has been shown that iron deficiency causes the uncoupling of mitochondria that can be the origin of an increase in mitochondria superoxide release. Furthermore, a decrease in iron apparently results in the reduction of the activity of iron-containing enzymes. Thus, any disturbance in iron metabolism may lead to the initiation of free radical overproduction. 

Despite a long-time studying of superoxide production by mitochondria, an important question is still debated does mitochondria produce superoxide under physiological conditions or superoxide release is always a characteristic of some pathophysiological disorders resulting in the damage of normal mitochondrial functions Uncertainties in this question arise due to the different results obtained with the use of respiratory inhibitors and different analytical methods. 

The possible involvement of free radicals in the development of hypertension has been suspected for a long time. In 1988, Salonen et al. [73] demonstrated the marked elevation of blood pressure for persons with the lowest levels of plasma ascorbic acid and serum selenium concentrations. In subsequent studies these authors confirmed their first observations and showed that the supplementation with antioxidant combination of ascorbic acid, selenium, vitamin E, and carotene resulted in a significant decrease in diastonic blood pressure [74] and enhanced the resistance of atherogenic lipoproteins in human plasma to oxidative stress [75]. Kristal et al. [76] demonstrated that hypertention is accompanied by priming of PMNs although the enhancement of superoxide release was not correlated with the levels of blood pressure. Russo et al. [77] showed that essential hypertension patients are characterized by higher MDA levels and decreased SOD activities. 

It has been proposed that a major source of oxygen radicals in sickle erythrocytes is mutant hemoglobin HbS. However, although HbS showed an accelerated autoxidation rate under in vitro conditions, its in vivo oxidative activity was not determined. Sheng et al. [401] suggested that the observed oxidation rate of HbS is exaggerated by adventitious iron. Dias-Da-Motta et al. [402] proposed that another source of enhanced superoxide production in sickle cells are monocytes in contrast, there is no difference in superoxide release by sickle... 

The efficiency of superoxide assays strongly depend on the nature of superoxide producers. Significant difficulties arise in the detection of superoxide in cells and tissue. Cytochrome c is unable to penetrate cell membranes and therefore, can be used only for the measurement of extracellular superoxide. Furthermore, SOD-inhibitable cytochrome c reduction is difficult to apply in nonphagocytic cells and tissue due to the complications of measuring low rates of superoxide release, direct reduction of cytochrome c by cellular enzymes, the reoxidation of reduced cytochrome by hydrogen peroxide, etc. [8], Moreover, in nonphagocytic cells superoxide is formed exclusively inside the cells and is not released outside as in phagocytes. These circumstances severely limit the number of analytical methods, which can be used for superoxide detection in vasculature. 

The kinetics of the reaction of 2 -deoxyuridin-L-yl radicals (11) with thiols, with superoxide release from the peroxyl radical (13) generated, have been reported. Radical (11) is produced by photolysis of precursor (10). When the radical is produced in the presence of thiols, (12) is formed. Second-order kinetics were found for the reactions with thiols. Peroxyl radical (13) is formed in the presence of oxygen. This undergoes heterolytic fragmentation to the superoxide anion O2 and cation (14), which ultimately leads to 2-deoxyribonolactone (15). 

Ryan, T. C., Weil, G. J., Newberger, P. E., Haugland, R., and Simons, E. R. (1990) Measurement of superoxide release in the phagovacuoles of immune complexes-stimulated human neutrophils. J. Immunol. Methods 130, 223-233. 

In spite of a relatively slow rate of superoxide release by peroxidases (Reaction (19), Table... 

Despite the well-studied respiratory burst it is now clear that superoxide is also released by a variety of non-phagocytic cells. For example human B-lympho-cytes, which have been transformed by Epstein-Barr virus, express a super-oxide-generating system similar in many respects to the NADPH-oxidase of neutrophils [80]. This oxidase in lymphocytes can be stimulated by cytokines, suggesting that superoxide release may be a normal function of these B-lympho-cytes [81], NADPH-oxidase also occurs in normal peripheral B-lymphocytes, but disappears from the cell surface during final differentiation to plasma cells. 

The NADPH binding site is on the cytosolic side of the membrane, whereas the superoxide release site is either extracellular or on the luminal side of the phagocytic vesicle. The enzyme acts as an ion pump, because it releases superoxide without an accompanying cation protons remain inside the cell, resulting in considerable membrane depolarization (Babior, 1992 Chanock etal., 1994). 

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