Fig. 5.2.
(A) Determination of mtROS triggered NADPH oxidase activation in isolated human neutrophils by electron paramagnetic resonance measurement. Freshly isolated human neutrophils (22x106 PMN/mL) were incubated in PBS with 300 µM Ca2+/Mg2+ and 10 mM DEPMPO for 15 min at 37 °C. Activators and inhibitors of phagocytic NOX were added as shown in the figure. The spectrum for phorbol ester (PDBu)-stimulated PMN is displayed at decreased intensity (1/10). All reactions below the red spectrum contained 20 µM myxothiazol. Incubations were with NOX inhibitor (VAS2870), intracellular calcium chelator (BAPTA-AM), cSrc kinase inhibitor (PP2) and PKC inhibitor (chelerythrine). All spectra were recorded at room temperature in 50 µl glass capillaries (Hirschmann Laborgeräte GmbH, Eberstadt, Germany). EPR conditions: B0 =3300 G, sweep =150 G, sweep time =60 s, modulation =3000 mG, MW power =10 mW, gain =9x102 using a Miniscope MS200 from Magnettech (Berlin, Germany). Representative spectra of mixed DEPMPO-OOH• and DEPMPO-OH• adduct for 2 independent experiments. Spectra were recorded as described previously [373]. With permission of Mary Ann Liebert, Inc. Copyright 2014. (B) Whole blood Hb-NO levels were determined by electron paramagnetic resonance spectroscopy as a read-out of iNOS activity in endotoxemic (lipopolysaccharide-treated) rats. The EPR measurements were carried out at 77 K using an X-band table-top spectrometer MS400 (Magnettech, Berlin, Germany). The instrument settings were as follows: 10 mW microwave power, 7000 mG amplitude modulation, 100 kHz modulation frequency, 3300 G center field, 300 G sweep width, 60 s sweep time and 3 scans. With permission of Springer-Verlag Berlin Heidelberg. Copyright 2015. (C) Aortic •NO formation was measured in untreated control and angiotensin-II infused hypertensive mice by EPR spin trapping using Fe(DETC)2. Each spectrum was measured from one murine aorta. The representative spectra below the bar graph are the mean of all measurements. EPR conditions: B0 =3276 G, sweep =115 G, sweep time =60 s, modulation =7000 mG, MW power =10 mW, gain =9x102 using a Miniscope MS400 from Magnettech (Berlin, Germany). The A23187-stimulated •NO signal was absent when the aorta were denuded, L-NAME (200 µM) was added, or when aorta from eNOS-/- mice were used (not shown). With permission of Mary Ann Liebert, Inc. Copyright 2014.
(a) Adapted from [63]. (b) Adapted from [371]. (c) Adapted from [63].