Attributable to intravascular superoxide release, as evident from a total block of this increase in the presence of SOD. Replacement of the lung by a fiber oxygenator to mimic oxygenation of the buffer fluid, as would occur in the lung, assured that no lung-independent oxidation of CPH was provoked by PMA, neither in the absence nor in the presence of FeCl2. Thus an overlapping effect of metal ions primarily being responsible for the oxygen-dependent effects seen in the presence of the lung as e.g. results from a Fenton reaction, can be excluded. The PMAinduced increase in the ESR signal was illustrated in our study to be attributable to the suggested BL-8040 chemical information pathway of NADPH oxidase stimulation, because it was prevented a) by the NADPH oxidase inhibitor apocynin as well as b) in mice lacking the NADPH oxidase subunit gp91phox (Nox-2). In contrast, rotenone, a mitochondrial complex I inhibitor, did not affect the PMA induced ROS release. This indicated that mitochondria-derived superoxide does not play a role in the oxygen-dependent ROS release induced by PMA. This finding is of particular interest, given the recent reports of mitochondria as possible sources of superoxide release [17]. Moreover, PMA caused an immediate pulmonary artery pressor response, which was also largely blocked by SOD, suggesting a direct vasoconstrictor effect of superoxide generated by PMA addition. This suggestion is in line with the inhibition of the vasoconstrictor response by the NADPH oxidase inhibitor apocynin. The fact that PMA stimulation of the lung induces a vasoconstrictor response via superoxide challenges previous studies suggesting that the PMA-induced vasoconstrictor response involves a Ca2+ sensitization by inhibition of myosin light chain phosphatase (for review see [47]). The superoxide-induced vasoconstriction in this pathway may involve intracellular calcium mobilization by enhancing cyclic ADP-ribose production [48], activation of RhoA/Rho kinase [49], or inactivation of NO [50] by superoxide. To investigate the oxygen-dependence of the PMA-induced superoxide release, we then stimulated the lungs with PMA in the presence of PubMed ID: different oxygen concentrations. Most interestingly, we detected peak PMA-evoked lung superoxide release when lungs were ventilated with 5 O2. This peak in superoxide releasecorrelated with the maximum PMA-evoked vasoconstrictor effect. The NADPH oxidases of endothelial cells, which have been shown to contain all NADPH oxidase subunits needed for superoxide generation as well as leukocytes are resident in the intravascular compartment, and are suggested as a possible source of the PMAinduced superoxide release. The ESR technology was not suitable for detecting significant hypoxia-dependent changes in superoxide release in unstimulated isolated rabbit lungs. However, since i) hypoxia caused an increased release of NADPH-dependent superoxide release when lungs were challenged with PMA and ii) that superoxide caused a vasoconstriction; it is tempting to speculate that such mechanisms may contribute to the regulation of HPV. Data from our laboratory have repetitively suggested that an NADPH oxidase-dependent increase in lung ROS release contributes to the initiation of HPV [8,21]. Thus, it is interesting that many studies investigating HPV in isolated lungs the pulmonary circulation was primed with angiotensin II to yield a sufficient hypoxic vasoconstrictor response [51-53]. Angiotensin II has also been shown to activate NADPH ox.