Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (·O2 −) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented ·O2 − release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

Expression of inducible NO synthase (iNOS) by macrophages is a prerequisite for the production of high output NO, which mediates many bactericidal and tumoricidal actions of these immune cells. The expression of iNOS in mammalian cells is governed predominantly by the transcription factor, NF-κB, which regulates the expression of many host defense proteins. In the present study, we characterize a novel, biphasic effect of NO on NF-κB activity in murine macrophages. This mechanism depends on the local concentration of NO and enables it both to up- and down-regulate the expression of host defense proteins including iNOS, cyclooxygenase-2, and IL-6. This biphasic activity of NO appears to play a pivotal role in the time course of activation of these immune cells and, by inference, in facilitating the initiation of a defense response against pathogenic stimuli and in its termination to limit tissue damage. This mechanism may explain at least in part the reported ability of NO to act in both a pro- and anti-inflammatory manner.

During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/ cyclosome-Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein-β-transducin repeat-containing protein (SCF-β-TrCP), which control the transient appearance and metabolic activity of the glycolysispromoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S 273 ) within the conserved recognition site for SCF-β-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-GluAsn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-β-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-tolate G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.

We have investigated in whole cells whether, at low oxygen concentrations ([O 2]), endogenous nitric oxide (NO) modulates the redox state of the mitochondrial electron transport chain (ETC), and whether such an action has any signaling consequences. Using a polarographic-and-spectroscopic-coupled system, we monitored redox changes in the ETC cytochromes b H, cc1, and aa3 during cellular respiration. T he role of nitric oxide (NO) as a signaling molecule involved in the physiology of the cardiovascular, pulmonary, and nervous systems has been clearly demonstrated (1). It is widely accepted that many of the signaling consequences of NO are mediated through activation of the biochemical target soluble guanylyl cyclase (2). In the last decade, however, another potential target has emerged, namely cytochrome c oxidase (CcO, complex IV), the mitochondrial enzyme responsible for reduction of O 2 into water in the final stage of the electron transport chain (ETC). NO reversibly inhibits CcO by competing with O 2 for the binuclear binding site (3-5). Moreover, activation of endothelial NO synthase (NOS) by bradykinin in respiring endothelial cells results in a decrease in the rate of O 2 consumption (VO 2 ), an effect that can be reversed by an inhibitor of NOS (6). The high affinity of CcO for NO suggests that the nanomolar concentrations of NO generated in tissues under basal conditions may play a role in the regulation of VO 2 (7).We have recently developed a polarographic-and-spectroscopic-coupled system based on visible light spectroscopy (VLS) to monitor changes in the redox states of the mitochondrial ETC cytochromes during cellular respiration (8). Using this system, we have confirmed previous studies showing an early reduction of cytochrome c at low O 2 concentration [O 2 ], without a change in VO 2 (9). This phenomenon has been postulated to be part of a mechanism to maintain VO 2 at decreasing [O 2 ], although there is no consensus as to the factors that control this process (10, 11). Increased generation of mitochondrial reactive oxygen species (ROS) at low [O 2 ] has previously been linked to the activation of various adaptive signaling pathways (12). However, at present, the mechanism responsible for the observed increase in ROS at low [O 2 ] is not clear (see ref. 13 for review). One possibility is that NO, via its contribution to the early reduction of ETC cytochromes, favors the generation of ROS. We have therefore studied the effects of endogenous NO on the ETC redox state in endothelial and monocytic cells and the subsequent signaling consequences, specifically the release of ROS and the activation of NF-B (14). Materials and MethodsReagents. Hepes, N-dodecyl-␤-D-maltoside, cytochrome c (from bovine heart mitochondria), N,N,NЈ,NЈ tetramethyl-p-phenylenediamine, sodium ascorbate, and dihydroethidium (DHE) were purchased from Sigma;CcO Activity. Experiments with the purified enzyme (from bovine heart mitochondria) were carried out using the VLS system described previously (8). Briefly, the sample was p...