BackgroundNeutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Δψm), which is usually generated by the respiratory chain complexes. We investigated the source of Δψm in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this Δψm for the generation of ATP.Methods and Principal FindingsIndividual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but Δψm was still decreased by inhibition of complex III, confirming the role of the respiratory chain in maintaining Δψm. Complex V did not maintain Δψm by consumption of ATP, as has previously been suggested for eosinophils. We show that complex III in neutrophil mitochondria can receive electrons from glycolysis via the glycerol-3-phosphate shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were lacking in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercomplex organisation while gaining increased aerobic glycolysis, just like neutrophils.ConclusionsWe show that neutrophils can maintain Δψm via the glycerol-3-phosphate shuttle, whereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors.

Abstract-Apoptosis-inducing factor (AIF), or programmed cell death 8 (Pdcd8), is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal apoptosis induced by oxidative stress. Conversely, in vitro, AIF has been demonstrated to have a proapoptotic role when, on induction of the mitochondrial death pathway, AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. To determine the role of AIF in myocardial apoptotic processes, we examined cardiomyocytes from an AIF-deficient mouse mutant, Harlequin (Hq). Hq mutant cardiomyocytes demonstrated increased sensitivity to H 2 O 2 -induced cell death. Further, Hq hearts subjected to ischemia/reperfusion revealed more cardiac damage and, unlike wild-type mice, the amount of damage increased with the age of the animal. Aortic banding caused enhanced hypertrophy, increased cardiomyocyte apoptotic and necrotic cell death, and accelerated progression toward maladaptive left ventricular remodeling in Hq mutant mice compared with wild-type counterparts. These findings correlated with a reduced capacity of subsarcolemmal mitochondria from Hq mutant hearts to scavenge free radicals. Together, these data demonstrate a critical role for AIF as a cardiac antioxidant in the protection against oxidative stress-induced cell death and development of heart failure induced by pressure overload. (Circ Res. 2005;96:e92-e101.)

ACE inhibitors improve endothelial dysfunction, possibly by blocking endothelial angiotensin production. Prorenin, through its binding and activation by endothelial mannose 6-phosphate (M6P) receptors, may contribute to this production. Here, we investigated this possibility as well as prorenin activation kinetics, the nature of the prorenin-activating enzyme, and M6P receptor-independent prorenin binding. Human umbilical vein endothelial cells (HUVECs) were incubated with wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala, thereby preventing cleavage by known proteases), M6P-free prorenin, and nonglycosylated prorenin, with or without M6P, protease inhibitors, or angiotensinogen. HUVECs bound only M6P-containing prorenin (K(d) 0.9+/-0.1 nmol/L, maximum number of binding sites [B(max)] 1010+/-50 receptors/cell). At 37 degrees C, because of M6P receptor recycling, the amount of prorenin internalized via M6P receptors was >25 times B(max). Inside the cells, wild-type and K/A-2 prorenin were proteolytically activated to renin. Renin was subsequently degraded. Protease inhibitors interfered with the latter but not with prorenin activation, thereby indicating that the activating enzyme is different from any of the known prorenin-activating enzymes. Incubation with angiotensinogen did not lead to endothelial angiotensin generation, inasmuch as HUVECs were unable to internalize angiotensinogen. Most likely, therefore, in the absence of angiotensinogen synthesis or endocytosis, M6P receptor-mediated prorenin internalization by endothelial cells represents prorenin clearance.

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