guanosine-triphosphate and 4-hydroxy-2-nonenal

We studied the influence of exogenously generated superoxide and exogenous 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the Acanthamoeba castellanii uncoupling protein (AcUCP). The superoxide-generating xanthine/xanthine oxidase system was insufficient to induce mitochondrial uncoupling. In contrast, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. In non-phosphorylating mitochondria, AcUCP activation by HNE was demonstrated by increased oxygen consumption accompanied by a decreased membrane potential and ubiquinone (Q) reduction level. The HNE-induced GTP-sensitive proton conductance was similar to that observed with linoleic acid. In phosphorylating mitochondria, the HNE-induced AcUCP-mediated uncoupling decreased the yield of oxidative phosphorylation. We demonstrated that the efficiency of GTP to inhibit HNE-induced AcUCP-mediated uncoupling was regulated by the endogenous Q redox state. A high Q reduction level activated AcUCP by relieving the inhibition caused by GTP while a low Q reduction level favoured the inhibition. We propose that the regulation of UCP activity involves a rapid response through the endogenous Q redox state that modulates the inhibition of UCP by purine nucleotides, followed by a late response through lipid peroxidation products resulting from an increase in the formation of reactive oxygen species that modulate the UCP activation.

Topics: Acanthamoeba castellanii; Aldehydes; Cysteine Proteinase Inhibitors; Guanosine Triphosphate; Ion Channels; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Oxidation-Reduction; Protozoan Proteins; Ubiquinone; Uncoupling Protein 1

Plant mitochondrial uncoupling protein (UCP) is activated by superoxide suggesting that it may function to minimize mitochondrial reactive oxygen species (ROS) formation. However, the precise mechanism of superoxide activation and the exact function of UCP in plants are not known. We demonstrate that 4-hydroxy-2-nonenal (HNE), a product of lipid peroxidation, and a structurally related compound, trans-retinal, stimulate a proton conductance in potato mitochondria that is inhibitable by GTP (a characteristic of UCP). Proof that the effects of HNE and trans-retinal are mediated by UCP is provided by examination of proton conductance in transgenic plants overexpressing UCP. These experiments demonstrate that the mechanism of activation of UCP is conserved between animals and plants and imply a conservation of function. Mitochondria from transgenic plants overexpressing UCP were further studied to provide insight into function. Experimental conditions were designed to mimic a bioenergetic state that might be found in vivo (mitochondria were supplied with pyruvate as well as tricarboxylic cycle acids at in vivo cytosolic concentrations and an exogenous ATP sink was established). Under such conditions, an increase in UCP protein content resulted in a modest but significant decrease in the rate of superoxide production. In addition, 13C-labeling experiments revealed an increase in the conversion of pyruvate to citrate as a result of increased UCP protein content. These results demonstrate that under simulated in vivo conditions, UCP is active and suggest that UCP may influence not only mitochondrial ROS production but also tricarboxylic acid cycle flux.

Topics: Aldehydes; Carrier Proteins; Citric Acid Cycle; Energy Metabolism; Gene Expression; Genes, Plant; Guanosine Triphosphate; Ion Channels; Lipid Peroxidation; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Models, Biological; Plant Proteins; Plants, Genetically Modified; Reactive Oxygen Species; Recombinant Proteins; Retinaldehyde; Solanum tuberosum; Superoxides; Uncoupling Protein 1

A comparison has been made between the effects of 4-hydroxy-2,3-trans-nonenal (HNE) and 4-hydroxy-2,3-trans-octenal (HOE), two lipid peroxidation products, on the basal and GTPgammaS-stimulated activities of phosphoinositide-specific phospholipase C (PL-C) of rat polymorphonuclear leukocytes. PL-C activity was determined in vitro by measuring the hydrolysis of [3H] phosphatidylinositol-4,5-bis- phosphate (PtdIns-P2) added as exogenous substrate to neutrophil plasma membranes. PL-C was activated by concentrations of HNE ranging from 10(-8) to 10(-6) M both in the presence and in the absence of 2 x 10(-5) M GTPgammaS; HOE stimulated the enzymatic activity between 10(-11) and 10(-8) M; maximal stimulation was given by 10(-11) M HOE plus GTPgammaS. The aldehyde concentrations able to accelerate PtdIns-P2 breakdown displayed a good correspondence with those which have been reported to stimulate the oriented migration of rat neutrophils. Pretreatment of neutrophils with pertussis toxin prevented the stimulation of PL-C by 10(-11) M HOE and by HOE plus GTPgammaS. Our results suggest that the chemotactic action of HNE and HOE might depend on the activation of PL-C; furthermore a regulatory G protein appears to be involved in the acceleration of PtdIns-P2 turnover by HOE.

Topics: Aldehydes; Animals; Enzyme Activation; Guanosine Triphosphate; Neutrophils; Pertussis Toxin; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Rats; Virulence Factors, Bordetella

4-Hydroxynonenal (HNE), a major lipid peroxidation product, displays several biological actions. Among them, the differentiation of human HL-60 cells and the stimulation of neutrophil oriented migration occur at concentrations which can be actually found in normal tissues and in body fluids. In spite of its chemotactic activity, HNE fails to increase neutrophil oxidative metabolism. The action of the aldehyde on cell migration appears to be mediated by a phosphoinositide specific phospholipase C. The acceleration of phosphatidylinositol turnover induced by 10 pM 4-hydroxyoctenal, another lipid peroxidation product, is prevented by the pretreatment of neutrophils with pertussis toxin. The mechanism of action of these 4-hydroxyalkenals appears to follow pathways common to other chemoattractants, but some differences can be found too. In particular HNE seems unable to stimulate phospholipase D activity. The action of 4-hydroxyalkenals and other lipid peroxidation products on transmembrane signalling systems and on phospholipid metabolism might regulate several cell functions, such as motility, proliferation and differentiation.

Topics: Aldehydes; Animals; Guanosine Triphosphate; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lipid Peroxidation; Male; Neutrophils; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Rats; Rats, Wistar; Tumor Cells, Cultured

Ischemia (one hour) and following reperfusion (up to one hour) of the small intestine induce biochemical changes which are indices for the formation and action of oxygen free radicals and which occur predominantly during the reperfusion period. But the villi and the epithelial cells show different patterns of damage, occurring both at the end of the ischemic period and during the reperfusion period. Although the quantitative morphological changes are increased during the reperfusion in comparison with the ischemic phase the quality of the pattern of structural damage is the same in both periods of the experiment. This pattern of the damage includes: 1. the neighbourhood of groups of villi with total ischemic-lytic dissolution of the villi, of villi with damage of the epithelial cells at the tip and at the lateral area and of normal villi; 2. the different degree of structural damage of neighbouring epithelial cells within one villus whose cells are either of regular structural or damaged at subcellular organelles including the plasma membrane or of those being necrotically destroyed and on the way of release into the luminal space; 3. a differentiation of the structural changes of the microvilli and other organelles within single and neighbouring epithelial cells. The biochemical findings on purine nucleotide metabolism and on the formation of oxygen free radicals as "mean values" of a homogenate from a large group of cells cannot reflect the morphological-ultrastructural changes of single villi or even single epithelial cells. The possible reasons for the mosaicism of the morphological changes during ischemia and reperfusion are discussed.

Topics: Adenosine Triphosphate; Aldehydes; Animals; Glutathione; Guanosine Triphosphate; Intestine, Small; Male; Microscopy, Electron; Rats; Rats, Wistar; Reperfusion Injury

Some aldehydes have previously been shown to alter the microtubular system in a concentration-dependent manner. This paper examines the effects of taxol on the impairment of in vitro functionality of microtubular protein and of cytoplasmic microtubules by C-9 aliphatic aldehydes. Taxol-induced polymerization was inhibited by aldehydes in a similar way to GTP-induced polymerization, even though to a different degree. The addition of taxol restored the ability of aldehyde-impaired tubulin to polymerize, but only in the presence of nonanal. Colchicine binding activity in the presence of taxol was slightly modified by aldehydes at 20 degrees C, while it increased at 37 degrees C, compared to controls. Immunofluorescence showed a low disruption of microtubules in fibroblasts incubated with aldehydes and pretreated with taxol, probably as a consequence of its stabilizing activity. Anyway, aldehydes studied behaved differently, indicating a structure-activity relationship.

Topics: 3T3 Cells; Aldehydes; Alkaloids; Animals; Binding Sites; Colchicine; Guanosine Triphosphate; Mice; Microtubule Proteins; Microtubules; Paclitaxel; Polymers; Structure-Activity Relationship; Temperature; Tubulin

Topics: Aldehydes; Animals; Dose-Response Relationship, Drug; Enzyme Activation; Guanosine Triphosphate; Liver; Male; Rats; Rats, Inbred Strains; Type C Phospholipases

4-Hydroxynonenal (HNE), a major aldehyde end-product of lipid peroxidation, induces in vitro a rapid stimulation of rat liver PIP2-phospholipase C. At physiological Ca2+ concentration the effect of the aldehyde is strongly potentiated by guanosine thiotriphosphate (GTP gamma S); GPT gamma S; at higher Ca2+ levels the acceleration of PIP2 breakdown induced by the aldehyde reaches very high values, but is no longer modulated by the presence of GTP gamma S. As the concentration of the aldehyde used (1 micromolar) can be actually reached in tissues, the effects shown in vitro are likely to occur in vivo.

Topics: Aldehydes; Animals; Calcium; Guanosine Triphosphate; Kinetics; Liver; Male; Rats; Rats, Inbred Strains; Type C Phospholipases