Effect of the level of free-radical lipid peroxidation on the ribonucleotide reductase activity of liver tissue

The dynamics of RNA and DNA synthesis as well as the activity of free radical processes in rat liver during the first 40 hours after partial hepatectomy was studied. It was shown that RNA synthesis activation follows the activation of lipid peroxidation (LPO), whereas the DNA synthesis activation follows the decrease of the LPO level. These facts are suggestive of the dependence of the ribonucleotide reductase (RNR) activity on free radical processes. This observation was confirmed by a RNR activity analysis of regenerating liver homogenates. The activity peak was shown to precede the peak of DNA synthesis. Evidence for free radical RNR suppression was also obtained in direct experiments, using intact animal liver homogenates, to which a natural antioxidant (tocopherol) was added.     

The inhibition stage of lipid peroxidation during stress

Stress is shown to induce at first the generalized inhibition of lipid peroxidation (LPO), and then the activation of LPO. In brain and blood serum of rats subjected to continuous footshock as well as to restraint stress LPO products decreased and superoxide scavenging activity increased during the initial period of stress, after 1 hour of footshock LPO indices nearly reached normal values, and after 2 hours of footshock the accumulation of LPO products and decrease of superoxide scavenging activity were seen. LPO inhibition was accompanied by accumulation of easy oxidizable brain phospholipids and by depletion of brain cholesterol, during LPO activation brain cholesterol content and cholesterol-phospholipid ratio increased. The content of LPO products--fluorescent Schiff bases in blood plasma of women suffering from algomenorrhea at first decreased (O-12 h) and then dramatically increased (12-24 h) after a onset of pain at the beginning of menstruation. The data suggest that the stage of LPO inhibition precedes its activation during stress.     

The role of lipid peroxidation in the mechanism of stress

A concept on the mechanism of stress response is substantiated proceeding from the data available in literature and obtained from the author's research made on the radiation stress model. The conception envisages that products of lipid peroxidation (LPO) appear as primary (under direct effect of a stress factor on tissues) and secondary (as a consequence of high- and long-term catecholamine++) mediators. Mobilization of stress-realizing systems in that process is regarded as an adequate response of the auto-oxidative++ system to the primary activation of LPO. Transformation of catecholamines into the factor of LPO stimulation (secondary) is a result of an increase in the relative role of the quinoid way to transform catecholamines in the case of their high concentration. Radical intermediates of the quinoid metabolism appear as LPO initiators. An important pathogenetic role of LPO activation in the stress mechanism substantiates expedience to use antioxidants as agents for prophylaxis and early treatment of stress-factor injuries.     

Role of lipid peroxidation in damage to serotonin receptors and development of epileptiform seizures during hyperoxia

Hyperoxia brought about substantial accumulation of primary and end products of lipid peroxidation (LPO) and a significant lowering of alpha-tocopherol content in rat brain tissues. Preinjection of animals with synthetic and natural antioxidants (4-methyl-2,6-ditretbutylphenol and alpha-tocopherol) prevented LPO activation and decreased the frequency of epileptiform seizures induced by hyperoxia. Administration of a mixture of unsaturated fatty acids led to an opposite effect. The changes in the properties of serotonin receptors were found to be dependent on the hyperoxia-induced LPO. These changes were marked by the reduced specific binding of serotonin with neuronal membranes of the rat brain cortex. The data obtained allowed the conclusion about the key role played by LPO activation in toxic action of hyperbaric activation on the brain.     

Carnosine prevents the activation of free-radical lipid oxidation during stress

Carnosine (beta-alanyl-L-histidine) injected to intact albino rats (20 mg/kg body weight) induces depletion of lipid peroxidation (LPO) products in brain and blood serum, an increase of superoxide scavenging activity in brain and serum, decrease of cholesterol: phospholipid ratio and increase of easy oxidizable phospholipid portion in brain lipid extracts. After painful stress (footshock during 2 hours) LPO products are accumulated in brain and serum, cholesterol: phospholipid ratio increases and the portion of easy oxidizable phospholipids decreases. Carnosine given before stress prevents LPO activation. Effects of carnosine and stress are not additive: LPO inhibition induced by carnosine is much more in rats subjected to stress.     

Damage to the sarcoplasmic reticulum of skeletal muscles in leukemia: role of lipid peroxidation]

Ischemia development was accompanied by inhibition of the enzymatic transport system (ETS) of Ca2+ (reduction of the Ca2+/ATP value and of the Ca2+-dependent ATPase activity), this correlating with the accumulation of primary and secondary molecular products of lipid peroxidation (LPO) in the sarcoplasmic reticulum membranes of the skeletal muscles, in vivo. Administration of antioxidants (2,6-ditretbutyl-4-methylphenol, alpha-tocopherol) prevented the LPO activation in the ischemic muscle and partially protected the ETS of Ca2+ from damage. The blood supply restoration after prolonged ischemia led to further ETS of Ca2+ inhibition against the background of unchanges LPO products level.     

The effect of paracetamol on oxidative damage in human peripheral lymphocytes

Unscheduled DNA synthesis (UDSox) and lipid peroxidation (LPO) induced by non-enzymatic activation of molecular oxygen (Fe2+ +H2O2) were measured in human peripheral lymphocytes from healthy volunteers. The effect of paracetamol (PC) in a final concentration range of 0.05-10 mmole/l on these oxidative processes and on DNA repair induced by MNNG (UDSmut) was investigated. The level of induced LPO was measured by the thiobarbituric acid assay, UDSox and UDSmut were determined by scintillometric measurement of incorporated [methyl-3H]thymidine into damaged DNA. PC at concentrations lower than 1 mmole/l significantly potentiates the non-enzymatically induced LPO and UDSox with the maximum of the activation being around 0.1 mmole/l. In contrast, PC at concentrations higher than 1 mmole/l exhibits an inhibitory effect on both LPO and UDSox. On the other hand, concentrations higher than 1 mmole/l significantly suppressed DNA-repair synthesis induced by MNNG.     

Relationship of tumor necrosis factor α expression with activation of sphingomyelinase and lipid peroxidation after removal of cholestatic factor

The goal of this work was to study the expression of tumor necrosis factor α (TNFα), sphingomyelin cycle activation, and lipid peroxidation (LPO) processes after the removal of a cholestatic factor in the liver subjected to different durations of cholestasis. Restored bile flow after a 9-day hepatic cholestasis normalized sphingomyelinase (SMase) activity and levels of TNFα and LPO products. The removal of a cholestatic factor after a 12-day cholestasis did not normalize the studied parameters: SMase activity and the levels of TNFα and LPO products remained much higher compared to control. A significant positive correlation between TNFα expression, SMase activity, and LPO rate has been revealed. The obtained data indicate that hepatocyte apoptosis after bile outflow restoration in late cholestasis can be due to the activation of the sphingomyelin cycle, LPO, and TNFα expression. The synergistic interaction can sharply increase the proapoptotic capacity of each of these factors since TNFα activates SMase and LPO, SMase activity depends on the LPO rate, while ceramide, an SMase-produced secondary messenger of apoptosis, can induce oxidative stress.     

Redox regulation of vascular prostanoid synthesis by the nitric oxide-superoxide system

Oxygen is involved in cell signaling through oxygenases and oxidases and this applies especially for the vascular system. Nitric oxide (*NO) and epoxyarachidonic acids are P450-dependent monooxygenase products and prostacyclin is formed via cyclooxygenase and a heme-thiolate isomerase. The corresponding vasorelaxant mechanisms are counteracted by superoxide which not only traps *NO but through the resulting peroxynitrite blocks prostacyclin synthase by nitration of an active site tyrosine residue. In a model of septic shock, this leads to vessel constriction by activation of the thromboxane A2-prostaglandin endoperoxide H2 receptor. This sequence of events is part of endothelial dysfunction in which the activated vascular smooth muscle counteracts and regenerates vessel tone by cyclooxygenase-2-dependent prostacyclin synthesis. Peroxynitrite was found to activate cyclooxygenases by providing the peroxide tone at nanomolar concentrations. Such new insights into the control of vascular function have allowed us to postulate a concept of redox regulation in which a progressive increase of superoxide production by NADPH-oxidase, mitochondria, xanthine oxidase, and even uncoupled NO-synthase triggers a network of signals originating from an interaction of *NO with superoxide.     

Role of endogenous free iron in activating lipid peroxidation in ischemia

Ischemia was simulated in rat liver perfused by physiological solution. The concentration of free iron and lipid peroxidation (LPO) products was measured 1, 2, 3, 4 and 5 hours after ischemia onset. The ESR method was used to measure free iron concentration. The LPO intensity was evaluated by the TBA test and by optical density at 232 nm. The content of free iron in cytoplasm increased in the course of ischemia with an increase in the concentration of LPO products. The content of free iron in the membranes remained unchanged. It is supposed that activation of LPO in ischemia may be caused by the appearance in the cytoplasm of a large amount of free iron. This iron can be liberated from ferritin in conditions of low oxygen concentration.     

Control of steroidogenesis in small and large bovine luteal cells

Evidence was cited to show that: (1) prostacyclin (PGI2) plays a luteotrophic role in the bovine corpus luteum and that products of the lipoxygenase pathway of arachidonic acid metabolism, especially 5-hydroxyeicosatetraenoic acid play luteolytic roles; (2) oxytocin of luteal cell origin plays a role in development, and possibly in regression, of the bovine corpus luteum; and (3) luteal cells arise from two sources; the characteristic small luteal cells at all stages of the oestrous cycle and pregnancy are of theca cell origin; the large cells are of granulosa cell origin early in the cycle, but a population of theca-derived large cells appears later in the cycle. Results of in vitro studies with total dispersed cells and essentially pure preparations of large and small luteal cells indicate that: (1) the recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system is involved in progesterone synthesis in the bovine corpus luteum; (2) activation of protein kinase C is stimulatory to progesterone synthesis in the small luteal cells; (3) activation of protein kinase C has no effect on progesterone synthesis in the large luteal cells; and (4) protein kinase C exerts its luteotrophic effect in total cell preparations, in part at least, by stimulating the production of prostacyclin. The protein kinase C system may cause down regulation of LH receptors in the large cells.     

Modification of an enzymic system of Ca2+ transport in sarcoplasmic reticulum during lipid peroxidation. In vivo damages in the development of pathological changes

The role of lipid peroxidation (LPO) in the damages of the enzymic system of Ca2+ transport in sarcoplasmic reticulum (SR) membranes of skeletal and cardiac muscles under conditions of vitamin E deficiency, ischemia and limb reoxygenation as well as in emotional-pain stress was investigated. It was shown that these processes are associated with activation of endogenous LPO in SR membranes "in vivo" and with simultaneous inhibition of Ca2+ transport, (i. e. decrease of the Ca2+/ATP ratio) and inactivation of Ca-ATPase. The degree of damage of the Ca2+ transport system was correlated with the concentration of LPO products accumulated in SR membranes "in vivo and during LPO induction by the Fe2+ + ascorbate system 'in vitro". Injection of natural and synthetic free radical scavengers (e. g. 4-methyl-2.6-ditretbutylphenol, alpha-tocopherol) to experimental animals resulted in practically complete suppression of LPO activation "in vivo" and in partial protection of the Ca2+-transporting capacity of SR membranes. A comparison of experimental results allowed to estimate the role of LPO in SR damage under pathological conditions. Model experiments with "contraction-relaxation" cycles including isolated components of muscle fibers (SR fragments and myofibrils) demonstrated that LPO induction in SR membranes by the Fe2+ + ascorbate system results in complete elimination of the relaxation step in myofibrils due to the loss of the SR affinity to decrease the concentration of Ca2+ in the incubation medium. This effect can be removed by free radical scavengers. The role of LPO in pathological changes of muscle contractility is discussed.     

The role of thrombin in the regulation of the endothelial prostaglandin production

Prostaglandin synthesis in endothelial cells may be initiated by the addition of exogenous substrate (arachidonic acid) or by addition of thrombin or the CA2+-ionophore A23187, which leads to prostacyclin formation from endogenous substrates. We noticed that endothelial cells produce more than twice the amount of prostacyclin when incubated with thrombin and arachidonic acid together than with arachidonic acid alone. In addition, it was found that the thrombin-induced conversion of endogenous substrates was inhibited by exogenous arachidonic acid. This means that the conversion of exogenous added arachidonic acid to prostacyclin was stimulated by thrombin. This activation of the enzymes involved in prostacyclin synthesis lasted about 5 min and could be inhibited by phospholipase inhibitors such as mepacrine and p-bromophenyl-acylbromide but not by the cAMP analogue dibutyryl cAMP, an inhibitor of arachidonic acid release from cellular phospholipids. These data demonstrate that, in addition to causing release of endogenous substrate, thrombin and the Ca2+-ionophore also activate the enzyme system involved in the further transformation of arachidonic acid.     

Prostacyclin inhibition of phosphatidic acid synthesis in human platelets is not mediated by protein kinase C

The activation of protein kinase C in human platelets by phorbol-12, 13- dibutyrate (PDBu) results in the phosphorylation of a 40,000 dalton protein. This phosphorylation is time- and concentration-dependent. Maximal phosphorylation is rapid and is not affected by indomethacin or prostacyclin. PDBu does not promote activation of the phosphodiesteratic cleavage (phospholipase C) of the inositol phospholipids and the subsequent formation of 1,2-diacylglycerol or its phosphorylated product, phosphatidic acid. If platelets exposed to PDBu are subsequently stimulated with thrombin, this stimulus does not initiate further 40,000 dalton protein phosphorylation but will promote the formation of phosphatidic acid and also the phosphorylation of a 20,000 dalton protein (myosin light chain). However, prostacyclin will prevent the subsequent stimulation of phosphatidic acid synthesis by thrombin in a concentration-dependent manner. The fact that prostacyclin can affect the response to thrombin, even in the presence of phorbol ester, supports the idea that the enzymes related to the formation of phosphatidic acid or inhibition of its synthesis are not related to the phosphorylated 40K protein.     

Platelet activation and prostacyclin release in essential hypertension.

To evaluate platelet activation thromboxane A2 (TxA2) and beta-thromboglobulin (beta TG) were used as markers and in addition we studied the biosynthesis of prostacyclin. Synthesis of TxA2 and prostacyclin was assessed by measurement of urinary metabolites. Fifteen untreated hypertensive patients (HT) and 15 age-matched normotensive controls (NT) were investigated at rest, during and after exercise. HT patients were re-examined after 3 months on enalapril. During basal conditions there was no difference in the excretion of Tx-M, PGI-M or beta TG between the groups. During strenuous exercise HT exhibit a significantly higher increase in prostacyclin synthesis (162%) compared to NT (76%). The levels of beta TG increased with 82% in the HT and 24% in the NT group, Tx-M increased with 27% and 23% respectively. Treatment with the ACE-inhibitor enalapril did not significantly alter these findings. These results indicate that there is no evidence of basal platelet activation in early essential hypertension. Strenuous exercise leads to some increase in Tx-M in both groups, with no pronounced differences between the groups. Hypertensive patients exhibit a significantly increased prostacyclin response to exercise which could be due to differences in vessel-wall reactivity. Enalapril seems to exert no effect on platelet activation or on prostacyclin biosynthesis.     

Long-term phorbol ester treatment dissociates phospholipase D activation from phosphoinositide hydrolysis and prostacyclin synthesis in endothelial cells stimulated with bradykinin

Bovine pulmonary artery endothelial cells (BPAEC) were prelabeled with [3H]choline or [3H]myristic acid to selectively label endogenous phosphatidylcholine. BPAEC were stimulated with ATP and bradykinin (BK), and phospholipase D (PLD) activation was detected as a 4-fold increase in [3H]choline in cells prelabeled with [3H]choline or as a 2- to 3-fold increase in [3H]phosphatidylethanol in cells prelabeled with [3H]myristic acid and stimulated in the presence of ethanol. Pretreatment of BPAEC with 0.1 microM phorbol 12-myristate 13-acetate (PMA) for 22 hr completely inhibited agonist-induced PLD activation, whereas prostacyclin synthesis and [3H]phosphoinositide ([3H]PIns) hydrolysis were enhanced in pretreated cells. Long-term PMA treatment thus dissociates agonist-induced PLD activation from [3H]PIns hydrolysis, and agonist-induced prostacyclin synthesis is not dependent upon PLD activation.     

HDL-induced cardiac prostacyclin synthesis: relative contribution of HDL apoprotein and lipid

The objective of this study was to determine if the apoprotein or lipid constituents of high density lipoproteins (HDL) mediate HDL-induced prostacyclin synthesis in the Langendorff-perfused rabbit heart. Acetylation, acetoacetylation, or partial removal by trypsin digestion of HDL apoprotein did not reduce the ability of the lipoprotein to stimulate cardiac prostacyclin synthesis. Delipidated apoproteins were less effective in stimulating cardiac prostacyclin synthesis in comparison to intact HDL. In contrast, protein-free lipid vesicles, made from HDL lipids, caused a pronounced stimulation of cardiac prostacyclin synthesis. These results suggest that HDL apoproteins, in their native state, are not essential for HDL-induced cardiac prostacyclin synthesis. The stimulation of cardiac prostacyclin synthesis by HDL may depend on the lipoprotein's lipid rather than on its apoprotein constituents.     

H2O2-mediated cross-linking between lactoperoxidase and myoglobin: elucidation of protein-protein radical transfer reactions

The H(2)O(2)-dependent reaction of lactoperoxidase (LPO) with sperm whale myoglobin (SwMb) or horse myoglobin (HoMb) produces LPO-Mb cross-linked species, in addition to LPO and SwMb homodimers. The HoMb products are a LPO(HoMb) dimer and LPO(HoMb)(2) trimer. Dityrosine cross-links are shown by their fluorescence to be present in the oligomeric products. Addition of H(2)O(2) to myoglobin (Mb), followed by catalase to quench excess H(2)O(2) before the addition of LPO, still yields LPO cross-linked products. LPO oligomerization therefore requires radical transfer from Mb to LPO. In contrast to native LPO, recombinant LPO undergoes little self-dimerization in the absence of Mb but occurs normally in its presence. Simultaneous addition of 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and LPO to activated Mb produces a spin-trapped radical electron paramagnetic resonance signal located primarily on LPO, confirming the radical transfer. Mutation of Tyr-103 or Tyr-151 in SwMb decreased cross-linking with LPO, but mutation of Tyr-146, Trp-7, or Trp-14 did not. However, because DBNBS-trapped LPO radicals were observed with all the mutants, DBNBS traps LPO radicals other than those involved in protein oligomerization. The results clearly establish that radical transfer occurs from Mb to LPO and suggest that intermolecularly transferred radicals may reside on residues other than those that are generated by intramolecular reactions.     

Prostacyclin-induced activation of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase in rat antral and fundic gastric mucosa.

The present investigations showed that after oral prostacyclin administration (100 micrograms/kg) as soon as the intracellular level of cAMP is elevated the activation of cAMP-dependent protein kinase follows in both parts (antrum and fundus) of rat gastric mucosa. The enzyme activation seems to be more significant in the fundic region which is in a complete agreement with the previously published results, i.e. the fundic mucosa reacts with de novo protein synthesis toward noxious agents (resulting finally in new cell formation), while the antral mucosa is more durable against damaging noxae. Taking into consideration all available data in the literature it seems that the intracellular effect of the exogenously administered prostacyclin in the gastric mucosa is a polyphasic effect, which contains the following consecutive steps: 1. Binding to the cell surface; 2. Effect on the intracellular second messenger system, (cAMP, cGMP); 3. Activation of the calmodulin system; 4. cAMP-dependent protein kinase activation; 5. DNA, RNA changes; 6. Influence on protein synthesis, and finally; 7. New cell formation.     

Mechanism of complement-induced stimulation of prostacyclin production by isolated rabbit peritoneum

The interaction between the complement system and prostaglandin synthesis has not thoroughly been explored, although both mediators are known to be involved in inflammatory reactions and endotoxic shock. When rabbit peritoneum, a rich source of prostacyclin forming activity was incubated in serum in which the complement system was activated (CVF, LPS, zymosan), the tissue produced significantly more PGI2, when compared with appropriate controls, indicating that by activation of the complement, factors were generated that stimulated PGI2 biosynthesis. Further results indicated that tryptic cleavage products of complement factor C3 and C5 also led to the appearance of PGI2 releasing principles with a molecular weight of about 7000-11000. The stimulation of PGI2 biosynthesis was explained by enhanced release of AA, and not due to increased activity of cyclo-oxygenase or PGI2 synthetase. Our results suggest that complement-derived products may promote the supply of prostaglandins at the site of inflammation.