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Biomedical subjects

R P Patel

Publications and source records attributed to R P Patel.

At least 37 records · Page 2Linked to original sources
PubMed1999

The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase.

Nitric oxide protects cells from oxidative stress through a number of direct scavenging reactions with free radicals but the effects of nitric oxide on the regulation of antioxidant enzymes are only now emerging. Using bovine aortic endothelial cells as a model, we show that nitric oxide, at physiological rates of production (1-3 nM/s), is capable of inducing the synthesis of glutathione through a mechanism involving gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase. This novel nitric oxide signalling pathway is cGMP-independent and we hypothesize that it makes an important contribution to the anti-atherosclerotic and antioxidant properties of nitric oxide.

Animals↗
PubMed1999

Molecular mechanisms of the copper dependent oxidation of low-density lipoprotein.

There is little doubt that oxidative modification of low-density lipoprotein (LDL) is an important process during atherogenesis. This conclusion has been derived in a relatively short period of time since the initial descriptions of LDL oxidation with a significant contribution from Professor Esterbauer and colleagues. In this short overview, we have described the mechanisms by which copper promotes LDL oxidation focussing on the importance of lipid hydroperoxides in this process. These mechanisms are discussed in the context of the ongoing debate as to relevance of metal dependent LDL oxidation in vivo and as a model reaction for assessing antioxidants.

Antioxidants↗
PubMed1999

Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH(2)-terminal kinase.

The c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, is a mitogen-activated protein kinase that determines cell survival in response to environmental stress. Activation of JNK involves redox-sensitive mechanisms and physiological stimuli such as shear stress, the dragging force generated by blood flow over the endothelium. Laminar shear stress has antiatherogenic properties and controls structure and function of endothelial cells by mechanisms including production of nitric oxide (NO) and superoxide (O(-)(2)). Here we show that both NO and O(-)(2) are required for activation of JNK by shear stress in endothelial cells. The present study also demonstrates that exposure of endothelial cells to shear stress increases tyrosine nitration, a marker of reactive nitrogen species formation. Furthermore, inhibitors or scavengers of NO, O(-)(2), or reactive nitrogen species prevented shear-dependent increase in tyrosine nitration and activation of JNK. Peroxynitrite alone, added to cells as a bolus or generated over 60 min by 3-morpholinosydnonimine, also activates JNK. These results suggest that reactive nitrogen species, in this case most likely peroxynitrite, act as signaling molecules in the mechanoactivation of JNK.

Animals↗
PubMed1998

A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure.

Muscle injury (rhabdomyolysis) and subsequent deposition of myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by approximately 80%. EPR and UV spectroscopy demonstrated that myoglobin was deposited in the kidneys as the redox competent ferric myoglobin and that it's concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of myoglobin-induced lipid peroxidation.

Animals↗
PubMed1998

Nitric oxide-dependent induction of glutathione synthesis through increased expression of gamma-glutamylcysteine synthetase.

The nitric oxide (NO) donors S-nitrosopenicillamine or DetaNONOate, which release NO at a rate of 0-15 nM sec-1, were exposed to rat aortic vascular smooth muscle cells for a period of 0-24 h. This treatment resulted in an increase in total glutathione levels of two- to threefold under conditions where no cytotoxicity was detected. The signaling pathways do not involve activation of protein kinase G Ialpha nor are they cGMP dependent. Oxidation of reduced glutathione (GSH) was found after exposure to NO for 3-4 h at rates of formation at or above 8 nM sec-1. Increased intracellular GSH was due to enhanced expression of the rate-limiting enzyme for GSH synthesis, gamma-glutamylcysteine synthetase. Since NO has been shown previously to protect cells against oxidative stress, we propose that the increase in GSH by NO is a potential mechanism for enhancing the antioxidant defenses of the cell. This result also has important implications for identifying redox-sensitive cell signaling pathways that can be activated by NO.

Animals↗
PubMed1997

Formation of the NO donors glyceryl mononitrate and glyceryl mononitrite from the reaction of peroxynitrite with glycerol.

Peroxynitrite (ONOO-), formed from the rapid reaction of superoxide (O2-.) with NO, is known to generate stable compounds capable of donating NO on reaction with thiols and molecules containing hydroxy groups. Using glycerol as a model compound for the reactions of ONOO- with biomolecules containing hydroxy groups, we separated the products and identified them by HPLC/MS. It was shown that both glyceryl mononitrate and glyceryl mononitrite were formed and released NO on incubation with copper and l-cysteine. The compounds were stable over a period of 4h when shielded from light and kept on ice. Slow spontaneous decomposition occurred in the buffer used for the bioassay, but this was not sufficient to explain the vasorelaxing properties of these NO donors. It is concluded that the stable organic nitrate and nitrite have the capacity to be metabolized by vascular tissues, resulting in vasorelaxation.

Animals↗
PubMed1997

The globin-based free radical of ferryl hemoglobin is detected in normal human blood.

Normal human venous blood was studied by electron paramagnetic resonance (EPR) spectroscopy at -196 degrees C. The EPR signal of free radicals in frozen blood is shown to have the same radiospectroscopic parameters and properties as the signal of the globin based free radical, .Hb(Fe(IV)=O), formed in the reaction of purified methemoglobin (metHb) with H2O2 and therefore has been assigned as such. The globin-based radicals and metHb exhibited significant variation (fluctuations) in different frozen samples taken from the same liquid blood sample. In any given sample a high concentration of free radicals was associated with a low concentration of metHb and vice versa, i.e. the fluctuations were always of opposite sense. No such fluctuations were observed in the concentration of two other paramagnetic components of blood, transferrin and ceruloplasmin. The time course of free radical formation and decay upon the addition of H2O2 to purified metHb was studied at three different molar ratios H2O2/metHb. This kinetic study together with the results of an annealing experiment allow us to propose a mechanism for the formation and decay of the globin-based radical in blood. Within this mechanism, the source of H2O2 in blood is considered to be dismutation of O-2 radicals produced via autoxidation of Hb. We postulate that the dismutation is intensified on the phase separation surfaces during cooling and freezing of a blood sample. The fluctuations are explained within this hypothesis.

Electron Spin Resonance Spectroscopy↗
PubMed1997

Reduction of Cu(II) by lipid hydroperoxides: implications for the copper-dependent oxidation of low-density lipoprotein.

The Cu(II)-promoted oxidation of lipids is a lipid hydroperoxide (LOOH)-dependent process that has been used routinely to assess the oxidizability of low-density lipoprotein (LDL) in human subjects. Metal-dependent redox reactions, including those mediated by copper, have been implicated in the pathogenesis ofatherosclerosis. Despite its widespread use and possible biological significance, key elements of the mechanism are not clear. For example, although it is evident that copper acts as a catalyst, which implies a redox cycle between the Cu(II) and Cu(I) redox states, the reductants remain uncertain. In LDL these could include alpha-tocopherol, amino acid residues on the protein and LOOH. However, both alpha-tocopherol and amino acid residues are probably consumed before the most rapid phase of lipid peroxidation occurs, suggesting that another reductant must be donating electrons to Cu(II), the most likely candidate being LOOH. This role has been disputed, since LDLs nominally devoid of LOOH are still capable of reducing Cu(II) to Cu(I) and thermodynamic calculations for this reaction are not favourable. Direct investigation of the role of LOOH as reductant has not been reported and in the present study, using simple lipid systems and LDL, we have re-examined this issue using the Cu(I) chelator bathocuproine. We have shown that Cu(II) may promote lipid peroxidation in liposomes, which do not contain either protein or alpha-tocopherol, and that this is associated with reduction to Cu(I). The data also indicate that an equilibrium between free Cu(II) and LOOH exists, which only in the presence of an oxidizable substrate, i.e. unsaturated fatty acids, is shifted towards formation of Cu(I) and lipid-derived peroxyl radicals. We propose that reduction of Cu(II) by LOOH is a necessary component in sustaining the propagation of lipid peroxidation and that the formation of peroxyl radicals and their products in a lipid environment is sufficient to overcome thermodynamic barriers to the reaction.

Copper↗
PubMed1997

The interplay of nitric oxide and peroxynitrite with signal transduction pathways: implications for disease.

Since the discovery that at least one form of endothelium derived relaxing factor is nitric oxide (NO), numerous studies have uncovered diverse roles for this free radical in a variety of physiological and pathophysiological processes. NO production, a process mediated by a family of enzymes termed NO synthases, has been detected in most cell types. Many of the effects of NO are thought to be mediated through its direct interaction with specific and defined cell signaling pathways. The nature of such interactions are highly dependent on the concentration of NO and cell type. Furthermore, specific NO derived reaction products, such as peroxynitrite, also have the potential to effect cell signal transduction events. As with NO, this can occur through diverse mechanisms and depends on concentration and cell type. It is perhaps not surprising that the reported effects of NO in different disease states are often conflicting. In this brief overview, a framework for placing these apparently disparate properties of NO will be described and will focus on the effects of NO and peroxynitrite on signaling pathways.

Animals↗
PubMed1996

An EPR investigation of human methaemoglobin oxidation by hydrogen peroxide: methods to quantify all paramagnetic species observed in the reaction.

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglobin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H2O2 was added at 3- and 10-fold molar excess to 100 microM metHb in 50 mM phosphate buffer, pH 7.4, 37 degrees C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H2O2 the EPR signals (-196 degrees C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (-196 degrees C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g parallel = 2.035 and g perpendicular = 2.0053), and the other an isotropic singlet (g = 2.0042, delta H = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic behaviour of both peroxyl (ROO.) and non-peroxyl (P.) free radicals were similar, we concluded that ROO. radicals are not formed from P. radicals by addition of O2. The time courses for both radicals showed a steady state during the time required for H2O2 to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 x 10(-3) s-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H2O2. The formation of the rhombic Fe(III) non-haem centres with g = 4.35 was found. Their yield was proportional to the H2O2 concentration used and the centres were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at -196 degrees C were used.

Electron Spin Resonance Spectroscopy↗
PubMed1996

Redox cycling of human methaemoglobin by H2O2 yields persistent ferryl iron and protein based radicals.

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H2O2, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H2O2 resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H2O2 showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H2O2. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalytic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10(4) fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: approximately 20 mol H2O2 and abolished the protein radical steady state. The biological implications for these results are discussed.

Catalase↗
PubMed1996

Formation of oxysterols during oxidation of low density lipoprotein by peroxynitrite, myoglobin, and copper.

Oxidation of low density lipoprotein (LDL) in the artery wall leads to the formation of cholesterol oxidation products that may result in cytotoxicity. Different mechanisms could contribute to LDL oxidation in vivo resulting in characteristic and specific modification of the cholesterol molecule. Alternatively, attack on cholesterol by chain propagating peroxyl radicals could result in the same distribution of oxidation products irrespective of the initial pro-oxidant mechanism. To distinguish between these possibilities we have monitored the formation of nine oxysterols during LDL oxidation, promoted by copper, myoglobin, peroxynitrite, or azo bis amidino propane. Regardless of the oxidant used, the pattern of oxysterol formation was essentially the same. The yields of products identified decreased in the order 7-oxocholesterol > 7 beta-hydroxycholesterol > 7 alpha-hydroxycholesterol > 5,6 beta-epoxycholesterol > 5,6 alpha-epoxycholesterol except in the case of peroxynitrite in which case a higher yield of 5, 6 beta-epoxycholesterol relative to 7-oxocholesterol was found. No formation of cholestane 3 beta, 5 alpha, 6 beta-triol, or the 24-,25-,27-hydroxycholesterols was seen. Concentration of 7-oxocholesterol levels in LDL was positively correlated with the degree of protein modification. Endogenous alpha-tocopherol in LDL or supplementation with butylated hydroxytoluene prevented oxysterol formation. Taken together these data indicate that the oxidation of cholesterol and protein in LDL occur as secondary oxidation events consequent on the attack of fatty acid peroxyl/alkoxyl radicals on the 7-position of cholesterol, and with amino acids on apoB. Furthermore, oxidant processes with atherogenic potential, such as peroxynitrite, copper, and myoglobin are capable of producing oxidized LDL containing cytotoxic mediators.

Amidines↗
PubMed1995

Pro-oxidant effects of cross-linked haemoglobins explored using liposome and cytochrome c oxidase vesicle model membranes.

The therapeutic use of cell-free haemoglobin as a blood substitute has been hampered by toxicological effects. A model asolectin (phosphatidylcholine/phosphatidylethanolamine) liposome system was utilized to study the pro-oxidant efficiency of several chemically modified haemoglobins on biological membranes. Lipid peroxidation, resulting from the interactions between haemoglobin and liposomes, was measured by conjugated diene formation and the maximal rates of oxygen uptake. Spectral changes gave insight into the occurrence of the ferryl iron species. The residual reactivity of oxidatively damaged haemoglobins with ligands during incubation with liposomes was assessed from rapid kinetic carbon monoxide-binding experiments. Liposomes in which cytochrome c oxidase was embedded show both haemoglobin and the enzyme to be oxidatively damaged during incubation. The functional state of cytochrome c oxidase was monitored in the presence and absence of a free radical scavenger. Once in contact, both unmodified and modified haemoglobins triggered and maintained severe radical-mediated membrane damage. Differences in the pro-oxidant activities among haemoglobins may be explained by either the differential population of their ferryl intermediates or disparate dimerization and transfer of haem into the membrane with subsequent haem degradation. This study may contribute to a better understanding of the molecular determinants of haemoglobin interactions with a variety of biological membranes.

Animals↗