Effect of 4-hydroxy-2(E)-nonenal on soybean lipoxygenase-1.

The oxidation of linoleic acid by soybean lipoxygenase-1 (LOX-1) was inhibited in a time-dependent manner by 4-hydroxy-2(E)-nonenal (HNE). Kinetic analysis indicated the effect was due to slow-binding inhibition conforming to an affinity labeling mechanism-based inhibition. After 25 min of preincubation of LOX-1 with and without HNE, Lineweaver-Burk reciprocal plots indicated mixed noncompetitive/competitive inhibition. Low concentrations of HNE influenced the electron paramagnetic resonance (EPR) signal of 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid (13-HPODE)-generated Fe3+-LOX-1 slightly, but higher concentrations completely eliminated the EPR signal indicating an active site hindered from access by 13-HPODE. HNE may compete for the active site of LOX-1 because its precursor, 4-hydroperoxy-(2E)-nonenal, is a product of LOX-1 oxidation of (3Z)-nonenal. Also, it was an attractive hypothesis to suggest that HNE may disrupt the active site by forming a Michael adduct with one or more of the three histidines that ligate the iron active site of LOX-1.

Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea.

The role of active oxygen species has been studied in spreading soft-rot lesions caused by the necrotrophic fungal pathogen Botrytis cinerea Pers.:Fr. in leaves of four genotypes of French bean (Phaseolus vulgaris L.). Large increases were observed for the aldehydic end-products of oxidative damage, malondialdehyde and 4-hydroxy-2-nonenal, as a result of infection in each of the genotypes studied. Similar increases were found in a stable free radical and g=4.27 Fe(III) signals, but not Mn(II) signals, in electron paramagnetic resonance spectra. These changes were accompanied by large decreases in ascorbic acid levels, with changes in the antioxidant glutathione being genotype dependent.

Electron spin resonance studies of nitrosyl haemoglobin in human liver, colon and stomach tumour tissues.

Iron nitrosyl haemoglobin (HbFeNO) gives well defined ESR spectra, and can be detected at room temperature, in contrast with most transition metal complexes of biological importance. This is because the unpaired electron remains strongly localised on the NO ligand. It is of importance because it proves the formation of nitric oxide, which unfortunately cannot be detected directly by ESR spectroscopy. We have studied a range of tissues taken from human liver, colon and stomach tumours which have been directly frozen to 77K and studied at 77K. The results show that formation of HbFeNO is rare in tissue adjacent to tumour tissue ("peripheral tissue"), but is always found in necrotic central regions, if present. However, in several cases, HbFeNO was also detected in tumour tissue which was not necrotic. Two factors contribute to the formation of this complex. One is the presence of "free" NO molecules in the cellular regions, and the other is the presence of deoxyferrohaemoglobin, since neither ferrihaemoglobin nor oxyhaemoglobin react to give this complex. [For systems containing myoglobin these comments include the possibility of the formation of nitrosylmyoglobin, which gives very similar ESR spectra.

Electron paramagnetic resonance spectroscopy of stable free radicals in the liver compared with ultrastructural and functional damage in a rat model of alcohol- and iron-overload.

1. Electron paramagnetic resonance spectroscopy was used to study free-radical signals in freeze-clamped frozen liver tissue from rats after a 1 year period of dietary supplementation with alcohol, iron, or alcohol and iron. In alcohol-fed, iron-fed and alcohol- and iron-fed animals, mild histological damage was seen on light microscopy and evidence of mitochondrial and nuclear injury was identified by electron microscopy. 2. Subcellular fractionation studies showed an increase in the activity of the peroxisomal marker catalase (P < 0.01) in alcohol-fed rats compared with controls, but a fall of 82% (P < 0.001) in alcohol- and iron-fed animals. The activity of the mitochondrial marker succinate dehydrogenase rose by 7% (not significant) in alcohol-fed animals and by 17% (not significant) in iron-fed animals, but fell by 94% (P < 0.001) in alcohol- and iron-fed animals, suggesting serious impairment of mitochondrial function. 3. Iron overload was substantial in the iron-fed animals and there was an excellent correlation between liver iron concentration and iron-derived signals by electron paramagnetic resonance spectroscopy (P < 0.001). A clear free-radical signal of g = 2.003-2.005 was detected in all liver samples, but there was no significant difference in the magnitude of this signal in any study group. 4. The absence of any increase in the stable free-radical signal, even in the presence of considerable hepatic damage, does not support the hypothesis that free radicals mediate alcoholic liver disease in this animal model, although the results cannot be taken as proof against this hypothesis.

Effects of 6-hydroxydopamine on pre- and post-junctional 5-HT1-like receptor-mediated responses in dog saphenous vein.

To investigate whether 5-HT1-like receptor-mediated inhibition of adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation occurs in nerves or smooth muscle of saphenous vein, infusions of 6-hydroxydopamine (6-OHDA) were administered to dogs with the aim of inducing sympathetic nerve damage. The effects of 6-OHDA on other 5-HT1-like receptor-mediated responses at the pre- and post-junctional level were investigated for comparison by studying 5-hydroxytryptamine (5-HT)-induced inhibition of 3H-noradrenaline release and contraction of smooth muscle respectively. Disruption of nerve function by 6-OHDA was revealed by the lack of catecholaminergic fluorescence and neurogenic contractile responses in saphenous veins from dogs treated with 6-OHDA. In addition, severe impairment of neuronal uptake mechanisms were apparent since basal efflux of 3H-noradrenaline, electrically-evoked release of 3H-noradrenaline and remaining 3H-noradrenaline content were considerably reduced. Some 3H-noradrenaline was taken up and released in 6-OHDA-treated tissues which is consistent with the existence of nerve varicosities resistant to the present dosing regime of 6-OHDA, an observation substantiated by electron microscopy studies showing inconsistent lesions of nerve terminals. 6-OHDA pre-treatment potentiated the smooth muscle contractile responses mediated by 5-HT1-like receptors as well as potentiating 5-HT-evoked inhibition of prostaglandin E2-stimulated cyclic AMP accumulation. It did not, however, affect 5-HT-induced inhibition of 3H-noradrenaline release. The present results suggest that inhibition of cyclic AMP accumulation by 5-HT occurs predominantly in smooth muscle.

The effects of chronic administration of adrenaline on the function and number of adrenoceptors in the rabbit.

Chronic (10-day) intravenous infusions of adrenaline (0.05 mumol/kg/h) were given to rabbits via osmotic minipumps implanted at the femoral vein. Blood pressure, heart rate, and plasma catecholamine concentrations were measured five times during the period of infusion. Tenfold elevations in circulating adrenaline levels were achieved within 24 h of commencing infusion and maintained throughout the study. This increase in plasma adrenaline was not accompanied by significant changes in blood pressure or heart rate. Rabbits were killed after 10 days: blood was withdrawn for platelet aggregation studies. Kidney, heart, and lung were also collected and alpha 2-adrenoceptor number on platelets and kidney measured using [3H]yohimbine. Beta adrenoceptors on platelets, lymphocytes, heart, and lung were quantified using [125I]iodocyanopindolol. Adrenaline infusion led to a significant reduction in platelet aggregation responses to adrenaline (0.001-100 microM), together with a decrease in alpha 2-adrenoceptor number on platelets, but no significant decrease in kidney alpha 2-adrenoceptors. A significant decrease in the density of beta adrenoceptors on heart and lung membranes was observed with no reduction in platelet and lymphocyte beta-adrenoceptor number. Thus adrenaline-induced down-regulation of adrenoceptors in the rabbit was dependent on the location and subtype of adrenoceptor.

Rapid and reversible desensitisation of vascular and platelet alpha 2 adrenoceptors.

The effects of intravenous infusion with the alpha2 adrenoceptor selective agonist alpha methylnoradrenaline on pressor responses to alpha adrenoceptor agonists, alpha2 adrenoceptor mediated platelet aggregation and adenylate cyclase were examined in conscious rabbits. Pressor responses to alpha methylnoradrenaline but not phenylephrine were decreased in a dose dependent manner during methylnoradrenaline infusion at all times examined. Recovery of these responses after stopping infusion was dependent on both the dose infused and the duration of the infusion. Alpha methylnoradrenaline infusion resulted in a dose and time dependent decrease in the pro-aggregatory response of platelet to adrenaline without any significant change in the response to ADP or in the number of [3H]yohimbine binding sites. The ability of PGE1 to stimulate adenylate cyclase was not influenced by alpha methylnoradrenaline infusions. However, reversal of this stimulation by adrenaline was decreased by relatively long (30 min) infusions of the highest dose of alpha methylnoradrenaline examined. It is concluded that alpha methylnoradrenaline infusions resulted in desensitisation of all the alpha2 adrenoceptor mediated responses examined. However the time course for the desensitisation apparently differed according to the response examined.

Changes in rabbit platelet alpha and beta adrenoceptor number and platelet aggregation.

Treatment with phenoxybenzamine caused a decrease in the number of alpha but not beta adrenoceptor ligand binding sites on platelets from male rabbits and thus a decrease in the ratio of alpha/beta adrenoceptor number. This was accompanied by decreased aggregation both in the presence and absence of propranolol. In contrast in female rabbits maturation and oestrogen treatment resulted in a decrease in both alpha and beta adrenoceptor ligand binding and no change in the alpha/beta adrenoceptor ratio or in the aggregatory response of the platelets to adrenaline in the absence of propranolol.

Cardiac and lymphocyte beta-adrenoceptors in perinephritis hypertension in the rabbit.

The effects of perinephritis hypertension on lymphocyte and cardiac beta-adrenoceptors in the rabbit were examined. Hypertensive animals 8-12 weeks after surgery had an increase in cardiac weight consistent with hypertrophy compared with sham-operated age-matched controls. Specific binding of [I125] iodocyanopindolol (ICYP) to cardiac ventricular membranes was reduced in the hypertensive animals (Bmax44 +/- 14 in hypertensives and 30 +/- 15 fmoles/mg protein in controls; p less than 0.01). However, weight-matched ventricles from a group of older sham-operated normotensive rabbits showed a similar cardiac beta-receptor number to that found in the hypertensive animals. There were no changes in affinity of the ligand for the binding site. The reduction in cardiac beta-receptor density was not accompanied by changes in the chronotropic or blood pressure responses to isoprenaline in the conscious animal. Specific ICYP binding to lymphocyte beta-receptors did not differ significantly between the hypertensive and age-matched normotensive animals. Lymphocyte beta-adrenoceptors thus may not always reflect changes in heart beta-adrenoceptors, and changes in receptor density may not be directly related to blood pressure or have identifiable functional significance.

Desensitization of platelet alpha 2-adrenoceptors after short term infusions of adrenoceptor agonist in man.

The effect of intravenous infusion of catecholamines and related drugs on human platelet alpha 2-adrenoceptor number and function was investigated. Short (60-120 min) infusions of catecholamines with alpha 2 agonist activity in vivo produced attenuation of the platelet responses to adrenaline in vitro. This desensitization was specific for the adrenaline induced aggregatory response. The maximum number of [3H]yohimbine binding sites on platelets was not altered by adrenaline infusion. The ability of adrenaline to reduce platelet cyclic AMP levels was significantly reduced after the infusions. Acute infusions of alpha 2-adrenoceptor agonists may alter the coupling of the platelet alpha 2-adrenoceptor to adenylate cyclase.