Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic isolated rat heart.

OBJECTIVE: A brief period of ischaemia (5 min) and reperfusion (5 min), prior to a longer period of ischaemia and reperfusion, has been shown to reduce the extent of injury (necrosis, arrhythmias, or postischaemic contractile malfunction) caused by a subsequent longer period of ischaemia and reperfusion. Adenosine has been identified as a factor in the protection afforded against regional tissue necrosis by such preconditioning. The aim of this study was to assess the role of adenosine in preconditioning induced protection of postischaemic function in the globally ischaemic isolated rat heart. METHODS: The ability of global ischaemia to precondition against postischaemic contractile malfunction was first confirmed in the isolated ejecting rat heart preparation. Hearts (n = 6 per group) were perfused aerobically (37 degrees C, paced at 350 beats.min-1) for 20 min, at the end of which contractile function was measured. This was followed by 10 min of Langendorff perfusion (control group) or 5 min of global ischaemia plus 5 min of Langendorff reperfusion (preconditioned group). The hearts were then subjected to 20 min of global ischaemia (37 degrees C) and 35 min of reperfusion (15 min Langendorff and 20 min ejecting); function was then reassessed. RESULTS: Postischaemic recovery of aortic flow was 26(SEM 8)% in the control group v 57(4)% in the preconditioned group (p < 0.05). To assess whether exogenous adenosine could mimic this protection, the experiments were repeated with the 5 min period of ischaemic preconditioning replaced by 5 min of aerobic Langendorff perfusion with adenosine-containing buffer (100, 50, or 10 mumol.litre-1). No protection of postischaemic function was observed in any of the adenosine treated groups. In further experiments, we assessed whether ischaemic preconditioning persisted in the presence of the A1/A2 adenosine antagonist, 8 (p-sulphophenyl) theophylline (8-SPT). Since pacing was not used in these studies, the ability of ischaemia to precondition the myocardium was again confirmed; the protocol was then repeated with 8-SPT (10 mumol.litre-1) present in the perfusate throughout. Although 8-SPT depressed recovery in both control and preconditioned hearts it failed to abolish the protective effects of ischaemic preconditioning. CONCLUSIONS: There is no evidence from these results to support the involvement of adenosine to any major extent in preconditioning induced protection of postischaemic contractile function in the isolated rat heart.

Effects of N-methyl hexanoylhydroxamic acid (NMHH) and myoglobin on endothelial damage by hydrogen peroxide.

OBJECTIVE: The aim was to investigate the interaction of the novel antioxidant N-methyl hexanoylhydroxamic acid (NMHH) with myoglobin in protecting endothelial cells against H2O2 mediated damage. METHODS: Cultured bovine aortic endothelial cells were exposed to 50-100 microM H2O2 for 10-60 min with and without NMHH and/or myoglobin, and immediate or delayed damage was assessed by lactate dehydrogenase release, 3H adenine uptake, a tetrazolium reduction assay, and microscopy. RESULTS: Brief exposure to low concentrations of H2O2 caused cell damage, for which the tetrazolium reduction assay was the most sensitive assay, and inhibited subsequent cell division. NMHH in concentrations from 50 to 200 microM protected against damage provided it was present at the time of adding H2O2, and the effect was markedly potentiated by 10 microM oxymyoglobin, which had little protective effect alone. CONCLUSIONS: NMHH is an effective antioxidant which is markedly potentiated by low concentrations of oxymyoglobin. Oxymyoglobin may potentiate NMHH by scavenging H2O2 through the rapid formation of ferrylmyoglobin, which is then reduced by NMHH. This synergism may be particularly relevant to the protection of myoglobin-rich cells such as myocytes.

Novel monohydroxamate drugs attenuate myocardial reperfusion-induced arrhythmias.

The novel monohydroxamates N-methyl hexanoylhydroxamic acid, N-methyl acetohydroxamic acid, and N-methyl butyrohydroxamic acid have antioxidant and iron chelating properties. They attenuated reperfusion-induced contractile dysfunction following long periods of ischaemia (50 min) in the isolated rat heart. Here we compare their effects and that of the trihydroxamate desferrioxamine on reperfusion-induced arrhythmias following short duration ischaemia (10 min). Isolated rat hearts were perfused by the Langendorff method, subjected to regional ischaemia and reperfusion. Arrhythmias induced during the first 5 min of reperfusion were quantified. Drugs (all at 150 microM) were introduced during the last 2 min of ischaemia and remained throughout reperfusion. Although the monohydroxamate- and desferrioxamine-treated hearts showed a reduction in the incidence of ventricular tachycardia and fibrillation, only the reduction in the incidence of sustained fibrillation ( > 3 min duration) in N-methyl acetohydroxamic acid--(27%), N-methyl hexanoylhydroxamic acid--(27%) and desferrioxamine-treated hearts (20%) was statistically significant (p < 0.05 vs control 73%; n = 15). There was a reduction in the severity of the arrhythmias, manifest as a significant increase in the duration of sinus rhythm in all the monohydroxamate-treated hearts, and a significant reduction (vs control 218 +/- 29 s; mean +/- SEM) in the duration of ventricular fibrillation in hearts treated with N-methyl acetohydroxamic acid (101 +/- 31 s) and desferrioxamine (112 +/- 30 s). This improvement was offset by an increase in the duration of ventricular tachycardia, in hearts treated with N-methyl acetohydroxamic acid, N-methyl butyrohydroxamic acid and desferrioxamine. These results suggest that these novel monohydroxamates, particularly N-methyl acetohydroxamic acid, attenuate reperfusion-induced arrhythmias in this model when introduced during the ischaemic period.

Non-oxidative modification of low density lipoprotein by ruptured myocytes.

In this study, the interaction of ruptured cardiac myocytes with low density lipoprotein (LDL) has been investigated and the consequent extent of uptake by macrophages. The results show that lysate released from ruptured myocytes is capable of inducing LDL oxidation and that the resulting modified form is recognised and degraded by macrophages. Peroxyl radical scavengers inhibit the LDL oxidation but not the macrophage uptake suggesting that LDL can be modified by mechanisms that are independent of oxidative processes by intracellular constituents of cardiac myocytes.

Effects of pulsed electromagnetic fields on Na+ fluxes across stripped rabbit colon epithelium.

The effect of pulsed electromagnetic fields on the electrical potential and two-way flux of Na+ across the epithelium of the rabbit colon in vitro was investigated. In control experiments the transepithelial mucosal-to-serosal and serosal-to-mucosal fluxes (Jm----s and Js----m) were constant over the experimental period. When the epithelium was at right angles to the applied electromagnetic field, the Jm----s flux of Na+ was reduced, whereas Js----m was enhanced. When the epithelium was rotated 180 degrees, Jm----s was enhanced, whereas Js----m was reduced. When the epithelium was mounted parallel to the magnetic field, both Jm----s and Js----m of Na+ were increased, the latter continuing to increase after the field was turned off. When the tissue was rotated 180 degrees, the same enhanced flux was observed, but now the Jm----s flux showed the greatest increase, which again occurred in the period after the field was turned off. The rate of decrease of transepithelial potential difference in all orientations was less than the control. Also, the conductance increased in orientations 2-4 and decreased in orientation 1 after the field had been applied. This suggests that pulsed electromagnetic fields can have a direct effect on the movement of Na+ across tissue and transepithelial potentials. The mechanism may depend on several factors, such as induced changes in certain ion pumps, the membrane potential, and the surface charge of cell wall proteins.

The effects of vasoactive intestinal polypeptide on the electrical activity of guinea-pig intestinal smooth muscle.

The effect of vasoactive intestinal polypeptide (VIP) on the electrical activity of the smooth muscle of the guinea-pig taenia coli has been examined using the single sucrose gap apparatus. VIP usually caused a slowly developing, long-lasting membrane hyperpolarisation, although sometimes it reduced the frequency of spontaneous spike discharge without hyperpolarising the membrane. In contrast, non-adrenergic, non-cholinergic nerve stimulation initiated rapid membrane hyperpolarisation followed by post inhibitory excitation. In the presence of apamin, the VIP response was little affected, whereas the responses to non-adrenergic, non-cholinergic nerve stimulation were either markedly antagonised or reversed to membrane depolarisation. Consideration is given to the possible role of either VIP or ATP as the neurotransmitter responsible for the inhibitory junction potential elicited in the guinea-pig taenia coli in response to non-adrenergic inhibitory nerve stimulation.

The effects of ascorbic acid and iron co-supplementation on the proliferation of 3T3 fibroblasts.

Exposure of 3T3 fibroblasts to FeII reveals a concentration-dependent inhibition of cell proliferation compared to control cells, the apparent threshold for this iron-mediated effect being 5 microM FeII. The inhibition of cell proliferation was accompanied by an enhancement of total malondialdehyde (MDA) levels (as detected directly by hplc) in the cells at higher iron concentrations. The co-supplementation of FeII with varying concentrations of ascorbic acid over the range 5 microM to 240 microM had no significant effect on the threshold for iron toxicity or lipid peroxidation. These results show that there is neither a significant exacerbation of the pro-oxidant effect of FeII nor any protective effect of ascorbate when cultures of 3T3 mouse fibroblasts are exposed to co-supplementation regimes of iron with ascorbic acid.

Effects of co-supplementation of iron with ascorbic acid on antioxidant--pro-oxidant balance in the guinea pig.

The relationship between intake of iron with ascorbic acid and their uptake into the plasma and liver of guinea pigs was studied. The influence on the antioxidant/pro-oxidant balance of liver microsomes was also determined. Animals were fed a standard pelleted diet low in iron and ascorbic acid for 35 days. The pellet diet was supplemented by oral dosing with a solution containing either maintenance dietary levels of ascorbic acid and iron, or one of three regimens that increased the dosage of these substances ten fold. There were no significant differences in animal growth rate or food intake between these regimens. Liver and plasma total ascorbate levels were significantly increased (p < 0.05) in animals receiving either ascorbic acid alone (liver 126 +/- 36 micrograms/g tissue wet wt. and plasma 51.7 +/- 17.0 microM; n = 9) or ascorbic acid and iron (105 +/- 18 micrograms/g and 40.3 +/- 15.3.0 microM; n = 8) compared to controls (84 +/- 36 micrograms/g and 15.3 +/- 8.5 microM; n = 11). Total iron levels in the liver (76.7 +/- 7.3 micrograms/g; control; n = 6) and plasma (2.4 +/- 0.03 mg/l; control) were not significantly raised in animals under these conditions of iron or ascorbate intake. Liver microsomes isolated from animals receiving iron had a greater susceptibility to oxidative stress in terms of malondialdehyde production during auto-oxidation compared to those from control animals under the same conditions. This effect was eliminated on combining ascorbic acid with the iron supplementation, suggesting that oral administration of vitamin C has a protective rather than a pro-oxidant effect under these circumstances.

Do iron and vitamin C co-supplementation influence platelet function or LDL oxidizability in healthy volunteers?

OBJECTIVE: To examine the effect of co-supplementation with iron and vitamin C on antioxidant status, platelet function and low density lipoprotein oxidation in normal healthy volunteers. DESIGN: The study was carried out with two groups of 20 subjects each acting as their own control, comparing presupplemention with postsupplemention. One group was supplemented with iron and the RDA level of vitamin C and the second group with iron and 260 mg/d vitamin C. SETTING: The International Antioxidant Research Centre, The Guy's, King's College and St Thomas's School of Biomedical Science, Guy's Campus, London. SUBJECTS: Forty normal healthy volunteers, recruited from the staff of the Medical School and Hospital in which two volunteers withdrew during the study. INTERVENTIONS: Subjects in both studies were randomly assigned to one of two groups (5 males and 5 females group) and received supplements containing iron (14 mg/d) and either 60 mg/d (Group A) or 260 mg/d (Group B) vitamin C for 12 wk. Blood samples were taken at 6 wk and 12 wk, and prior to supplementation and analysed for iron and antioxidant status (transferrin bound iron, vitamin C and E, and beta-carotene levels) in both studies. Samples from the first study were analysed for the susceptibility of LDL isolated from plasma to Cu2+-induced oxidation and samples from the second for platelet function. RESULTS: Transferrin-bound iron was significantly increased (P < 0.05) at 12 wk, in Group A subjects (from 14.9+/-5.3 micromol/1 to 19.5+/-2.3 micromol/l; mean+/-s.d.; n=19), whereas those in Group B showed a significant increase (P < 0.05) after 6 wk (from 15.8+/-4.5 micromol/l to 20.4+/-6.6 micromol/l; n = 19) which decreased at 12 wk (16.3+/-5.0 micromol/l). Plasma total ascorbate significantly increased from an initial level of 59.3+/-21.3 micromol/l to 87.6+/-29.0 micromol/l after 6 wk and 81.7+/-11.4 micromol/l after 12 wk following the Group B supplementation, but only after 12 wk in Group A (from 64.0+/-24.8 micromol/l to 77.2+/-13.2 micromol/l). Plasma alpha-tocopherol concentrations were significantly increased after 6 wk and 12 wk with both levels of supplementation (from 24.2+/-5.71 micromol/l Group A and 23.4+/-5.3 micromol/l Group B to 26.3+/-5.5 micromol/l and 25.71+/-4.7 micromol/1 respectively at 12wk). The mean lag phase to oxidation of low density lipoprotein (LDL) was significantly increased in subjects in Group B after 12 wk ingestion of iron and 260 mg vitamin C (from 80.0+/-14.8 min to 97.2+/-16.9 min; n = 9). Platelet sensitivity to ADP-induced aggregation was significantly decreased (P < 0.05) by 12 wk in Group A (from EC50 2.3 < or = 1.3 microM to 3.7+/-2.2 microM; n = 10), whereas those receiving higher vitamin C showed a significant decrease (P < 0.05; from EC50 1.9+/-0.6 microM to 3.1+/-1.8 microM) after 6wk which subsequently increased towards presupplemental levels (2.6+/-1.6 microM). Platelets from the latter subjects showed a significant reduction in ADP-induced ATP secretion at both 6wk and 12 wk. CONCLUSION: The results show modest beneficial effects on LDL oxidation and platelet function following supplementation with iron and vitamin C. No evidence for pro-oxidant effects was observed.

Serum total antioxidant activity after myocardial infarction.

Serum total antioxidant activity (TAA), albumin and uric acid were measured on admission, and for the next 2 days in 56 patients suffering myocardial infarction, 20 of whom received streptokinase. The 'antioxidant gap', the difference between the serum TAA and the sum of the serum albumin and uric acid activity, was calculated. No significant changes in serum total antioxidant activity were observed in either group of patients between admission, day 1 and day 2. However, a decline in the 'antioxidant gap' after myocardial infarction was associated with a significantly higher mortality.

Comparison of N-methyl hexanoylhydroxamic acid, a novel antioxidant, with desferrioxamine and N-acetyl cysteine against reperfusion-induced dysfunctions in isolated rat heart.

The effects of a novel free radical scavenger, N-methyl hexanoylhydroxamic acid (NMHH) on ischaemia and reperfusion-induced dysfunction were compared with those of desferrioxamine (DFO) and N-acetyl cysteine (NAC) in isolated Langendorff-perfused rat heart. NMHH (150 microM) produced significant improvement in recovery of left ventricular developed pressure (LVDP) as compared with that of control hearts (35 +/- 8%) when included in the perfusate both before ischaemia and on reperfusion (68 +/- 6%) and on reperfusion alone (61 +/- 6%). In contrast, neither DFO (52 +/- 9%) nor NAC (54 +/- 12%), at the same concentration, produced a significant increase in recovery of LVDP when applied on reperfusion alone. Only DFO (77 +/- 5%), not NAC (54 +/- 12%), had a significant effect on recovery of LVDP when added to the perfusate before ischaemia and reperfusion. NAC applied before ischaemia produced significant improvement in recovery of heart rate (HR). The improved recovery of LVDP obtained with NMHH applied on reperfusion as compared with that obtained with controls and equimolar concentrations of DFO or NAC suggests that the novel hydroxamate NMHH is the more effective in attenuating reperfusion injury of contractile function in this model.

The effects of N-methyl butyrohydroxamic acid and other monohydroxamates on reperfusion-induced damage to contractile function in the isolated rat heart.

The effects of three novel methyl substituted monohydroxamates N-methyl butyrohydroxamic acid (NMBH), N-methyl acetohydroxamic acid (NMAH) and N-methyl benzohydroxamic acid (NMBzH) against reperfusion induced contractile dysfunction were investigated in the isolated Langendorff-perfused rat heart. All these drugs produced an improved recovery of left ventricular developed pressure (LVDP) compared to control hearts in the order NMBH* (65 +/- 8%) > NMAH (59 +/- 8%) > NMBzH (48 +/- 3%) > control (35 +/- 3%) (mean +/- s.e.), however only the recovery obtained in NMBH treated hearts was significantly different from control hearts (*p > 0.05, Dunnett's test). Both NMAH (98 +/- 10%) and NMBH (84% +/- 8) produced a significant improvement in the recovery of heart rate (control 48 +/- 13%). There was no significant improvement of coronary flow, and NMBzH-treated hearts showed a significant reduction in recovery. The improved recovery in both LVDP and heart rate obtained with NMBH suggests this drug may be effective in attenuating reperfusion-induced contractile dysfunction in the isolated rat heart. Further, a comparison of the structures of the hydroxamates described in this study with the results obtained with desferrioxamine, (a trihydroxamate), and N-methyl hexanoylhydroxamic acid (NMHH) in other studies suggests that the nature of the alkyl chain attached to the carbonyl group of the hydroxamate may contribute to the efficacy of monohydroxamates in attenuating this type of myocardial injury in this model.

Kinetics and Na independence of amino acid uptake by blood side of perfused sheep choroid plexus.

The kinetic constants and sodium independence of amino acid uptake by the basolateral face (blood side) of the isolated perfused choroid plexus of the sheep were investigated. Uptake of 3H-labeled L-alanine, glycine, L-glutamine, L-leucine, L-glutamate, and L-lysine was not significantly inhibited when the sodium level of the perfusate was lowered to less than 6 mM. The cerebrospinal fluid (CSF) secretion rate was, however, reduced by 65.6%. The Michaelis constants for L-serine, glycine, L-phenylalanine, L-glutamate, and L-arginine were measured and varied between 25.4 microM for L-arginine and 2.6 microM for L-glutamate. The Vmax for the amino acids varied by about the same range as Km, with L-serine having the highest Vmax of 28.8 nmol.min-1.g-1, and L-phenylalanine having the lowest at 4.6 nmol.min-1.g-1. At normal plasma concentrations of 30-155 microM for individual amino acids, the carriers for glycine, L-phenylalanine, and L-glutamate would be almost fully saturated (greater than 90%), while those for L-serine and L-arginine would be greater than 60% saturated. This restricted uptake of amino acids by the basolateral face of the choroid plexus, coupled to clearance of these substances out of the CSF (5), could account for the low levels in the CSF compared with that in the plasma.

The effects of (R) alpha-methyl histamine on the isolated guinea pig aorta.

The effects of the H3 agonist (R) alpha-methyl histamine ( (R) alpha-MeHA) on the isolated guinea pig aorta were investigated. At all the concentrations of (R) alpha-MeHA used, a relaxation of the aorta, which had been preconstricted with KC1 (50mM), was produced. This response was abolished by the H3 antagonist mixture of cimetidine (100 microM) and impromidine (10 microM). In contrast the same concentrations of histamine produced vasoconstriction which could be abolished by the H1 antagonist pyrilamine (3 microM). The results show that (R) alpha-MeHA produces a vasodilation response which is most probably mediated by H3 receptors and provides further evidence of a functional role for H3 receptors in the control of the cardiovascular system.

The effects of iron and vitamin C co-supplementation on oxidative damage to DNA in healthy volunteers.

The effects of co-supplementing healthy volunteers with iron (14 mg/day ferrous sulphate) and vitamin C (either 60 mg/day or 260 mg/day as ascorbic acid) on levels of oxidative DNA damage in white blood cells were studied. The subjects were divided into two groups: one group of 20 volunteers with a higher mean initial level of plasma vitamin C (71.9 +/- 14.0 mumol/l) and a second group of 18 volunteers with a lower mean level (50.4 +/- 25.8 mumol/l). In the first group there was a significant rise in several oxidative DNA base damage products and in total oxidative DNA damage in DNA extracted from white blood cells, but not in 8-hydroxyguanine, after 6 weeks of supplementation. However, after 12 weeks levels returned approximately to normal. In the group with the lower initial level of plasma ascorbate, presupplemental levels of oxidative DNA damage were higher and decreased on supplementation with iron and ascorbate. Since oxidative DNA damage has been suggested as a risk factor for the development of cancer, the implications of increased levels in well-nourished subjects after iron/ascorbate supplementation are disturbing in view of the frequent use of dietary supplements containing both iron salts and ascorbate.