Effect of long-term therapy with fasidotril, a mixed inhibitor of neprilysin and angiotensin-converting enzyme (ACE), on survival of rats after myocardial infarction.

OBJECTIVE: Two hormonal systems with opposite effects are activated in congestive heart failure: the renin-angiotensin system that promotes vasoconstriction, cardiac hypertrophy and salt retention, and the atrial natriuretic factor (ANF), which has vasorelaxant and natriuretic effects. It could be of therapeutic interest to associate prevention of angiotensin II formation, by inhibition of angiotensin I-converting enzyme (ACE), with potentiation of the ANF effects, by inhibition of neprilysin (NEP). METHODS: The effects of long-term therapy with fasidotril, a mixed NEP/ACE inhibitor, were assessed in rats submitted to coronary artery ligation. Twenty-four hours after ligation, 172 rats were assigned to either placebo or fasidotril therapy (180 mg/kg/day, orally) for 40 weeks. The date of spontaneous death was recorded, myocardial infarct size was determined and rats were classified as having small, moderate or large infarcts. RESULTS: In rats with moderate infarcts, fasidotril prolonged survival, 50% of the control rats dying during the 40-week observation period compared with 30% of treated rats (P = 0.04, log-rank test)). In rats with large infarcts, mortality was significantly reduced during the initial 25 weeks of therapy, during which 23.5% of animals died compared to 53.8% in untreated rats (P = 0.015). Cardiac hypertrophy was significantly attenuated by fasidotril for the three infarct sizes. Plasma renin activity was not increased by therapy, which presumably reflected the inhibition of renal renin secretion by endogenous ANF. Fasidotril therapy had no significant effects on arterial blood pressure and heart rate. CONCLUSION: In addition to its beneficial effects on survival and cardiac hypertrophy, the lack of hypotensive effect of fasidotril is of interest by reducing the risk of renal hypoperfusion and differentiates the mixed inhibitor from selective ACE inhibitors.

Protective action of 1,3-butanediol in cerebral ischemia. A neurologic, histologic, and metabolic study.

1,3-Butanediol (BD) is converted in the body to beta-hydroxybutyrate, and previous studies have shown that hyperketonemia had beneficial effects in experimental models of generalized hypoxia. The aim of this study was to determine if BD would reduce brain damage following cerebral ischemia. A transient forebrain ischemia of 30-min duration was induced by the four-vessel occlusion technique in control and BD-treated rats (25 mmol/kg, i.p.; 30 min prior to ischemia). BD treatment led to significant improvement of neurologic deficit during the 72-h recovery period and reduced neuronal damage in the striatum and cortex but not in the CA1 sector of the hippocampus. Evaluation of cerebral energy metabolism before and at the end of the ischemic period showed that the treatment did not change the preischemic glycolytic and energy metabolite levels but attenuated the ischemia-induced metabolic alterations. It increased energy charge, phosphocreatine, and glucose levels, and reduced lactate accumulation. The decrease in brain lactate concentration might account for the beneficial effects of BD by minimizing the neuropathological consequences of lactic acidosis.

Ischemia-induced brain iron delocalization: effect of iron chelators.

Tissue damage in cerebral ischemia may be produced by acidosis-induced delocalization of intracellular iron which acts as a catalyst in oxidative reactions. Acidosis was induced either by homogenization and incubation of rat cortical homogenates in acidified buffers or by submitting hyperglycemic rats to complete ischemia, a procedure that leads to intracellular lactic acidosis. The level of low molecular weight species (LMWS) iron was measured after filtration of tissue homogenates through a 10,000 Mr ultrafiltration membrane. When cortical tissue was homogenized in buffer pH 7, the level of LMWS iron was equal to 0.21 microgram/g. It was significantly enhanced by acidification of the homogenization medium, reaching 0.34 microgram/g at pH 6 and 0.75 microgram/g at pH 5. When the tissue was homogenized in water, the LMWS iron level reached 0.17 microgram/g in normoglycemic rats and 0.38 microgram/g (p < .05) in hyperglycemic rats. Both aerobic incubation of homogenates for 1 h at 37 degrees C and inclusion of EDTA in the homogenization medium led to further increases in the iron level. In order to demonstrate the deleterious role of iron in brain ischemia, the effect of treatment with bipyridyl, an iron-chelating agent, was assessed by measuring regional brain edema by the specific gravity method, 24 h following induction of thrombotic brain infarction. The treatment significantly attenuated the development of brain edema, reducing the water content of the infarcted area by about 2.5%. Taken together, these results support the hypothesis that a significant component of brain ischemic injury involves an iron-dependent mechanism.

Effect of intracellular iron loading on lipid peroxidation of brain slices.

The effect of artificially elevated cell iron content on oxygen-derived free radical production was assessed in brain slices by use of an iron ligand, 8-hydroxyquinoline (HQ). The iron complex Fe(3+)-HQ exhibited a high lipid solubility evidenced by n-octanol/water partition coefficient and was avidely taken up by brain slices. The catalytically active form of Fe3+ within the complex was evidenced by measuring the rate of ascorbate oxidation. Lipid peroxidation was assessed by measuring the thiobarbituric acid-reactive substances (TBARS) in brain homogenates or slices exposed to two doses of Fe(3+)-HQ (10 microM/20 microM, 100 microM/200 microM) or Fe(3+)-citrate (10 microM, 100 microM). Addition of the iron complexes to homogenates or slices resulted in a dose-dependent increase in lipid peroxidation. In homogenates, the effects were grossly similar with both complexes, whereas in slices the effects of Fe-HQ were significantly higher than those of Fe-citrate. Lipid peroxidation persisted in washed slices preexposed to Fe-HQ, but not in slices preexposed to the hydrophilic iron complex Fe-citrate. Fe-HQ-induced lipid peroxidation in slices was enhanced in the presence of H2O2, an effect that was not seen using Fe-citrate. Addition of Fe-HQ to brain homogenates in the presence of salicylic acid resulted in the production of 2,3-dihydroxybenzoic acid and the effect was potentiated in the presence of H2O2. This model of iron cell loading may be useful for evaluating the efficacy of antioxidant drugs.

Effect of alpha-adrenoceptor antagonists (phentolamine, nicergoline and prazosin) on reperfusion arrhythmias and noradrenaline release in perfused rat heart.

Rat isolated hearts were perfused through the left atrium with a modified Krebs-Henseleit solution or mounted on a Langendorff perfusion system. The hearts were prelabelled with [3H]-noradrenaline [( 3H]-NA) and the left main coronary artery was ligated for 10 min after which reperfusion followed. The liberation of [3H]-NA and the development of ventricular tachycardia and fibrillation were monitored throughout. During the occlusion period, ventricular arrhythmias did not occur and heart rate was not significantly altered in the control series. In contrast, reperfusion was followed by ventricular fibrillation and ventricular tachycardia in all the hearts in the control series (Langendorff or 'working' models). The alpha-adrenoceptor antagonists phentolamine (7.1 X 10(-6) M and 7.1 X 10(-5) M) and nicergoline (3.1 X 10(-6) M) diminished or prevented reperfusion arrhythmias. However, prazosin (5.2 X 10(-6) M) was not effective. The lower concentration of phentolamine did not alter the pattern of [3H]-NA release, whereas, high doses of phentolamine and nicergoline increased the release of [3H]-NA. Prazosin (5.2 X 10(-6) M) caused a very marked increase in release of [3H]-NA but was not antiarrhythmic. A 'membrane-stabilizing' effect seems the most appropriate explanation for these antiarrhythmic effects of alpha-antagonist agents.

Effect of acidosis and anoxia on iron delocalization from brain homogenates.

Cortical homogenates were prepared from rat brain in Krebs-Ringer phosphate media adjusted to pH 7, 6 or 5 and incubated for 1 hr under aerotic or anaerobic conditions in the presence of dipyridyl, an iron chelator. Low molecular weight species (LMWS) iron was measured spectrophotometrically after passing of the homogenates through a 10,000-Mr ultrafiltration membrane. Following aerobic incubation, LMWS iron reached 1.24 micrograms/g tissue at pH 7, and increased 1.7-fold at pH 6 and 3.1-fold at pH 5. Anoxia enhanced significantly the amount of ultrafiltrable iron at the three pH values, the LMWS iron level being increased by 190% at pH 7, by 113% at pH 6, and by 77% at pH 5. Addition of the ultrafiltrates to brain membranes caused significant rises in the production of lipid peroxides assessed by the thiobarbituric acid test, indicating that LMWS iron was in a form capable for catalysing oxygen-derived free radical-mediated lipid peroxidation. It was concluded that decompartmentalization of intracellular iron may be an important factor in the initiation of peroxidative damage to ischemic cells.

Effect of acidosis on lipid peroxidation in brain slices.

Acidification of the incubation medium markedly increased lipid peroxidation of cortical brain slices. Lactic acidosis caused a more extensive lipid peroxidation than did phosphoric acidosis (+35% at pH 6 and +81% at pH 5), probably due to the rapid diffusion of the protonated form of lactic acid across cell membranes. These results support the hypothesis that free radical mechanisms may be involved in the cytotoxicity of acidosis.

Diuretic and natriuretic responses in rats treated with enkephalinase inhibitors.

Rat atrial natriuretic factor (125I-rANF, 99-128) is hydrolysed by pure enkephalinase (EC 3.4.24.11) in vitro at a rate similar to that of 125I-hANF. Trichloroacetic precipitated radioactivity was significantly elevated in the kidneys of rats pretreated with acetorphan, an enkephalinase inhibitor, and receiving 125I-rANF, indicating that the exogenous hormone was protected against degradation. A single oral administration of acetorphan elicited diuretic and natriuretic effects in conscious normotensive rats and natriuretic effects in spontaneously hypertensive rats, effects which were not accompanied by significant changes in kaliuresis. The diuretic and natriuretic effects were still observed in conscious normotensive rats after three days of repeated administration of the drug. In conscious or anesthetized rats in which volume expansion was elicited by hydroelectrolytic loads, the initial rate of urinary elimination of water and sodium was nearly doubled by treatment with enkephalinase inhibitors. This effect was prevented by coadministration of an ANF antiserum, which suggests that the effect was mediated by endogenous ANF. These various observations suggest that enkephalinase inhibitors protect endogenous ANF from degradation and thereby enhance the typical renal effects of the hormone.

Neurologic and cytologic outcome following repeated ischemia. Effect of pentobarbital.

We examined clinical recovery from repeated brain ischemic insults that have been reported to affect cytologic outcome. Brain ischemia was induced in the rat by four-vessel occlusion. A 30-min ischemia was given as a single insult or induced in animals made ischemic 24 h earlier by a 10-min insult but exempt both of brain hypoperfusion and neurologic deficit in spite of a partial necrosis of the CA1 sector of hippocampus. Repeated ischemia was associated with a significantly poorer clinical outcome as indicated by an increase in percentage of rats that exhibited postischemic seizure activity combined with the percentage of unconvulsive rats exhibiting neurologic deficits after 72 h of reperfusion (81% vs. 50% after a single 30-min ischemia). Examination of hippocampal damage showed that neurons surviving the first ischemia did not acquire resistance to the second ischemia. Pentobarbital given from start of overt seizures (30 to 60 mg/kg, IP, thrice daily) was able to stop convulsions and to antagonize processes involved in ischemia-induced neuronal death of CA1 hippocampus.

Effect of chronic clonidine treatment on the turnover of noradrenaline and dopamine in various regions of the rat brain.

The turnover of noradrenaline (NA) and dopamine (DA) was estimated in various rat brain regions by measuring the depletion of the amines after inhibition of their biosynthesis by alpha-methyltyrosine. Acute treatment with clonidine (0.1 mg/kg) reduced NA turnover in the brain stem, hypothalamus and rest of the brain but had no effect on DA turnover in the corpus striatum and rest of the brain. After chronic clonidine treatment (0.1 mg/kg, twice daily for 156 days), NA turnover was not affected by an additional injection of clonidine in the brain stem or in the hypothalamus but was still markedly reduced in the rest of the brain. In addition, DA turnover was reduced in the corpus striatum and rest of the brain, an effect which was also observed after a single injection of a high dose of clonidine (1 mg/kg). These findings suggest that a chronic administration of clonidine may cause regionally differential changes in the sensitivity of central NA receptors.

Photochemically induced, graded cerebral infarction in the mouse by laser irradiation evolution of brain edema.

Cerebral infarction was produced in mice by intravenous injection of a photosensitive dye, rose bengal (10 mg/kg), and by focal illumination of the intact skull surface for 3 min with a laser source, operating at 570 nm with power levels of 2, 5, 10, and 20 mW. Location of infarction was made using 2,3,5-triphenyltetrazolium chloride and the time course of edema in the irradiated cerebral hemisphere was evaluated by measuring water, sodium, and potassium content 4, 24, and 72 hr after irradiation. With power levels of 2 and 5 mW, the infarct was restricted to the cortex, whereas with power levels of 10 and 20 mW, it extended to subcortical regions. Increases in water and sodium and decrease in potassium content were maximal 24 hr after irradiation and depended on the power level. Highly significant correlations were found, 24 hr after irradiation, between the power level and the changes in water and ion concentrations. With reproducible, incremental grades of damage being desirable for pharmacological trials, the model may be suited for the testing of therapeutic agents.

Dopamine and norepinephrine turnover in various regions of the rat brain after chronic manganese chloride administration.

Rats were treated with MnCl2 X 4H2O (1 mg/100 g/day, i.p.) for a period of 4 months. The turnover of dopamine (DA) and norepinephrine (NE) was measured in several brain regions (brain stem, hypothalamus, corpus striatum and "rest of the brain") by the decay in endogenous DA and NE after inhibition of tyrosine hydroxylase by alpha-methylparatyrosine. Monoamine oxidase (MAO) activity and manganese levels were also estimated. Manganese treatment produced a decrease in DA level and turnover in the corpus striatum but not in the rest of the brain. An increase in contents of NE was observed both in the brain stem and hypothalamus. NE turnover was found to be increased in the brain stem, decreased in the hypothalamus and unaltered in the rest of the brain. MAO activity was not significantly altered in all the brain regions studied. These results which show that chronic administration of manganese may cause regionally different changes in catecholamine turnover were discussed in relation to the accumulation of manganese in the brain regions and to other metabolic changes associated with manganese toxicity.

Enkephalinase (EC 3.4.24.11) inhibitors: protection of endogenous ANF against inactivation and potential therapeutic applications.

Atrial natriuretic factor (ANF) is a cardiac hormone exerting potent cardiovascular and renal effects but its poor intestinal absorption and rapid inactivation have prevented so far its therapeutic utilisation. However inhibition of endogenous ANF metabolism progressively emerges as a novel therapeutic approach in cardiovascular and renal disorders. The critical role played by enkephalinase (membrane metalloendopeptidase, EC 3.4.24.11) in ANF inactivation was deduced from the effects of inhibitors. These compounds not only protect partially exogenous ANF from hydrolysis by some tissue preparations in vitro but also, in vivo, they increase the half-life of the exogenous hormone in plasma and, even more markedly, its recovery in intact form in kidney, a major target organ. In addition, enkephalinase inhibitors increase by two- to three-fold the circulating level of endogenous ANF, even when the latter is already markedly elevated, such as in patients with chronic heart failure. Finally, enkephalinase inhibitors induce a series of ANF-like responses such as natriuresis, diuresis or increase in cGMP excretion which are attributable to the hormone. These pharmacological observations, as well as preliminary clinical trials, suggest that enkephalinase inhibitors may represent a novel class of therapeutic agents with potential applications in congestive heart failure, essential hypertension and various sodium-retaining states.

Effects of promethazine on ischemic and reperfusion arrhythmias in rat heart.

The effects of the H1-receptor antagonists promethazine, mepyramine, and chlorpheniramine on ischemic and reperfusion arrhythmias were studied in the isolated perfused rat heart. Promethazine reduced both ischemic and reperfusion arrhythmias (2 x 10(-6)M-7.5 x 10(-6)M). Mepyramine and chlorpheniramine decreased these arrhythmias but at concentrations about 10 times higher. The H2-blockers cimetidine and ranitidine had no antiarrhythmic effect. Promethazine also: (i) increased release of noradrenaline by the heart; and (ii) increased coronary flow in the reperfusion period and in some mildly ischemic zones. It is proposed that promethazine exerts most of its antiarrhythmic effects by a nonspecific mechanism, possibly membrane stabilization; in addition, enhanced coronary flow may play a role.

PAF-acether-induced mortality in mice: protection by benzodiazepines.

Intraperitoneal injection to mice of platelet-activating factor (PAF) induces severe shock symptoms, leading to death. The effects of various drugs which are potential specific or non-specific PAF inhibitors were tested. Salbutamol, a beta 2-agonist, showed a partial protection, whereas the specific PAF antagonists CV 3988 or BN 52021 induced an important beneficial action against PAF-induced lethal effects. The effects of drugs related to benzodiazepines were also studied: alprazolam administered p.o. prior to PAF challenge provides almost complete protection against PAF toxicity. Thus PAF-induced death in mice represents a useful model of systemic anaphylaxis; moreover, studies of benzodiazepine-related compounds may be interesting for investigating the mechanisms of the biological actions of PAF.