Effect of pyracetam on fatigue of inspiratory muscles in cats

The aim of the work was to study the influence of pyracetam on respiratory muscles fatigue and ventilatory disorders caused by inspiratory resistive load in cats. The experiments have show that after the use of pyracetam in conditions of fatigue total bioelectric activities of inspiratory muscles and of the phrenic nerve and transdiaphragmal pressure restore; duty cycle, respiratory rate and tidal volume per minute decrease. The conclusion is drawn that pyracetam in the dose 300 mg/kg, in intravenous administration, compensates inspiratory muscle fatigue at the expense of its central mechanism of action.     

Multiple mechanisms underlying nootropic drug-induced enhancement of neuronal responses mediated by activation of glutamate NMDA-type receptors

Nootropic drugs of various chemical structure (pyracetam, dimethylaminoethanol, ethymisol, and ambocarb) possessing antiamnesic activity were found to increase depolarization evoked by L-aspartate and the EPSP late components evoked by dorsal roots stimulation (DR-EPSP). Experiments were performed on motoneurons of the lumbar region of isolated frog spinal cord superfused with Mg²⁺-free saline. When ionic channels of NMDA-glutamate receptors were blocked with ketamine and Mg²⁺, the nootropic substances did not affect DR-EPSP and motoneuronal depolarization evoked by L-glutamate. Butamid, a protein kinase A inhibitor, blocked the ethymisol and pyracetam effects, whereas polymixin B, a protein kinase C inhibitor, blocked the ambocarb effect. Trifluoroperazine, a calmodulin inhibitor, blocked the effect of dimethylaminoethanol. Proline inhibited only the pyracetam effect. These results indicate that the NMDA-potentiating activity of the above nootropic drugs may be based on both intracellular phosphorylation of NMDA receptor-channel complexes, as mediated by protein kinases, and allosteric enhancement of affinities of these complexes to excitatory amino acids.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 179–184, May–June, 1993.     

Characteristics of the antihypoxic action of piracetam

Pyracetam was found to inhibit free-radical lipid peroxidation, slow oxygen consumption in the liver mitochondria and increase hemoglobin oxygen-binding properties. It is assumed that the ability of pyracetam to inhibit lipid peroxidation is a mechanism which determines its antihypoxic effect.     

Synchronization dynamics in the neuronal work of the neocortex and hippocampus during learning before and after the administration of nootropics and narcotics

The basal difference in action of the studied drugs was that nootropics (phenybut in a dose of 40 mg/kg and pyracetam in a dose of 200-400 mg/kg) did not change the initial action of pain reinforcement on synchronism in responses of the cortical neurones of alert nonimmobilized rabbits by inhibitory type (coincidence of the presence and absence of impulse activity) towards its decrease, while narcotics of various types (ethanol in a dose of 4-6 mg/kg, morphine-like opiate DAGO and opioid peptide DADLE in doses of 250 mkg/kg) eliminated the action of pain reinforcement on synchronism in responses of the cortical neurones both by inhibitory and activation (time of coincidence only of the presence of impulse activity) types. These and other drugs mainly weakened the initial action of both the inhibitory and reinforced light flashes of synchronism in neurones activity both by inhibitory and activation types. There was no constant parallelism between changes of synchronization and the frequency of the cortical impulses.     

Neuro-and psychotropic activity of <Emphasis Type="Italic">N</Emphasis>-uronoylamino acids and <Emphasis Type="Italic">N</Emphasis>-uronoylpeptides

Peculiarities of the rat behavior were studied in a series of experimental stress models after a systemic administration of new N-uronoyl derivatives of amino acids. The psychotropic effect was shown to be determined by the nature of the amino acid fragment. N-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyraneuronoyl)-glycylglycine exhibited an anxiolytic effect more pronounced than that of pyracetam, whereas N-(1,2:3,4-di-O-isopropilidene-α-D-galactopyranuronoyl)-glycylglutamic acid has antidepressant action stronger than that of amitriptyline. Mechanisms for the psychotropic effects of the examined derivatives are discussed.     

Activity of marker enzymes and status of the erythrocyte membrane lipid matrix in stress and during its correction using medications]

On the basis of experiments it is shown that chronic stress at the exhaustion stage is followed by activation of lipid peroxidation processes and inhibition of the antioxidant system. Products of peroxide lipid oxidation are accumulated, which in its turn may induce pathological changes in the ratio of different lipid fractions and disturbance in the functional activity of membranes. Under the stress, when a preventive average therapeutic dose of the nicotinic acid derivatives (lithonite, nicogamol, OMI-17) and GABA derivatives (pyracetam, pycamilon) are introduced to rats, activity of marker enzymes remains practically at the control level. A normalizing effect of these drugs on the parameters studied is connected with its expressed membrane-stabilizing effect and non-specific defense of the cytomembranal lipid matrix. Individual peculiarities in pharmacological activity of these drugs are indicated.     

Neuronal responses mediated by activation of the non-NMDA receptors: Potentiation by nootropes

Experiments on superfused slices of rat hippocampus showed that the nootropic drugs pyracetam, ethymizol, ambocarb, and nooglutil increase the amplitude of populational EPSP (pEPSP) of neurons of the dentate gyrus evoked by electrical stimulation of the perforant pathway (PP). Nootropes exert no effect on the process of presynaptic glutamate liberation from the PP axons, but increase the chemosensitivity of the postsynaptic AMPA/kainate receptors mediating EPSP generation in the dentate gyrus neurons. Inhibitors of protein kinase (A-buthamide) and guanylatecyclase (methylene blue) do not modify the effects of nootropes. The nootrope-induced potentiation of pEPSP does not develop against the background of the application of calmodulin inhibitor W-7. In the presence of protein kinase inhibitor C, polymixin B, nootropes reversibly depress pEPSP in the dentate gyrus neurons. Blocking of the NMDA receptor ionic channels by ketamine and of the voltage-dependent T-type calcium channels by Ni²⁺ does not significantly modify the effects of nootropic drugs. A blocker of Ca²⁺-ATPase of the Ca²⁺ stores sodium orthovanadate, potentiates the effects of nootropes. Dantrolene, which disrupts Ca²⁺ liberation from the non-mitochondrical depots, blocks the effects of nootropes and diminishes pEPSP depression evoked by nootropes in the presence of polymixin B. On the basis of presented data, it is supposed that nootropic drugs assist Ca²⁺ liberation from the neuronal depots and activate calmodulin-dependent protein kinase and protein kinase C. Protein kinases phosphorylate the intracellular domains of the AMPA/kainate receptors, and this process results in an increase in their sensitivity to excitatory amino acids.Neirofiziologiya/Neurophysiology, Vol. 26, No. 5, pp. 365–372, September–October, 1994.     

Habituation of caddies Chaetopteryx villosa larvae to vibrational stimulus and effect of piracetam on it

Habituation to a vibrational stimulus was studied in intact caddies Chaetopteryx villosa Fabr. of the last age before pupation and of the larvae placed for 72 h into pyracetam solution (100 mg/l). The vibrational stimulus was shown to produce a passive-defensive reaction: the caddies were pulled into the house and stood still. In the course of the experiment the time of stay in the house decreased statistically significantly in both groups. Pyracetam did not affect the defensive reaction at the initial moment of the first vibrational stimulus, so both in the experimental and in the control groups the number of contracted larvae was equal and all of them remained in the house for the first seconds of vibration. However, the compound increased the number of larvae that did not respond to the next vibrational stimulus and decreased the number of larvae that remain in the house throughout all 10 s of action by accelerating on the whole the habituation. Hence, on this model, the mnemotropic effect of the standard nootrope has been realized, which earlier was established by other methods in mammals, the lower vertebrates, and the higher invertebrates. This indicates that this model also is perspective for comparative studies of effect of nootropic substances on the higher nervous activity.     

Neurochemical mechanisms underlying NMDA-independent long-term post-tetanic potentiation of synaptic transmission

In experiments performed on rat transversial slices of the rat dorsal hippocampus, we found that high-frequency tetanic stimulation of the mossy fibers (MF) and short-term action of 1 μM kainic acid on the slices resulted in long-term potentiation of the population spikes evoked inCA3 pyramidal neurons by single stimuli applied to the MF. The tetanus-and kainate-induced potentiations of synaptic transmission were accompanied by a decrease in the degree of paired facilitation at a 50-msec-long interstimulus interval; they were additive, prevented by 10 μM CNQX, a competitive antagonist of AMPA/kainate receptors, and insensitive to 100 μM ketamine, a noncompetitive antagonist of NMDA-glutamate receptors. Both types of potentiation were enhanced by 10 μM (1S, 3R)-ACPD, an agonist of metabotropic glutamate receptors, as well as by 1 μM pyracetam or 50 μM dichlothiazide, substances weakening AMPA/kainate receptor desensitization. The effects produced by high-frequency tetanic stimulation of the MF and by kainic acid were prevented by 50 μM polymixin B, a protein kinase C blocker, and weakened by 10 μM trifluoroperazine, a calmodulin inhibitor, or 1 μM pirenzepine, an M1 acetylcholine receptor blocking agent. In total, the above data suggest that the tetanus- and kainate-induced potentiations of transmission in the synapses formed by the MF and dendrites ofCA3 pyramidal neurons are due to the combined activation of pre-synaptic high-affinity kainate-preferring receptors, located in the membranes of the MF varicosities, and post-synaptic phosphoinositide metabolism-coupled metabotropic glutamate receptors and 1 and M1 acetylcholine receptors. This activation results in a significant increase in the activity of epsilon-form protein kinase C, phosphorylation of protein substrates involved in vesicular glutamate release from the MF varicosities, and long-term enhancement of presynaptic glutamate release.     

Cutting edge: mechanism of enhancement of in vivo cytokine effects by anti-cytokine monoclonal antibodies

Inhibitory anti-cytokine mAbs are used to treat cytokine-mediated disorders. Recently, however, S4B6, an anti-IL-2 mAb that blocks IL-2 binding to IL-2Ralpha, a receptor component that enhances affinity but is not required for signaling, was shown to enhance IL-2 agonist effects in vivo. We evaluated how S4B6 enhances IL-2 effects and whether a similar mechanism allows mAbs to IL-4 to enhance IL-4 effects. Induction of T cell proliferation by IL-2/S4B6 complexes did not require complex dissociation and was IL-2Ralpha independent. S4B6 increased IL-2 agonist effects by increasing in vivo half-life, not by focusing IL-2 onto cells through Fc receptors. In contrast to IL-2/S4B6 complexes, anti-IL-4 mAb enhancement of in vivo IL-4 effects required IL-4/anti-IL-4 mAb complex dissociation. Thus, agonist effects observed with high doses of anti-IL-2 mAb are most likely only applicable for mAbs that maintain cytokine half-life without blocking binding to receptor signaling components.     

Comparison of the effects of amiodarone and ipodate on the rat heart

Chronic treatment of rats with amiodarone has been shown to produce hypothyroid-like effects such as a reduction in body and heart weight and increased synthesis of the low ATPase V3-cardiac isomyosin (Bagchi, Brown, Schneider and Banerjee 1987). In this report, we have tested the hypothesis that amiodarone causes these effects through the inhibition of intracellular production of triiodothyronine (T3) from thyroxine (T4) by comparing the effects of amiodarone with those of ipodate, a potent inhibitor of T4 to T3 conversion. Separate groups of rats were given dietary ipodate and amiodarone respectively for six weeks. Both agents increased serum T4 and T4/T3 ratios, a finding consistent with the inhibition of peripheral T4 to T3 conversion. However, ipodate failed to produce hypothyroid-like effects on body weight, heart weight and isomyosin transitions similar to those found in the amiodarone group. These data indicate that the hypothyroid-like effects of amiodarone on the rat heart are not due to the inhibition of intracellular generation of T3 from T4.     

Effects of 4-Aminopyridine on Extracellular Concentrations of Glutamate in Striatum of the Freely Moving Rat

4-aminopyridine (4-AP) is a voltage-sensitive K⁺-channel blocker extensively used in in vitro experiments as a depolarizing agent for the release of glutamate (GLU). This research investigated whether 4-AP could be used in in vivo experiments using microdyalisis. For that, the effects of 4-AP on the extracellular concentrations of glutamate (GLU), glutamine (GLN), taurine (TAU) and citrulline (CIT) in striatum of the freely moving rat were investigated. The effects of 4-AP were compared with those produced by perfusion with a high K⁺ (100 mM) medium. Intrastriatal perfusion with 4-AP (1, 5 and 10 mM) produced no effects on extracellular [GLU], [TAU] and [CIT], but decreased extracellular [GLN]. Perfusion with a high K⁺ (100 mM) medium increased extracellular [GLU] and [TAU], decreased extracellular [GLN], and had no effects on [CIT]. To test whether the lack of effects of 4-AP on extracellular [GLU] was due to GLU uptake mechanisms, 4-AP was perfused after a previous inhibition of GLU uptake with L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). Under the effects of PDC (1 mM), 4-AP (1 mM) had no effects on extracellular [GLU], [TAU] and [CIT], but decreased extracellular [GLN]. These results show that 4-AP decreased extracellular [GLN] but failed to produce a significant release of GLU in striatum of the freely moving rat. Thus, 4-AP can not be used as a depolarizing agent for stimulating the release of GLU in in vivo studies using microdialysis.     

Pharmacological characterization using selected antagonists of the leukotriene receptors mediating contraction of guinea-pig trachea

The effects of six leukotriene (LT) antagonists on LTC4-, D4- and E4-induced contraction of isolated guinea-pig tracheal spirals were examined. Concentration-response effects of the leukotrienes were determined by cumulative addition in the presence of indomethacin (5 microM) alone for LTE4, or with 10 mM of either glutathione or L-cysteine to inhibit metabolism of LTC4 or LTD4, respectively. Concentration-response curves to the LTs were obtained in the absence and presence of Wy-45,911, Wy-44,329, FPL-55,712, Ly-171,883, Wy-48,252 and ICI-198,615 representing three structurally different chemical groups of LT antagonists. At 30 microM, the antagonists produced little or no antagonism of LTC4-induced contractions. Analysis of the Schild plots for antagonism of LTD4 and E4 suggested two receptors for the agonist effects of LTD4 and a single receptor for the agonist effects of LTE4. Comparison of pA2 values for Wy-45,911, FPL-55,712, LY-171,883 and Wy-48,252 provided evidence that LTE4 is acting at the antagonist high affinity LTD4 receptor to produce contractile effects. From the data, we conclude that there are three LT receptors (one for LTC4 and two LTD4 subtypes) through which exogenously applied LTs evoke contraction of the isolated guinea-pig trachea.     

Acute pulmonary hemodynamic effects of intravenous copper sulfate: role of alpha-adrenergic system

We investigated the acute pulmonary hemodynamic effects of intravenous copper sulfate (CuSO4) infusion and its mechanism of action in six groups of conscious sheep (total 40). After 300 mg CuSO4 alone, mean pulmonary artery pressure (Ppa) increased from 10.3 to 22.5 Torr and pulmonary artery wedge pressure (Ppaw) from 3.5 to 7.6 Torr, whereas systemic arterial pressure (Psa) increased from 95 to 102 Torr. Cardiac output (Qp) decreased from 4.7 to 3.3 l/min. Pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) increased to 320 and 160% of base line, respectively. The hemodynamic changes correlated well with serum copper, which increased from a base-line value of 0.12 to 3.5 mg/dl after the CuSO4. Serum dopamine beta-hydroxylase increased from 3.2 U/l before CuSO4 injection to 5.7 after its administration, signifying activation of adrenergic nervous system. H1-histamine receptor blockade with chlorpheniramine failed to prevent the effects of CuSO4. Pretreatment with methysergide, a serotonin antagonist, partially attenuated the effects of CuSO4. Phenoxybenzamine, an alpha-adrenergic receptor blocker, and 6-hydroxydopamine, a catecholamine depleting agent, completely blocked the effects of CuSO4. beta-Adrenergic receptor blockade with propranolol enhanced the effects of CuSO4. We conclude, that, in conscious sheep, acute infusion of CuSO4 caused a marked reversible increase in PVR with a slight transient increase in SVR, and this pulmonary hypertension was produced by stimulation of the alpha-adrenergic nervous system.     

Evidence that Formation of Protoanemonin from Metabolites of 4-Chlorobiphenyl Degradation Negatively Affects the Survival of 4-Chlorobiphenyl-Cometabolizing Microorganisms

A rapid decline in cell viability of different PCB-metabolizing organisms was observed in soil microcosms amended with 4-chlorobiphenyl. The toxic effect could not be attributed to 4-chlorobiphenyl but was due to a compound formed from the transformation of 4-chlorobiphenyl by the natural microflora. Potential metabolites of 4-chlorobiphenyl, 4-chlorobenzoate and 4-chlorocatechol, caused similar toxic effects. We tested the hypothesis that the toxic effects are due to the formation of protoanemonin, a plant-derived antibiotic, which is toxic to microorganisms and which has been shown to be formed from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway. Consistent with our hypothesis, addition to soil microcosms of strains able to reroute intermediary 4-chlorocatechol from the 3-oxoadipate pathway and into the meta-cleavage pathway or able to mineralize 4-chlorocatechol by a modified ortho-cleavage pathway resulted in reversal of this toxic effect. Surprisingly, while direct addition of protoanemonin influenced both the viability of fungi and the microbial activity of the soil microcosm, there was little effect on bacterial viability due to its rapid degradation. This rapid degradation accounts for our inability to detect this compound in soils amended with 4-chlorocatechol. However, significant accumulation of protoanemonin was observed by a mixed bacterial community enriched with benzoate or a mixture of benzoate and 4-methylbenzoate, providing the metabolic potential of the soil to form protoanemonin. The effects of soil heterogeneity and microcosm interactions are discussed in relation to the different effects of protoanemonin when applied as a shock load and when it is produced in small amounts from precursors over long periods.     

PF4 antagonizes retinal neovascularization via inhibiting PRAS40 phosphorylation in a mouse model of oxygen-induced retinopathy

Retinal neovascularization (RNV) is a common pathology of blinding proliferative retinopathies. The current treatments to RNV, however, are hindered by limited efficacy, side effects, and drug resistance. A naturally-occurring cytokine in retina that is amicable to immune system and possesses robust anti-neovascular function would facilitate to overcome the hurdles. In this study, retinas from a mouse model of oxygen-induced retinopathy (OIR) underwent a protein array to screen the naturally-occurring cytokines that may antagonize RNV. Among the 62 angiogenesis-associated cytokines, platelet factor 4 (Pf4) stood out with the most prominent upregulation and statistical significance. Moreover, an intravitreal injection of mouse Pf4 demonstrated dramatic anti-vaso-obliteration and anti-neovascularization effects dose dependently in the OIR model; whereas human PF4 inhibited the proliferation, migration, and tubulogenesis of monkey retinal vascular endothelial cells treated with VEGF and TNF-α. These previously undescribed angiostatic effects of PF4 in OIR retinas and retinal vascular endothelial cells support translation of this naturally-occurring chemokine into a therapeutic modality to RNV supplementary to the anti-VEGFs. Mechanistically, a phosphorylation array and western blots indicated that downregulation of proline-rich Akt substrate of 40 kDa (Pras40) and its phosphorylation were necessary for Pf4's anti-neovascular effects in the OIR retinas. Indeed, overexpression of the wildtype Pras40 and the mutant version with deficient phosphorylation abolished and mimicked the Pf4's angiostatic effects in the OIR retinas, respectively. The similar effects were also observed in vitro. This study, for the first time, links PF4's anti-RNV function to an intracellular signaling molecule PRAS40 and its phosphorylation.     

Cardiac inotropic effects of leukotriene C4 and prostaglandin I2 in the unanesthetized American bullfrog, Rana catesbeiana

The cardiovascular effects of leukotriene (LT) C4 and prostaglandin (PG) I2 were compared in the unanesthetized American bullfrog, Rana catesbeiana. Bullfrogs were instrumented to measure mean arterial pressure, peak ventricular pressure, its derivative (VP + dP/dt), and heart rate. Two hours after recovery from anesthesia, intravenous injections of LTC4 or PGI2 were tested over a dose range from 0.003 to 3 micrograms/kg body weight (bw). Both eicosanoids decreased mean arterial pressure, systolic ventricular pressure, and its derivative (VP + dP/dt). The effects of LTC4 and PGI2 on all parameters were similar at doses below 3 micrograms/kg bw. However, at 3 micrograms/kg bw, LTC4 had more potent negative inotropic effects than PGI2. Both compounds increased heart rate at 0.3 microgram/kg bw, but at 3 micrograms/kg bw PGI2 caused greater increases than LTC4. The hypotensive and negative inotropic effects of LTC4 were blunted in animals pretreated with indomethacin (4 mg/kg bw) to prevent endogenous prostaglandin and thromboxane synthesis, whereas the cardiovascular effects of PGI2 were unaffected by the blockade. The data show that both eicosanoids have similar qualitative effects on blood pressure and cardiac performance. However, the effects of LTC4 may be partially mediated by release of endogenous cyclooxygenase products, possibly PGI2. These results suggest that the bullfrog, an animal with no coronary arteries, is a useful model for comparative studies of cardiac actions of eicosanoids which are independent of effects mediated by changes in coronary vascular resistance.     

Potentiation by ammonia of the metabolic effects of pent-4-enoate in isolated rat hepatocytes.

The metabolic effects of pent-4-enoate were studied in isolated rat hepatocytes; 1 mM-pent-4-enoate did not significantly inhibit gluconeogenesis from lactate, alanine and glycerol, but significantly decreased glucose synthesis from pyruvate. The addition of 1 mM-NH4Cl led to a drastic inhibition of glucose synthesis from all these substrates. In hepatocytes incubated with 10 mM-alanine and 1 mM-oleate, pent-4-enoate at 0.05-1 mM slightly inhibited glucose synthesis and ketogenesis. The addition of ammonia resulted in a dramatic potentiation of the metabolic effects of pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of ATP and acetyl-CoA were also decreased by the addition of ammonia, as were lactate/pyruvate ratio and beta-hydroxybutyrate/acetoacetate ratio. These data suggest that ammonia seriously interferes with the cellular metabolism of pent-4-enoate and leads to a dramatic potentiation of its effects.     

Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects

Previous immunohistochemical studies of the long-term effects of the noradrenergic neurotoxin DSP-4 have demonstrated a remarkably selective vulnerability of norepinephrine (NE) axons of the locus coeruleus (LC). NE axons originating in non-LC NE neurons appear to be largely resistant to the neurotoxic action of DSP-4. We conducted this study to evaluate the acute effects of DSP-4 on NE axons in four different brain regions: cerebral cortex, cerebellum, ventral forebrain, and hypothalamus. NE levels were determined by high-performance liquid chromatography (HPLC) 6 and 24 hr and 14 days after DSP-4 administration. NE axons in these brain regions were visualized in brain sections at 6 and 24 hr after drug treatment, using a specific antiserum to NE. HPLC assays revealed profound reductions of NE levels in cerebral cortex and cerebellum, but only minor decreases in ventral forebrain and hypothalamus. NE immunohistochemistry showed dramatic differences in the acute effects of DSP-4 on NE axon staining: nearly complete loss of staining in cortex and cerebellum, in contrast to an almost unchanged staining pattern in ventral forebrain and hypothalamus. This study demonstrates that NE immunohistochemistry is a valuable tool to assess the acute effects of DSP-4 on NE axons in different brain regions. The results provide the first direct evidence that NE axons are not uniformly acted on by DSP-4 and suggest that the acute effects of DSP-4 are restricted to LC axons.     

Cellular stresses profoundly inhibit protein synthesis and modulate the states of phosphorylation of multiple translation factors.

We have examined the effects of widely used stress-inducing agents on protein synthesis and on regulatory components of the translational machinery. The three stresses chosen, arsenite, hydrogen peroxide and sorbitol, exert their effects in quite different ways. Nonetheless, all three rapidly ( approximately 30 min) caused a profound inhibition of protein synthesis. In each case this was accompanied by dephosphorylation of the eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and increased binding of this repressor protein to eIF4E. Binding of 4E-BP1 to eIF4E correlated with loss of eIF4F complexes. Sorbitol and hydrogen peroxide each caused inhibition of the 70-kDa ribosomal protein S6 kinase, while arsenite activated it. The effects of stresses on the phosphorylation of eukaryotic elongation factor 2 also differed: oxidative stress elicited a marked increase in eEF2 phosphorylation, which is expected to contribute to inhibition of translation, while the other stresses did not have this effect. Although all three proteins (4E-BP1, p70 S6 kinase and eEF2) can be regulated through the mammalian target of rapamycin (mTOR), our data imply that stresses do not interfere with mTOR function but act in different ways on these three proteins. All three stresses activate the p38 MAP kinase pathway but we were able to exclude a role for this in their effects on 4E-BP1. Our data reveal that these stress-inducing agents, which are widely used to study stress-signalling in mammalian cells, exert multiple and complex inhibitory effects on the translational machinery.